Impaginato 139 Adv. Hort. Sci., 2021 35(2): 139­150 DOI: 10.36253/ahsc­10641 Responses of different quality parame­ ters of Chia to arbuscular mycorrhiza and plant growth regulator H.A. Ashour (*), S.E.A. Esmail, A.B. El­Attar Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University, Giza, Egypt. Key words: AMF, hormones, nutritional values, Salvia hispanica, seed yield. Abstract: Field experiment was conducted to evaluate the influence of arbuscu­ lar mycorrhiza fungi (AMF) and foliar spray of plant growth regulators (PGRs) and their interaction on vegetative growth, seed yield and yield attributes and some biochemical criteria of chia (Salvia hispanica L.), in a split plot design with three replications. Plants grown in absence or presence of AMF were sprayed every 2 weeks with benzyl adenine (BA), CPPU [N­(2­chloro­4­pyridinyl)­N’­ phenylurea], common name forchlorfenuron, and Naphthalene acetic acid (NAA) at 50, 20 and 50 ppm respectively, while control plants were sprayed with tap water. The results revealed that, inoculation with AMF generally caused significant augmentation in all studied growth, yield and yield attribut­ es, total chlorophylls and carbohydrates content in leaves, augmentation in nutritional values of seeds like carbohydrates %, macronutrient, micronutri­ ents, proteins %, total flavonoids, oil % compared to non­inoculated plants. In absence or presence of AMF, application of PGRs generally caused significant increases in the studied parameters compared to control. The interaction between NAA and AMF was more effective since gave higher increases in the studied parameters. It can be concluded that, cultivation of chia plant in pres­ ence of mycorrhiza with foliar application of NAA at 50 ppm is recommended for enhancing growth, and nutritional values of seed yield. 1. Introduction Salvia hispanica, ordinarily known as chia, is an annual herbaceous plant which belongs to Lamiaceae family. It is native to southern Mexico and Northern Guatemala; the word of Chia comes from the Nahuatl word “chian” with means oily. The name Salvia hispanica was specified by the Swedish botanist Carl Linnaeus, who discomfited the wild­growing plant coming from the new world with a regional plant from Spain. It grows up to 1­m tall with leaves of about 4­8 cm long and 3­6 cm wide. Chia flowers are white or purple containing oval seed mottle­colored with brown, gray, black, and white with size ranging from 1 to 2 mm. it grows naturally in tropical and subtropical environments; it is optimally established from 400 to 2500 m and considered to be a short­day plant with a threshold of (*) Corresponding author: hossam.ahmed@agr.cu.edu.eg Citation: ASHOUR H.A., ESMAIL S.E.A., EL­ATTAR A.B., 2021 ­ Responses of different quality parameters of Chia to arbuscular mycorrhiza and plant growth regulator. ­ Adv. Hort. Sci., 35(2): 139­150 Copyright: © 2021 Ashour H.A., Esmail S.E.A., El­Attar A.B. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 March 2021 Accepted for publication 30 March 2021 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-10641 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2021 35(2): 139­150 140 12­14 h. Chia acquired acceptance owing to the high nutritional value of its seeds. The main components of seeds are polyunsaturated fatty acids Omega­3 (PUFA ω3) (58­64% of total lipids) and Omega­6 (ω6), protein with a ratio (16­24%), carbohydrates (26­ 41%) lipids (31­35 %), and fiber (34­56 %). in addition to some minerals, vitamins and high level of antioxi­ dants (Baginsky et al., 2016; Sosa et al., 2016; Marcinek and Krejpcio, 2017). Arbuscular mycorrhiza fungi (AMF) are a category of soil microorganisms which form a symbiotic asso­ ciation with plants. Not only it could enhance uptake of mineral elements and water by plants that pro­ mote plant growth, but also increase crop yield, qual­ ity properties and active ingredients (Dupre et al., 2008; Singh et al., 2010). The positive effect of inocu­ lation with AMF on physiological and biochemical changes in different medicinal and aromatic plants has been reported in a number of species such as enhancing growth performance and seed yield (Gashgaril et al., 2020; Bilalis et al., 2020), increase photosynthetic pigments and carbohydrates content (Amiri et al., 2017), increased nutrient status in plant orangs (Chaudhary et al., 2008), protein content (Ouzounidou et al., 2015), antioxidants activity (Golubkina et al., 2020), fixed oil (Moghith, 2019), promoted concentration of essential oil (Chaudhary et al., 2008; Al­Amri et al., 2016), enhanced primary and secondary metabolism and bioactive compounds (Gashgaril et al., 2020 ). Plant growth regulators (PGRs) have been defined as one of the major factors affecting plants growth and their primary and secondary metabolites, NAA is an organic compound that is synthetic plant hor­ mone in the auxin family. it is known to enhance cell elongation, cell division, elongation of shoot, vascu­ lar tissue, photosynthesis, RNA synthesis, membrane permeability and water uptake is also involved in many physiological processes such as fruit set, delayed senescence, leaf chlorophyll content, stimu­ lates flowering and increases yield (Davies, 1987). Foliar application of NAA on different medicinal and aromatic plants have been reported to improve growth and yield attributes, photosynthetic pig­ ments, total carbohydrate and oil yield (Rohamare et al., 2013), enhance nutrient status in plant orangs, essential oil %, polyphenols and flavonoids content and antioxidant activity (Atteya and El Gendy, 2018). Moreover, it has been reported for ameliorating the harmful effects of salinity (Abou El­ghit, 2015). Cytokinins include benzyl adenine (BA) that promotes cellular elongation and division (Krug et al., 2006). Foliar spray of BA has been reported to increase the growth and development of medicinal and aromatic plants (Matter, 2016; Moussa, 2019). It was reported to improve photosynthetic pigments content, total carbohydrate oil percentage and oil yield (Abdel­ Rahman and Abdel­Kader, 2020), contents of macronutrient and micronutrient, total phenols and total flavones (Abdel­Hamid, 2020), enhance protein concentration (Prins et al., 2013) and antioxidant activity (Sidkey, 2020). Likewise, CPPU is one of PGRs. It is a cytokinin like substance that has strong cytokinin activity by stimu­ lating fruit set and fruit quality, it plays a role in cell division and cell wall elongation (Nickell, 1985; Zhang and Whiting, 2011). It has been reported to improve growth and yield attributes or protein percentage of medicinal plants (Abbas and Zahwan, 2016). Although the valuable roles of AMF and PGRs on medicinal and aromatic plants and their useful influ­ ence on enhancing growth and production has been formerly evaluated. However, there are not enough available data about their activity on the growth and production of Chia plants. Therefore, the objective of this study was to evaluate the impact of AMF and foliar spray of PGRs (BA, CPPU and NAA) and their interactions on vegetative growth, seed yield and yield attributes, and some chemical compositions of Salvia hispanica plants. 2. Materials and Methods Open field experiment was carried out during the two successive seasons of 2018 and 2019 at Experimental area of the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza Governorate (latitude 30°01’13.44”N, longitude 31°12’30.24”E, altitude 22 m a.s.l.). The aim of this work was to evaluate the effects of foliar application of different plant growth regulators (PGRs) such as 6­ BA (6­Benzylaminopurine) benzyl adenine, CPPU [N­ (2­chloro­4­pyridyl)] and NAA (Napthaleneacetic Acid) in absence or presence of arbuscular mycor­ rhizal fungi (AMF) on vegetative growth, seed yield and its attributes and some biochemical parameters of Chia (Salvia hispanica L.) plant. Experimental procedure Seeds of Salvia hispanica plants were obtained from experimental farm of Faculty of Pharmacy, Ashour et al. ‐ Responses of different quality parameters of Chia to AMF and PGR 141 Cairo University. On 1st October and 15th October (in the two seasons, respectively), seeds were sown in the nursery at the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza. After 45 days old from seeds sowing (on the 15th of November, and1st December, in both seasons, respectively), uniform seedlings, with an average plant height of 15­18 cm, were transplanted in the experimental open field with a distance of 70 cm among rows, 50 cm spacing between plants in plots (3.5×3.5 m). The physical and chemical properties of the exper­ imental soil were determined according to Jackson (1973), and the data are recorded in Table 1. AMF inoculum contained roots, hyphae, spores and growth media from a pot culture of onion plants colonization with Glomus mosseae NRC31 and Glomus fasciculatum NRC15, and were obtained from Agricultural Microbiology Department, National Research Center. Inoculum material consisted of 275 spores g­1 oven dry bases in addition to the coloniza­ tion roots pieces (the infectivity 104 propagola). AMF inoculation treatments was achieved by mixing 5 g of it with 10 g of chia seed before sowing and repeated monthly after transplanting by injection inoculum material into the soil