Impaginato 111 1. Introduction The degree of success in any technology employ- ing plant cell, tissue or organ culture is related to quite a few major factors. A significant factor is the choice of nutritional components and growth regula- tors (Gamborg, 1991). A plant tissue culture medium is composed of necessary and optional components required for plant growth, which vary according to the plant species, cultivar, or explant type that is used and must be experimentally defined for each particular case. Moreover, all the nutrients in a medi- um should be present in optimum concentrations to ensure the best possible growth of explants (George, 1993). Under in vitro conditions, an intact plant requires macronutrients, micronutrients, plant growth regulators, vitamins, amino acids and other nitrogen supplements and sugars (Gamborg, 1991). Another important component in plant tissue culture media is the carbon source because it supplies ener- gy to the plants, especially when they are not ready to photosynthesize their own food during the early stage of tissue culture (Al-Khateeb, 2008 b). Carbon source can be in the form of simple or complex sug- ars (Akter et al., 2007). Normally, sucrose is used as the carbon source in plant tissue culture. A range of other organic additives have been used in plant tis- sue culture to promote the growth of the plants, including coconut milk, banana pulp, potato homogenate and juice, honey, date palm syrup, corn extract, papaya extract, guar gum, and isubgol (Islam et al., 2003; Jain and Babbar, 2005; George et al., 2008; Murdad et al., 2010; Nambiar et al., 2012). Such additives are commonly known as organics with undefined compositions (Torres, 1989). The advan- tages of adding such organic materials to medium have already been reported by some researchers, for example, the organic additives help to produce more PLBs, shoots and leaves in orchid (Akter et al., 2007), increase the size of date palm somatic embryos (Al- Khateeb, 2008 a), and also promote growth and development of asymbiotic seeds and regeneration Adv. Hort. Sci., 2016 30(2): 111-118 DOI: 10.13128/ahs-19137 The usefulness of apricot gum as an organic additive in grapevine tissue culture media S. Khorsha, M. Alizadeh (*), K. Mashayekhi Horticulture Department, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Golestan, P.O. Box 386, Gorgan, Iran. Key words: apricot gum, callus culture, carrot, grapevine, rooting, stevia, tissue culture. Abstract: The growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. In addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. Hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. The proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. The growth parameters of treated samples were measured and compared to gum-free medium. Earlier callus initia- tions with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. The usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot mul- tiplication and rooting parameters. Due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. Overall, the gum at the rate of 4.0 g/L was found to be a logical concen- tration with respect to encouraging response in all three species. Owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. However, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media. (*) Corresponding author: mahdializadeh@gau.ac.ir Received for publication 9 May 2016 Accepted for publication 25 May 2016 Copyright: © 2016 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Adv. Hort. Sci., 2016 30(2): 111-118 112 of Cymbidium plantlets (Tawaro et al., 2008). The reasons for application of organic additives into cul- ture medium, in addition to being a natural source of carbon, are because they contain natural vitamins, phenols, fiber, hormones, and also proteins (Gnasekaran et al., 2010). It was mentioned in a report by Al-Khateeb (2008 a) that organic additives contained not only sugar but also other nutrients such as proteins, lipids and minerals. In recent years, plant-derived polymers have evoked remarkable attention in various industries due to their diverse applications as food emulsifiers, stabilizers and thickeners, pharmaceuticals, cosmet- ics, textiles, and in art. They are also used as gelling agents in gels and bases in suppository (Nussinovitch, 1996). These polymers are biocompatible, biodegrad- able and are preferred to semi synthetic and synthet- ic excipients because of their lack of toxicity, low cost, soothing action, and non irritant nature (Deogade et al., 2012). Environmental-friendly pro- cessing and local availability, especially in developing countries, are considered additional advantages for application of these natural products (Jain and Babbar, 2005). The Rosaceae family, Prunus genus, consists of peach, plum, apricot, cherry, and almond trees, all of which can produce exudate gums. Herbal exudate gums normally secrete from bark, branch and fruit of trees due to their protection impact against mechanical damage or microbial attacks. Gum secretion may also occur due to adaptation to climate of some trees, called physiological gummosis (Simas et al., 2008; Simas-Tosin et al., 2009). A large number of