in roots area from five sides at 5 g/seedling. PGRs treatments were initiated after 15 days from the transplanting, by foliar spraying the plants every 2 weeks with Sytonine 4%® [commercial name, con­ sists of 6­ BA (6­Benzylaminopurine) benzyl adenine 4 %], Cytovac® (CPPU) and Fast tonic® [commercial name, consists of Napthaleneacetic Acid 25% + Sodium nitrophenolate 0.6%]. The three commercial products were obtained from Bio Green Company, the regional representative of the Jordanian compa­ ny, Elite Company for the manufacture of agricultural fertilizers development, Egypt. The concentrations of BA and NAA were 50 ppm for each one, while CPPU was applied at 20 ppm and the untreated control plants were sprayed with tap water. Freshly prepared solutions of PGRs (50 mL containing Tween 20 at 1 mL/L (0.1%) as surfactant agent) were sprayed using plastic automizer until run off point. The common horticulture practices (such as irrigation, manual weeds control, fertilization) were carried out when needed. The experimental design was split plot design with eight treatments [2 AMF (absence or presence) x 4 PGRS concentrations (including the control)] with 3 replicates, each consisting of 16 plants (2 plants from each treatment). AMF assigned to the main plots in a randomized complete blocks design and PGRs con­ centrations were allocated at random in sub­plots. Measurement of vegetative growth and yield para‐ meters Vegetative growth parameters were recorded after 90 days from transplanting (On 15th of February to 1st March). In both seasons plant samples were taken for measurements growth characters in terms of plant height (cm), number of leaves/plant, stem diameter (cm, at 5 cm above the soil surface), num­ ber of branches/plant, fresh and dry weights of the herb (g/plant), root length (cm), number of root/plant and fresh and dry weights of the roots (g/plant). Whereas, at harvesting stage yield and yield attributes including number of inflorescence/ plant, number of seeds/plant, weight of seeds (g /plant), weight of 1000 seeds (g), and seeds yield (Kg/Fed.) were recorded. Seeds yield per feddan was obtained according to the equation: [Weight of seeds (g/plant) × number of plants/fed]/1000 Number of plants/fed= (100×100×4200)/ (50×70) = 12000 plant/fed. Some chemical parameters were determined in the seeds as carbohydrates %, some macro & micro elements, proteins %, phenols, flavonoids content Parameter 2018 2019 Soil texture Clay Clay Clay[%] 40.50 41.60 Silt [%] 35.10 34.00 Fine sand[%] 21.00 20.00 Coarse sand [%] 3.40 4.40 Field capacity [% V] 67.30 69.37 pH 7.12 7.19 EC [dS/m] 1.67 1.55 CEC [meq/100g] 39.40 35.72 Organic matter [%] 1.60 1.75 CaCO3 [%] 1.65 1.75 K+ [ppm] 65.85 67.95 Mg++[ppm] 39.83 40.94 Available N [ppm] 93.35 98.75 Available p [ppm] 20.25 22.35 Available Fe [ppm] 2.11 2.19 Available Mn [ppm] 3.12 3.99 Available Zn [ppm] 1.59 1.53 Table 1 ­ Some physical and chemical characteristics of experi­ mental soil during the two seasons pH= soil acidity, EC= Electrical conductivity, CEC= cation exchange capacity, CaCO3= calcium carbonate. Adv. Hort. Sci., 2021 35(2): 139­150 142 and antioxidant activity. Chemical analysis Total chlorophylls in fresh leaf samples were determined by using chlorophyll meter (Model SPAD 502 Minolta Co. Japan) as described by Netto et al. (2005). The total carbohydrates content in leaves and seeds (% of dry matter) was determined in dried samples according to Dubois et al., (1956). A known weight (0.1 g) of the dried samples was completely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. The solu­ tion was decolorized and the filtrate was completed to 100 ml with distilled water. A known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. The optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. The standard curve of glucose was used to calculate the total carbohydrates concentration in the extract. Dried seeds samples were digested to extract nutri­ ents and the extract was analyzed to determine con­ centrations of N, P, K, Ca, Mg (% of dry seeds), Fe and Zn (ppm) which were determined according to Estefan et al. (2013). Nitrogen concentration was determined by using the micro­Kjeldahl method. Phosphorus was determined calorimetrically by using the chlorostannous molybdophosphoric blue colour method in sulphuric acid. Potassium was determined by using the flame photometer apparatus (CORNING M 410, Germany). The concentrations of Ca, Mg, Fe and Zn were determined using atomic absorption spectrophotometer with air­acetylene and fuel (Pye Unicam, model SP­1900, US). Protein content in seeds was estimated by multiplying N values by 5.71 (conversion factors). Total phenolics content was determined by using the Folin Ciocalteau’s reagent colorimetric method while total flavonoids content was estimated by the aluminum chloride colorimetric method and results are expressed as milligram Catechin equivalent per gram of seeds dry weight extract (mg CE/g DW) (John et al., 2014). The antioxi­ dant activity of seeds extract and standard antioxi­ dant was assessed on the basis of the radical scav­ enging effect on DPPH (2, 2­diphenyl­1­picrylhy­ drazyl) free radicals (Brand­Williams et al., 1995). Fixed oil %: the clean air dried seeds of chia were separately crushed in a Willey mill, then extracted in Soxhlet apparatus, samples of 10 g of seeds were moved into Soxhlet apparatus in 100 ml of N­hexane and the extraction period extended to 6 hours (30­36 syphon cycle approx.). The N­hexane extract was dried over anhydrous sodium sulfate, then filtered and the oil was obtained by distillation under vacu­ um. Fixed oil % was calculated according to the equa­ tion: Fixed oil % = Extracted fixed oil weight (g) ̸ seeds sample weight (g) ̸ × 100. Statistical analysis The means of all obtained data were subjected to statistical analysis of variance (ANOVA) in split plot design. Combined analysis of the two growing sea­ sons was carried out. Means of data were compared by using Duncan’s multiple range tests at 5% level Snedecor and Cochran (1989). 3. Results and Discussion Vegetative growth parameters It is evident from data in Table 2 and 3 that under the same treatments of PGRs, chia plants grown in presence of AMF had significant increase in studied vegetative growth parameters (viz., plant height, number of leaves, stem diameter, number of branch­ es/plant, fresh and dry weights of herb, root length, number of roots, root fresh and dry weights) com­ pared to those grown in absence of AMF. The obtained increases in vegetative growth attributes due to AMF inoculation are in agreement with reports of several researches on medicinal and aro­ matic plants including Artemisia annua (Chaudhary et al., 2008), Salvia officinalis (Geneva et al., 2010; AbdelKader et al., 2014), Corianderum sativum (Al­ Amri et al., 2016; Oliveira et al., 2016), Origanum majorana (Engel et al., 2016), Pelargonium grave‐ olens (Amiri et al., 2017), Salvia miltiorrhiza (Yang et al . , 2017), Salvia hispanica (Moghith, 2019), Artemisia dracunculus and Hyssopus officinalis (Golubkina et al., 2020) and Foeniculum vulgare (Mohamed, 2020). The stimulatory influence of AMF on vegetative growth traits could be explained by AMF form symbi­ otic relationship with the host increase of root sur­ face, which led to promote root uptake of nutrients. Thus, it could significantly augment growth parame­ ters of tested plant (Jian­heng et al. , 2016). Furthermore it was indicated that the ability of AMF to enhance the availability of essential elements macro­ and micronutrients in the rhizospheric soil that induce its uptake and the accumulation in plant (Gashgaril et al., 2020). Ashour et al. ‐ Responses of different quality parameters of Chia to AMF and PGR 143 Results in Table 2 and 3 also indicate that, in absence or presence of AMF, treating plants with dif­ ferent concentrations of PGRs resulted in significant increase in tested vegetative growth parameters compared to control. Among PGRs, application of NAA (at 50 ppm) appeared to be the most effective one particularly in presence of AMF since recorded the highest values in most cases. The results of the pronounced increase in growth parameters due to application of NAA are in accordance with findings of previous studies on medicinal plants (Alam et al., 2012; Rohamare et al., 2013; Danesh­Talab et al., 2014; Abou El­ghit, 2015; Rostami and Movahedi, 2016; Dheeraj and Saravanan, 2018). Moreover, numerous studies reported increase in growth attrib­ utes of medicinal plants owing to either BA (Matter, 2016; Moussa, 2019; Abdel­Rahman and Abdel­ Kader, 2020 and Abdel­Hamid, 2020) or CPPU treat­ ments (Abbas and Zahwan, 2016). The stimulation effect of NAA on morphological Table 2 ­ Mean square for the effect of Arbuscular mycorrhiza fungi (AMF), plant growth regulators (PGRs) and their interaction on veg­ etative growth, yield and yield parameters of Salvia hispanica *, **, *** significant at P≤0.05, P≤0.01, P≤0.001, respectively. Traits Source of variation Treatment Error Cv AMF (A) PGRs (B) A × B A B A B Plant height (cm) 233.750 ** 514.534 *** 24.278 *** 1.182 1.037 1.322 1.239 No. of leaves 213.010 ** 194.372 *** 5.844 * 2.042 2.222 4.527 4.723 Stem diameter (cm) 0.375 ** 0.124 *** 0.011 * 0.004 0.005 