complex natural additives can be very effective in providing an undefined mixture of organ- ic nutrients and growth factors. In the context of car- bohydrates, gums are usually considered to be non- starch, water-soluble polysaccharides with commer- cial importance. Gums are typically more or less sticky in nature and are translucent and amorphous substances which are degradation products of the cell wall of woody species which exude from trees. Natural gums (gums obtained from plants) are hydrophilic carbohydrate polymers having high mole- cular weights, generally composed of monosaccha- ride units joined by glucosidic bonds (Khorsha, 2014). While thorough reports on gum structure (Saniewski et al., 2001; Lluveras-Tenorio et al., 2012), its rheo- logical properties (Wang et al., 2008), and its applica- tions in different industries (Verbeken et al., 2003) were previously reported, the influence of such nat- ural products as organic addenda in plant tissue cul- ture media has yet to be studied. Therefore, the pre- sent investigation was conducted to evaluate the effectiveness of apricot gum as an organic additive on growth and in vitro regeneration of three differ- ent plant species: carrot (as a model plant), stevia (as herbaceous species) and grapevine (as woody plant). The results of the current study should be applicable to other plant species following minor modifications. 2. Materials and Methods The present research work was conducted in the plant tissue culture laboratory of the Horticulture department, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. The physiological effects of apricot gum on regeneration capacity and growth rate of three different plant species i.e. carrot (as model plant), stevia (as herbaceous plant) and grapevine (as woody plant) were examined. The apri- cot gum was obtained dried on bark and shoots of apricot trees immediately after secretion, collected from a commercial apricot orchard (Gonabad, Khorasan, Eastern Iran) through scraping it from bark surfaces. The bark residues were removed from the collected mass and the clean, pure gum was pow- dered prior to addition to the culture medium. Plant materials and in vitro culture establishement Carrot. Healthy, undamaged roots of carrot (Daucus carrota cv. Nantes), 3-4 cm diameters long, were selected and washed thoroughly with normal tap water (30 min) and surface sterilized with ethanol (70% v/v for 40 s) followed by sodium hypochlorite solution (35% v/v plus two drops of tween-20 for 15 min). The clean, sterilized roots were cut transverse- ly, employing a scalpel, to prepare root explants as slices (10×10 mm and 1mm thickness). Each explant was prepared in such a way to consist of xylem, sec- ondary phloem and a small part of cambium tissues (Hall, 1991). Stevia. Stevia (Stevia rebaudiana) mother plants were procured from Golsaran-e-Shomal Corporation, as tissue cultured pot plants and were transferred to our laboratory. Single node explants were disinfected with HgCl2 (0.1% for 6 min) and washed three times with sterilized distilled water inside a laminar hood cabinet. The single node explants were cultured and in vitro culture was established following an already standardized protocol (Taherian, 2012). Grapevine. Single node explants of grapevine (Vitis vinifera cv. Laal) were utilized following the pre- Khorsha et al. - Apricot gum as an organic additive in grapevine tissue culture media 113 viously standardized micropropagation protocol developed by Alizadeh et al. (2010). Laal is an Iranian, commercially grown variety of grapevine. Single node explants (2-4 cm length) were pre-washed in 0.1- 0.2% commercial detergent (JAM dish-washing liquid, Iran) followed by stirring in Mancozeb (2 g/L) solution for 45 min. The explants were surface disinfected using 60% (v/v) NaOC1 solution (5% available chlo- rine) for 30 min. After four to five rinses in sterile dis- tilled water, single node cuttings (2-4 cm) were inoc- ulated for in vitro culture establishement. Culture media and incubation conditions Basal MS (Murashige and Skoog, 1962) medium was used during the whole experiment for all three plant species. The various media along with their growth regulators for each plant species are reported in Table 1. The pH was adjusted to 5.8 prior to the addition of 0.8% agar, and the media were auto- claved at 121°C and 15 PSI for 15 min. Carrot root cultures were kept in growth chamber in darkness and low light intensity for 7-14 days and the explants were investigated for callus formation. The callus mass was sub-cultured on fresh medium supple- mented with apricot gum. The stevia and grapevine single node cultures were incubated at 25±2°C under continuous light (50 μmol m-2 s-1). The established and sprouted cultures were sub-cultured as double- node explants on proliferation media (Table 1) sup- plemented with different concentrations of apricot gum. Measured parameters Due to the utilization of explants with different origins, dissimilar parameters were recorded in each species. In the case of carrot callus cultures, days to callus initiation, callus color, firmness, volume and fresh/dry weight were recorded 30 days after caulo- genesis. In