7.036 8.386 No. of branches/plant 37.500 *** 36.301 *** 1.701 ** 0.035 0.189 1.685 3.921 Herb fresh weight (g) 27.307 *** 18.106 *** 2.681 *** 0.003 0.090 0.433 2.412 Herb dry weight (g) 25.627 *** 21.694 *** 1.288 *** 0.002 0.072 0.609 3.991 Root length (cm) 145.042 *** 96.486 *** 12.042 *** 0.087 0.746 1.979 5.808 No. of roots/plant 32.667 *** 34.375 *** 1.167 * 0.135 0.448 4.576 8.322 Root fresh weight (g) 38.760 *** 20.386 *** 1.412 *** 0.020 0.030 2.531 3.068 Root dry weight (g) 4.770 ** 5.794 *** 0.228 ** 0.050 0.035 7.233 6.039 No. of inflorescence /plant 145.042 * 70.972 *** 7.903 * 0.510 1.313 4.844 7.767 No. of seeds / plant 18897.29 *** 57551.63 *** 283.104 *** 1.439 5.467 0.219 0.426 Weight of 1000 seeds (g) 1.321 *** 0.371 ** 0.001 * 0.145 0.061 20.73 13.45 Weight of seeds (g/plant) 13.024 ** 40.227 *** 0.556 * 0.290 0.367 7.02 7.89 Seeds yield (Kg/Fed.) 1875.494 ** 5792.741 *** 80.027 * 5.041 21.110 2.44 4.99 Table 3 ­ Vegetative growth parameters of Salvia hispanica as affected by the interaction between plant growth regulators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two seasons) Treatment Plant height (cm) No. of leaves Stem diameter (cm) No. of branches/ plant Herb fresh weight (g) Herb dry weight (g) Root length (cm) No. of roots/ plant Root fresh weight (g) Root dry weight (g)AMF PGRs ­ 0 67.53±0.44 h 23.67±0.60 d 0.57±0.07 d 7.00±0.29 f 9.77±0.03 f 3.53±0.15 g 8.50±0.060 f 4.00±0.58 e 2.67±0.07 g 1.57±0.09 f BA at 50 ppm 78.50±0.50 f 27.17±0.33 c 0.77±0.03 c 9.93±0.35 d 11.83±0.07 cd 5.87±0.12 e 12.33±0.67 d 7.50±0.29 c 4.07±0.03 f 2.30±0.06 e CPPU at 20 ppm 81.83±0.67 e 28.00±0.58 c 0.87±0.03 c 11.10± 0.06 c 11.67±0.23 d 6.63±0.28 d 14.00±0.50 c 7.50±0.29 c 4.83±0.15 e 3.27±0.09 d NAA at 50 ppm 88.50±1.15 c 35.50±0.29 b 0.83±0.03 c 11.37±0.105 c 12.33±0.19 c 6.67±0.13 d 14.83±0.73 c 8.50±0.50 c 5.93±0.03 d 3.50±0.06 cd + 0 70.83±0.68 g 26.67±0.60 c 0.80±0.02 c 8.00±0.29 e 10.4 3±0.09 e 4.37±0.07 f 10.67±0.44 e 5.17±0.17 d 3.87±0.15 f 2.03±0.2 e BA at 50 ppm 90.67±0.60 b 34.00±0.58 b 1.00±0.06 b 13.30±0.15 b 13.67±0.12 b 8.17±0.15 c 17.17±0.17 b 9.67±0.44 b 6.53±0.13 c 3.60±0.1 c CPPU at 20 ppm 86.17±0.33 d 35.00±1.00 b 1.03±0.03 b 13.60±0.20 b 13.80±0.10 b 8.70±0.10 b 17.83±0.17 b 10.33±0.73 b 8.00±0.06 b 3.93±0.12 b NAA at 50 ppm 93.67±0.73 a 42.50±0.29 a 1.20±0.06 a 14.50±0.15 a 16.23±0.32 a 9.73±0.09 a 23.67±0.33 a 11.67±0.44 a 9.27±0.12 a 4.63±0.03 a Each value represents the mean ± standard error of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan multiple rang test. 144 Adv. Hort. Sci., 2021 35(2): 139­150 attributes may be due its ability to increase mem­ brane permeability and water uptake which accom­ panied by elements absorption, synthesis of chloro­ phyll and carbohydrates contents that achieving the highest dry weight of plants (Atteya and El Gendy, 2018). Yield and yield attributes Data in Table 4 revealed that under the same treatments of PGRs, the values of yield and yield attributes (viz., number of inflorescence /plant, num­ ber of seeds/plant, weight of seeds, weight of 1000 seeds, and seeds yield) were significantly higher in plants grown in the presence of AMF than corre­ sponding values in the absence of AMF. The present increase in number of inflorescence/plant due to AMF inoculation is supported by the results of previ­ ous report (Engel et al., 2016). Moreover, recent study (Mohamed, 2020) on Foeniculum vulgare revealed that AMF inoculation significantly increased seeds yield parameters. Exogenous application of PGRs in absence or pres­ ence of AMF resulted in significant increase in yield and yield parameters compared to control. Among PGRs, NAA was the most effective one particularly when interacted with presence of AMF. In this con­ cern, application of NAA at 50 ppm has been report­ ed to increase yield attributes (Rohamare et al., 2013; Danesh­Talab et al., 2014; Khudus et al., 2017; Venkatesan and Shakila, 2017). Other study (Kassem et al., 2011) found that, foliar spray of NAA at 75 mg/l or CPPU at 10 mg/l caused significant increase in yield and yield attributes of Ziziphus jujuba com­ pared with the control. Moreover, increases in num­ ber of inflorescence /plant owing to application of NAA are in accordance with findings of earlier studies (Atteya and El Gendy, 2018; Dheeraj and Saravanan, 2018). While increasing yield and its attributes due to either BA application are in harmony with the find­ ings of various studies (Mousa et al., 2001; Matter, 2016; Abdel­Rahman and Abdel­Kader, 2020) or CPPU (Abbas and Zahwan, 2016). The augmentation in yield and its attributes due to PGRs treatments may be due to its role in enhanced absorption of nutrients which promoted photosynthesis rate and translocation of photosyn­ thates and other metabolites to the sinks that lead­ ing to increase yield and its attributes in present study (Alam et al., 2012). Total chlorophylls and total carbohydrates Data in figure 1 indicate that under the same treatments of PGRs, plants grown in the presence of AMF had significantly higher values of total chloro­ phylls in leaves and total carbohydrates in leaves and seeds compared to corresponding values in the absence of AMF in most cases. These results run par­ allel with those obtained by earlier reports on medic­ inal plants that reported increase in chlorophyll and carbohydrates content in plants inoculated with AMF compared to controls (Amiri et al., 2017; Gashgaril et al., 2020; Moghith, 2019; Mohamed, 2020). Results also show that, the recorded values were significantly increased in plants grown in absence or presence of AMF as a result of spraying tested PGRs compared to control. In most cases, plants sprayed with NAA in the presence of AMF had the highest val­ ues of the tested components. The results of increase Table 4 ­ Yield and yield attributes of Salvia hispanica as affected by the interaction between plant growth regulators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two seasons) Each value represents the mean ± standard error of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan multiple rang test. Treatments No. of inflorescence/ plant No. of seeds/ plant Weight of seeds (g/plant) Weight of 1000 seeds (g) Seeds yield (Kg/Fed.) AMF PGRs ­ 0 9.17±0.44 f 409.17±0.58 h 3.43±0.20 f 1.23±0.03 e 41.20±2.45 f BA at 50 ppm 12.67±0.88 e 477.17±1.11 f 7.47±0.01 d 1.69±0.09 d 89.64±0.14 d CPPU at 20 ppm 12.33±0.33 e 573.67±1.48 d 7.32±0.38 d 1.73±0.15 cd 87.88±4.52 d NAA at 50 ppm 15.00±0.58 d 622.17±1.21 c 9.53±0.15 bc 1.76±0.18 bcd 114.40±1.79 b + 0 11.00±0.58 e 448.50±0.76 g 4.64±0.03 e 1.70±0.21 d 55.68±0.36 e BA at 50 ppm 17.17±0.6 c 546.83±1.72 e 8.57±0.13 c 2.16±0.17 abc 102.84±1.54 c CPPU at 20 ppm 19.00±0.58 b 638.33±1.15 b 9.71±0.26 b 2.19±0.11 ab 116.48±3.17 b NAA at 50 ppm 21.67±0.67 a 673.00±1.15 a 10.74±0.18 a 2.23±0.192 a 128.84±2.22 a Ashour et al. ‐ Responses of different quality parameters of Chia to AMF and PGR 145 of total chlorophylls or total carbohydrates due to application of NAA are similar to those obtained by previous studies (Alam et al., 2012; Rohamare et al., 2013; Rostami and Movahedi, 2016; Venkatesan and Shakila, 2017; Atteya and El Gendy, 2018), whereas the obtained increase due to either BA treatments are in close conformity with the findings of previous reports (Matter, 2016; Moussa, 2019; Abdel­Hamid, 2020; Abdel­Rahman and Abdel­Kader, 2020) or CPPU treatments (Kassem et al., 2011; Abbas and Zahwan, 2016). Content of macronutrients in seeds Results of chemical analysis of dried seeds of Salvia hispanica plants (Table 5 and 6) disclosed that, under the same treatments of PGRs the uptake and accumulation of macronutrients in seeds of plants inoculated with of AMF were significantly higher in most cases compared to non­inoculated plants. The only exception to this general trend was observed in the case of P and K% as in the presence of AMF with control treatments they recorded insignificantly high­ er values compared to corresponding values in the absence of AFM. The obtained results are in agree­ ment with those obtained by various researchers that reported the potential effects of AMF on the accumulation of macronutrients in medicinal plant orangs (Chen and Zhao, 2009; Karagiannidis et al., 2011; AbdelKader et al., 2014; Vafadar et al., 2014; Oliveira et al., 2016; Yang et al., 2017; Moghith, 2019; Mohamed, 2020). The enhanced mineral absorption by AMF inocu­ lated plants could be elucidated by the efficiency of AMF to boost mineral affinities, reduce the critical concentration of elements absorption, augment the area of uptake and decrease the area of diffusion (Gashgaril et al., 2020). Data in Table 6 also indicate that, in absence or presence of AMF foliar spraying with any concentra­ tions of PGRs resulted in significant increase in macronutrient in seeds in most cases, compared to control. With some exceptions recorded in absence of AMF as foliar application of CPPU resulted in insignificantly higher values of P% than the control, also in absence of AMF, foliar application of three tested PGRs resulted in insignificantly higher values of Ca % compared to control. Among PGRs, applica­ tion of NAA was the most effective treatment espe­ cially in presence of AMF. The results of increasing