the case of stevia and grapevine, initially the in vitro cultures were established as explained in above. Then, the double-node explants procured from in vitro proliferated cultures were inoculated on their respective proliferation media supplemented with different concentrations of apricot gum. The parameters such as days taken to bud sprouting, number of leaves, leaf area, total chlorophylls and cartenoides, intermodal length, shoot length, days to root initiation, number of roots, root length, rooting percentage and root/shoot fresh and dry weights were measured in both stevia and grapevine regener- ated cultures. Experimental design and data analysis The present experiment was conducted as a com- plete randomized design with four replications. The percentage data were transformed using root square method (√ % + 0.5) prior to analysis. The results were analyzed using SAS software (SAS Institute Inc., 2003) and the mean values were compared by least signifi- cant difference (LSD) test in p<0.01 probability. 3. Results The present research was undertaken to ascertain the likely positive effects of apricot gum as an organic additive to plant tissue culture media. The gum was primarily supplemented to carrot callus cultures and, owing to its positive response, was tested in two other plant species, stevia and grapevine, for which in vitro culture propagation protocols were already standardized in our laboratory (Alizadeh et al., 2010; Taherian, 2012). Iran is the world’s second producer of apricot (FAOSTAT, 2012) and apricot gum is readily available throughout the country at a very low price. In order to optimize the concentrations of the apricot gum, some preliminary tests were performed and the rate of 2.0-6.0 g/L was found to be a logical range because the higher concentration formed a dense solution that was difficult to dispense in culture ves- sels. Furthermore, in high concentration, it negatively interacted with agar solidification (Khorsha, 2014). Carrot callus growth The application of gum surprisingly enhanced the performance of in vitro callus initiation and further growth of carrot explants. Furthermore, it signifi- cantly reduced the time taken to callus initiation and among the treatments caulogenesis occurred even less than two weeks following inoculation of the explants on medium supplemented with 6.0 g/L apri- cot gum. The callus initiation in control explants was found to start normally after 5 weeks, therefore, pre- cocious callus formation (nearly 20 days earlier than control) is considered a promising result for in vitro application of apricot gum. In addition, callus mor- phological traits such as callus volume, color, and tis- sue firmness were also affected by gum treatments Table 1 - Different culture media and growth regulators for each plant species Plant species In vitro establishment Shoot proliferation Rooting Carrot 2,4-D (1.0 mg/l) - - Stevia Hormone-free medium IBA (2.0 mg/l) IBA (2.0 mg/l) Grapevine BA (2.0 mg/l) + NAA (0.2 mg/l) IBA (2.0 mg/l) IBA (2.0 mg/l) Adv. Hort. Sci., 2016 30(2): 111-118 114 (Table 2). The callus mass produced on control medi- um (30 days after inoculation) were light brown in color and had intermediate firmness. However, with gum application the color was greenish and the tis- sue mass volume was increased corresponding to gum concentration (Fig. 1). The callus fresh weight was also improved in all the treatments, however the dry weight was the same and was determined to be lower than control media (Table 3). It is worth noting that the treatment with 6.0 g/L apricot gum may be toxic for carrot callus; however callus fresh weight was not statistically significant compared to 2 g/L level (Table 3). Due to the positive and promising results with car- rot callus cultures, the effect of apricot gum was evaluated on in vitro proliferation of an herbaceous medicinal plant, stevia, for which its proliferated cul- tures were already established in our laboratory. Stevia shoot multiplication and rooting Stevia is an herbaceous plant with rapid in vitro proliferation. In our laboratory, stevia cultures were normally sub-cultured at three- to four-week inter- vals. The growth parameters of stevia shoot prolifer- ation and rooting four weeks after inoculation are shown in Table 4. It is clear that apricot gum added to stevia medium was particularly beneficial. The gum significantly reduced the time taken to root initi- ation and the micro-cuttings inoculated on medium containing gum (6.0 g/L) achieved root initiation in less than a week (6.5 days only), while the control plantlets came into rooting after at least 10 days. Root length also increased in the presence of gum, however, the number of roots were not considerably different among treatments. In vitro plantlet length and leaves were reduced on gum-supplemented media, but instead the leaf area and lateral shoots were enhanced (Table 4). It seems the lower concen- trations of apricot gum (2.0 and 4.0 g/L) led to better vegetative response in stevia cultures. A similar trend was also recorded with respect to stevia leaf pig- ments. Thus, at the rate of 2.0 g/L, gum was more effective in pigmentation and production