macronutrients accumulation due to NAA treatments are accordance with findings of previous studies (Alam et al., 2012; Atteya and El Gendy, 2018), while the obtained increase in macronutrients due to BA is similar to those described in numerous reports (Matter, 2016; Moussa, 2019; Abdel­Hamid, 2020; Abdel­Rahman and Abdel­Kader, 2020). Content of micronutrients in seeds As shown in figure 2 that under the same treat­ ments of PGRs plants inoculated with AMF had signif­ icantly higher values of Fe and Zn in their seeds than those grown in the absence of AMF. These results are in the same line of the findings of earlier authors (Chaudhary et al., 2008; Golubkina et al, 2020). Data in figure 2 also showed that, in the absence or presence of AMF foliar application of any concen­ Fig. 1 ­ Total chlorophyll in leaves (A), total carbohydrates in lea­ ves (B), and total carbohydrates in seeds (C) of Salvia hispanica as affected by the interaction between plant growth regulators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two sea­ son). Column with different letters indicate a significant difference at 5% level. Vertical bars indicate to standard error (SE) of three replicates. Adv. Hort. Sci., 2021 35(2): 139­150 146 trations of PGRs resulted in significant increase in tested micronutrients (Fe and Zn) in seeds compared to control. The increases in the recorded values were more evident in the presence of AMF mostly with application of NAA. Such increase in Fe and Zn con­ tent due to BA treatments is in good agreement with those elicited by prior works (Baydar and Erdal, 2004; Abdel­Hamid, 2020). Total protein Data in Table 7 displayed that under the same PGRs treatments total protein percentage was signifi­ cantly higher in plants grown in the presence of AMF than those grown in the absence of AMF. These results are in conformity with that recorded by previ­ ous studies (Ouzounidou et al., 2015; Al­Amri et al., 2016). Increasing protein due to AMF may be due to Table 5 ­ Mean square for the effect of Arbuscular mycorrhiza fungi (AMF), plant growth regulators (PGRs) and their interaction on some chemical constituents of Salvia hispanica *, **, *** significant at P≤0.05, P≤0.01, P≤0.001, respectively; NS= Not significant at p=0.05. Traits Source of variation Treatment Error Cv AMF (A) PGRs (B) A × B A B A B Total chlorophylls in leaves (SPAD) 36.630 ** 46.448 *** 0.517 * 0.782 0.428 2.53 1.87 Total carbohydrates in leaves (%) 16.368 ** 25.279 *** 0.242 * 0.167 0.878 3.94 9.02 Total carbohydrates in seeds (%) 5.042 *** 1.914 ** 0.221 * 0.007 0.037 1.58 3.61 N (%) 0.855 *** 0.824 *** 0.003 * 0.001 0.004 1.21 2.39 P (%) 0.014 ** 0.008 *** 0.001 * 0.00 0.00 3.14 4.01 K (%) 0.005 * 0.006 ** 0.001 * 0.000 0.001 8.20 11.28 Mg (%) 0.001 NS 0.002 * 0.000 * 0.001 0.001 13.82 9.71 Ca (%) 0.013 * 0.001 * 0.001 * 0.001 0.000 6.49 3.20 Fe (ppm) 62.210 * 32.204 *** 2.316 * 1.148 1.473 1.66 1.88 Zn (ppm) 29.704 * 67.935 *** 0.778 * 2.251 0.445 3.34 1.48 Total protein (%) 27.907 *** 26.881 *** 0.083 * 0.038 0.145 1.22 2.39 Total phenol (μg CE/g) 0.917 * 1.774 ** 0.119 * 0.158 0.204 9.36 10.65 Total flavonoid (μg CE/g) 60.770 * 63.393 *** 2.161 * 0.621 0.473 1.83 1.60 Antioxidant (DPPH IC50 (µg/ml) 8.143 ** 23.270 *** 1.989 * 0.619 0.843 1.19 1.38 Fixed oil % 93.102 * 139.584 *** 0.509 * 2.833 2.978 6.39 6.55 Table 6 ­ Macronutrients in seeds of Salvia hispanica as affected by the interaction between plant growth regulators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two seasons) Each value represents the mean ± standard error of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan multiple rang test. Treatment N (%) P (%) K (%) Mg (%) Ca (%) AMF PGRs ­ 0 2.21±0.03 f 0.33±0.01 d 0.19±0.02 f 0.21±0.01 d 0.40±0.01 c BA at 50 ppm 2.40±0.02 e 0.37±0.01 c 0.23±0.02 d 0.25±0.02 b 0.43±0.02 bc CPPU at 20 ppm 2.73±0.08 c 0.35±0.00 cd 0.21±0.01 e 0.24±0.00 c 0.43±0.01 bc NAA at 50 ppm 3.080±0.01 b 0.41±0.01 b 0.25±0.02 c 0.26±0.03 b 0.42±0.00 bc + 0 2.60±0.02 d 0.36±0.00 cd 0.20±0.01 f 0.23±0.01 c 0.45±0.01 ab BA at 50 ppm 2.77±0.03 c 0.41±0.01 b 0.27±0.02 ab 0.27±0.01 a 0.47±0.01 a CPPU at 20 ppm 3.15±0.02 b 0.43±0.00 b 0.25±0.01 c 0.25±0.01 b 0.47±0.01 a NAA at 50 ppm 3.40±0.01 a 0.46±0.01 a 0.28±0.02 a 0.27±0.01 a 0.48±0.01 a Ashour et al. ‐ Responses of different quality parameters of Chia to AMF and PGR 147 its role on inducing NH4 + and NO3 − absorption, and assimilation of these molecules into free amino acids that are involved in protein synthesis (Gashgaril et al., 2020). The data in Table 7 also exhibited that in the absence or presence of AMF, spraying of plants with PGRs resulted in significant increase in total protein content compared to control. Among the tested PGRs NAA was superior in its effect predominately in the presence of AMF. The results of increasing pro­ tein content due to application of BA are in good agreement with those elicited by Prins et al., 2013, while such increase owing to CPPU treatments is coincided with those obtained by Abbas and Zahwan, 2016. Total phenol content (TPC), Total flavonoid content (TFC) in in seeds The data presented in Tables 7 showed that under the same treatments of PGRs TPC and TFC in seeds were higher in plants inoculated with AMF compared to non­inoculated plants; however such increase was statically insignificant in the case of TPC compared to corresponding values in absence of AMF. The results of increasing TPC or TFC due to inoculation with AMF are analogy with that recorded by earlier workers (Amiri et al., 2017; Gashgaril et al., 2020). Data outlined in Table 7 also indicate that in the absence or presence of AMF TPC and TFC in seeds were significantly higher in seeds of plants foliar sprayed with any concentrations of PGRs compared to control, with superiority of BA for increasing TPC and NAA for TFC especially in the presence of AMF. The present augmentations in TPC or TFC owing to NAA are in harmony with those obtained by previous author (Atteya and El Gendy, 2018), whereas increas­ ing in tested components due to BA are in the same line with the findings of earlier study (Abdel­Hamid, Fig. 2 ­ Fe (A) and Zn (B) in seeds of Salvia hispanica as affected by the interaction between plant growth regulators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF) (mean of two season), column with different letters indicate a significant difference at 5% level. Vertical bars indicate to standard error (SE) of three replicates. Table 7 ­ Total protein, total phenol, total flavonoid and antioxidants in seeds as affected by the interaction between plant growth regu­ lators (PGRs) in absence (­) or presence (+) of Arbuscular mycorrhiza fungi (AMF), (mean of two seasons) Each value represents the mean ± standard error of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan multiple rang test. Treatments Total protein (%) Total phenol (μg CE/g) Total flavonoid (μg CE/g) Antioxidant (DPPH IC50 (µg/ml)AMF PGRs ­ 0 12.62±0.18 f 3.18±0.12 c 37.56±0.06 f 63.14±0.60 d BA at 50 ppm 13.68±0.13 e 4.15±0.49 ab 40.74±0.13 d 66.18±0.58 c CPPU at 20 ppm 15.57±0.45 c 4.55±0.03 a 43.18±0.05 c 67.54±0.02 bc NAA at 50 ppm 17.57±0.06 b 4.31±0.04 ab 44.22±0.07 bc 66.34±0.6 c + 0 14.87±0.10 d 3.68±0.34 bc 39.3±1.15 e 64.18±0.58 d BA at 50 ppm 15.82±0.17 c 4.83±0.09 a 45.33±0.13 b 66.25±0.43 c CPPU at 20 ppm 17.97±0.12 b 4.56±0.19 a 46.74±0.08 a 68.31±0.17 ab NAA at 50 ppm 19.41±0.07 a 4.70±0.32 a 47.06±0.01 a 69.12±0.57 a Adv. Hort. Sci., 2021 35(2): 139­150 148 2020; Sidkey, 2020). Antioxidant activity in seeds As shown from data listed in Table 7 that under the same treatments of PGRs antioxidant activity in seeds were higher in plants inoculated with AMF compared to corresponding values of non­inoculated plants, however such increase was insignificant in most cases. The results of increasing antioxidant activity due to mycorrhizal treatments is supported by the results of other authors (Geneva et al., 2010; Amiri et al., 2017; Golubkina et al., 2020; Gashgaril et al., 2020). Data recorded in Table 7 also indicate that in the absence or presence of AMF antioxidant activity was significantly higher in seeds of plants sprayed with PGRs concentrations compared to control, with supe­ riority of NAA especially in the presence of AMF, since recorded the highest values compared to con­ trol. The obtained increase in antioxidant activity due to NAA are in harmony with those obtained by previ­ ous author (Atteya and El Gendy, 2018), while the augmentation due to BA are in harmony with the finding of recent study (Sidkey, 2020). Fixed oil percentage It is obvious from data listed in figure 3 that under the same treatments of PGRs, the values of fixed oil % for plants grown in presence of AMF was significantly higher than corresponding values in the absence of AMF. The results of increasing fixed oil % owing to inoculation with AMF are in sequence with the find­ ings of earlier author (Moghith, 2019). Data in figure 3 also showed that, in the absence or presence of AMF in general application of PGRs resulted in significant increase in fixed oil % compared to control. These increases in the recorded values were more evident in the presence of AMF particular­ ly with application of NAA. 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