of dark green leaves. Overall, it may be stated that apricot gum had a positive influence on most of the in vitro traits measured in stevia tissue-cultured plants. Grapevines shoot multiplication and rooting It has previously been reported that shoot prolif- eration and rooting occur simultaneously in grapevine (Alizadeh et al., 2010), hence, in the pre- sent experiment, it was possible to measure related Fig. 1 - The effect of apricot gum on in vitro growth and prolifer- ation of carrot callus cultures 30 days after inoculation (left to right: Control, 2.0, 4.0, 6.0 g/l apricot gum). Means in the same row followed by different letters are signifi- cantly different at P<0.01 using LSD-Test. Qualitative traits Apricot gum concentration (g/l) Control 2.00 4.00 6.00 Callus volume <30% 30-50% 50-100% ≥100% Callus color Light brown Beige to light green Beige to greenish Beige to pistachio green Tissue firmness +++ +++ ++++ ++++ Table 2 - The qualitative traits of carrot callus as affected by gum application 30 days after inoculation Table 3 - The growth parameters of carrot callus as affected by gum application 30 days after inoculation Qualitative traits Apricot gum concentration (g/l) Control 2.00 4.00 6.00 Days to callus initiation 35.75 a 29.80 b 19.60 c 13.70 d Callus fresh weight (g) 0.121 c 0.269 cb 1.01 a 0.364 b Callus dry weight (g) 21.54 a 10.08 b 11.32 b 13.66 b Means in the same row followed by different letters are signifi- cantly different at P<0.01 using LSD-Test. Growth trait Apricot gum concentration (g/l) Control 2.00 4.00 6.00 Plantlet length (cm) 20.00 ab 10.95 bc 22.72 a 8.60 c Number of shoots 2.25 b 3.75 ba 3.25 ba 5.25 a Number of leaves 68.25 a 27.00 b 36.75 b 47.5 ab Average leaf area (cm3) 0.06 c 1.75 a 1.16 ab 0.68 bc Days to root initiation 10.75 a 10.25 a 7.25 a 6.50 a Number of roots 35.00 a 29.50 a 35.00 a 17.50 a Root length (cm) 0.56 b 2.21 a 1.86 a 0.85 b Chlorophyll a (mg.g F.W.) 7.50 c 14.45 a 10.96 b 10.68 b Chlorophyll b (mg.g F.W.) 4.33 ab 5.43 a 2.77 b 3.87 ba Total chlorophyll (mg.g F.W.) 11.91 b 19.97 a 13.76 b 14.72 b Carotenoid (mg.g F.W.) 3.15 b 5.95 a 4.25 b 4.53 ba Table 4 - The growth parameters of stevia shoot proliferation and rooting 28 days after inoculation +++ indicates medium hadness, ++++ indicates medium hardness and fragile callus tissue. Khorsha et al. - Apricot gum as an organic additive in grapevine tissue culture media 115 parameters collectively in a single medium (shoot proliferation cum rooting medium supplemented with apricot gum). Furthermore, the results of our grapevine experiment were more obvious and noticeable than for the other species. These distinct effects on grapevine shoot proliferation and rooting are reported in figure 2 and Table 5. In the case of rooting parameters, the gum considerably reduced the time taken to root initiation (6 days against 14 days in control explants) and the rooting percentage was also improved, reaching nearly 100 % in the case of 6.0 g/L gum-supplemented media. Root length also increased: the greatest length was found in medium containing 4.0 g/L gum. However, all gum treatments showed longer roots compared to control explants. A similar trend was also observed in stevia samples (Table 4). Shoot proliferation was vigorously enhanced following application of gum to the media (Fig. 2 and 3). Thanks to the positive effects of apricot gum, longer vines with more lateral shoots, intern- odes and greater leaf area were achieved (Fig. 3). Although all three concentrations of gum were found to be effective with regard to the aforementioned vegetative traits as compared to gum-free medium, the efficiency of 4.0 g/L was evident. 4. Discussion and Conclusions Cultured plant tissues need a continuous supply of carbohydrates from the medium to encourage growth and survival in vitro (Kozai, 1991). Therefore, sugars such as sucrose, glucose, and sorbitol are gen- erally added as a carbon source (Kadota and Niimi, 2004). Furthermore, reliable callus proliferation and subsequent plant regeneration are important for effi- cient micropropagation and genetic manipulation of plant tissues (Khorsha, 2014). Carbohydrates play an important role in in vitro cultures as an energy and carbon source, as well as an osmotic agent (George et al., 2008). There are numerous reports on the effects of various carbon sources on in vitro callus growth and regeneration. For example, Huang and Huang (1999) demonstrated that sorbitol acts as a carbon source, providing energy for callus growth and plantlet regeneration. Other studies suggest that it acts only as an osmotic regulator to adjust osmotic pressure in calli (Al-Khayri and Al-Bahrany, 2002; George et al., 2008). The capacity of various carbohy- Fig. 2 - The effect of apricot gum on in vitro growth perfor- mance of grapevine explants. Fig. 3 - The effect of apricot gum on in vitro shoot proliferation (above) and rooting (below) of grapevine. Table 5 - The effect of apricot gum on certain growth parame- ters of grapevine 30 days after inoculation Growth trait Apricot gum concentration (g/l) Control 2.00 4.00 6.00 Appearance of the first leaf 17.25 a 7.00 b 5.00 c 4.75 c Number of shoots 1.00 c 1.00 c 2.5 a 2.00 b Number of leaves 3.00 b 4.75 b 9.5 a 9.50 a Number of roots 20.25 b 32.5 b 77.25 a 65.75 a Internode length (cm) 0.94 c 1.02 bc 1.41 a 1.29 ab Means in the same row followed by different letters are signifi- cantly different at P<0.01 using LSD-Test. Adv. Hort. Sci., 2016 30(2): 111-118 116 drates to support growth of Japanese morning glory callus was examined and it was found that sucrose was the most effective compound but glucose, fruc- tose, trehalose, maltose, cellobiose, raffinose and soluble starch were also found to be significant (Hisajima and Thorpe, 1985). Apricot gum and callus growth and proliferation Gums are typically more or less sticky in nature and translucent and amorphous. Chemical analysis of apricot gum has already been performed by Lluveras- Tenorio et al. (2012). They found total sugars (60%), galactose (43%), mannose (4%), arabinose (44%), xylose (7%) and ramnose (1%) in apricot gum. It is clear that such composition may be different in each lot, from one tree to another, and the results may not be reproducible in different tissue culture labora- tories. However, the present study was performed in five replications and positive responses were found with carrot callus growth. The callus volume pro- duced on media fortified with 4.0-6.0 g/L apricot gum was at least three to four times that of gum-free medium (Fig. 1). The enhanced callus growth induced by apricot gum may be considered a significant logi- cal reason for application of gum in plant tissue cul- ture media, especially in species for which indirect regeneration (plant regeneration from callus) is to follow. Apricot gum and shoot/root proliferation Different types of basal media used in plant tissue culture vary with regard to the concentration of macro and microelements, greatly affecting the in vitro growth and multiplication of shoots. Apart from this, type and concentration of carbon sources in the medium affect the physiology and differentiation of tissues (Lipavska and Konradova, 2004) while serving as osmotic and energy source (George et al., 2008). In our experiment, the utilization of apricot gum caused reasonable shoot proliferation both in stevia and grapevine (Table 4, Fig. 3), not to mention its very low cost and economic advantages, making it useful in commercial tissue culture laboratories seek- ing economical and feasible protocols for in vitro propagation of horticultural crops. There are some reports on the utilization of such low-priced and organic materials as a potential plant source with a high amount of sucrose and other sugars which could possibly be used as an alternative carbon source, for example molasses (Dhamankar, 1992), sugar cane juice (Buah et al., 2011), and date palm syrup (Al-kha- teeb, 2008 a). Furthermore, in a review presented by Yaseen et al. (2012), factors determining the efficacy of a carbon source including its type, concentration, and their mutual interaction are reported. In our study, apricot gum was exploited, although the appli- cation of almond gum or sweet cherry gum would also be possible, since these types of gum are also readily available throughout the country at similar price. However, there may be some differences in in vitro responses of gums from different origins. With regard to concentration, for tissue culture media, 4.0-6.0 g/L may be considered as a feasible range, because beyond this dose the solubility of gum would be difficult even with heat treatments. Moreover, in high concentrations the culture media cannot be solidified properly (Khorsha, 2014). The mutual interaction between gum and explants is also an important matter. Although, in the present experi- ment all three plant species showed positive responses for most of the measured parameters, however grapevine in vitro shoot proliferation was greatly enhanced in gum-supplemented media (Fig. 3). Rooting is considered a difficult step in micro- propagation of many woody plants (George, 1993) and it is regulated by a number of physiological, bio- chemical, and genetic factors (Pawlicki and Welander (1995). Generally, rooting occurs in an auxin- enriched medium. On the other hand, Hassan et al. (2009) mentioned that sucrose at the highest level considerably increased rooting of palm tissue cul- tures. The results of the present research on rooting parameters of stevia and grapevine correspond with those of Al-Khateeb (2008 b) on date palm. He observed an enhanced root formation as the sugar concentration increased (60 g/L and above). In conclusion, the present study considered addi- tion of apricot gum as a complex organic addendum in tissue culture of carrot callus, stevia and grapevine. It was found that addition of gum not only was without negative effects, but its usefulness was definitely confirmed. Apricot gum significantly increased in vitro growth and proliferation of carrot callus tissues. Furthermore, shoot/root vegetative parameters of gum-fortified media were actually promising in stevia and grapevine. Overall, gum at the rate of 4.0 g/L was found to be a logical concen- tration with respect to the positive response in all three species. 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