ACTA BOT. CROAT. 76 (2), 2017 163 Acta Bot. Croat. 76 (2), 163–170, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0006 ISSN 0365-0588 eISSN 1847-8476 The ameliorative effects of 24-epibrassinolide on shoot organogenesis inhibition occurring under NaCl-stressed conditions in cultures of cotyledon and hypocotyl explants of tomato (Lycopersicon esculentum Mill.) Emel Yılmaz-Gokdogan*, Betul Burun Faculty of Science, Department of Biology, University of Mugla Sitki Kocman, 48000- Mugla, Turkey Abstract – In this study, possible effects of 24-epibrassinolide (24-epiBL) pretreatment against NaCl stress were investigated using in vitro shoot organogenesis from cotyledon and hypocotyl explants in M-28 tomato hybrid cultivar. The cotyledon and hypocotyl explants of 10-day sterile seedlings were treated with 1 µM and 2 µM 24-epiBL solutions (prepared with 70% acetone) for 30 seconds and applied explants were cultured on MS medium supplemented with 2 mg/l 6-benzylaminopurine (BAP) + NaCl (0, 20, 40, 60, 80 and 100 mM). It was determined that the regeneration percentage as well as the shoot number/explant, the shoot length and the leaf number/shoot derived from both explants suffered NaCl stress after 30 days. 24-epiBL pretreatment against NaCl stress showed ameliorative effects and hypocotyl explants gave better results than cotyledon explants for these growth parameters. Different stages of shoot organogenesis from hypocotyl explants ap- plied with 24-epiBL under salt stress were observed with scanning electron microscopy. As a result, it was shown that 24-epiBL treatment against NaCl stress may play an effective role in salt tolerance in M-28 to- mato hybrid cultivar. Keywords: 24-epibrassinolide, in vitro culture, NaCl stress, shoot organogenesis, tomato. * Corresponding author, e-mail: emelyilmazgokdogan@gmail.com Introduction Salinity in soil and irrigation water is one of the envi- ronmental problems that have the most negative impact on agricultural productivity in arid and semi-arid areas (Mo- hamed et al. 2010). High salinity influences plant growth and development due to degradation of or damage to cellu- lar macromolecules such as lipids, proteins and nucleic acid (Aly et al. 2012). In vitro plant tissue culture techniques are very useful and economical tools for the study of the physiological ef- fects of NaCl at cellular level under controlled conditions, and they give us valuable information concerning the re- sponses of plants to NaCl stress (Cano et al. 1998, Mercado et al. 2000). Additionally, tissue culture can dissolve vari- ous limitations triggered by salt stress (Osman et al. 2010). In vitro techniques may be used quickly to screen a lot of genotypes in plant culture under stress conditions (Aazami et al. 2010). Various tissue techniques can be utilized to de- rive NaCl tolerant cell lines and to improve tolerance to salt stress in several plants such as tomato (El-Enany 1995). The organogenesis process is shoot or plantlet forma- tion from cell, tissue or callus, and there is a positive inter- action between plantlet formation and callus development in tomato (Noaman and Ahmad 2004). The success of the regeneration response in tomato was reported to depend on genotype, explants, and plant growth regulators in culture media (Bhatia et al. 2004, Yilmaz and Burun 2014). There have been some studies about negative effects on direct and indirect regeneration of tomato explants affected by the stress of rising NaCl concentration (Mercado et al. 2000, Hassanein 2004, Mohamed et al. 2011). Brassinosteroids, which exist naturally in plants, are a new class of steroidal plant hormones (Fujioka et al. 1998). They affect numerous physiological processes such as cell elongation and proliferation when applied exogenously at nanomolar or micromolar concentrations (Clouse 1996). In addition to their roles in plant development, brassino- steroids have preventative effects against salt stress and various abiotic stress factors and have given satisfactory re- sults in reducing the effects of environmental stress in many horticulture crops (Surgun et al. 2012). YILMAZ-GOKDOGAN E., BURUN B. 164 ACTA BOT. CROAT. 76 (2), 2017 In vitro shoot regeneration from explants such as hypo- cotyl, cotyledon, leaf and shoot tip has already been achieved in tomato and other economically important plants in saline conditions (Mercado et al. 2000, Hassanein 2004, Noaman and Ahmad 2004, Mohamed et al. 2011). Howev- er, there is no literature concerning the study of the possible effects of brassinosteroids on shoot regeneration under salt stress. The purpose of this study is to investigate for the first time the effect of short-term exogenous 24-epiBL pretreat- ment against salt stress using in vitro shoot organogenesis in tomato. Material and methods Plant material and in vitro culture Tomato M-28 hybrid cultivar was used as plant material in this study and its seeds were obtained from Agrotek Seed Agriculture Industry and Trade Limited Company. For sur- face sterilization, the seeds were soaked in 2.25% NaOCl (50% diluted Na-hypochlorite) for 5 minutes and then thor- oughly washed with sterile distilled water three times (Yilmaz-Gokdogan and Burun 2015). These seeds were germinated on half-strength Murashige-Skoog (1962) (½ MS) medium supplemented with 20 g L–1 sucrose and 7 g L–1 agar. The cotyledon (5 mm × 5 mm) and hypocotyl (5 mm) explants of 10-day sterile seedlings were treated with 1 µM and 2 µM 24-epibrassinolide (24-epiBL) solutions prepared with 70% acetone for 30 seconds and the control explants were exposed to only 70% acetone application. 24-epiBL applied explants were transferred to MS medium including 2 mg L–1 6-benzylaminopurine (BAP) and increas- ing concentrations (0, 20, 40, 60, 80 and 100 mM) of NaCl (Yilmaz and Burun 2014). pH of MS medium containing 30 g L–1 sucrose was adjusted to 5.8 and then 7 g L–1 agar was added. The sterilization of MS medium was carried out in an autoclave at 121 °C and under 1 atm pressure for 15 minutes. All the culture vessels were kept under a 16 hour photoperiod (45 µE m–2 s–1 supplied by daylight fluorescent tubes) at 25 °C ± 2 in a culture room. For each application, 20 cotyledon and 20 hypocotyl explants were used and each treatment was repeated at least two times in the study. The regeneration percentage (%), the shoot number/explants, the shoot length (mm) and the leaf number/shoot derived from both explants were evaluated after four weeks. Scanning electron microscopy examination (SEM) The different stages of direct and indirect shoot organo- genesis from hypocotyl explants in the various samples were investigated by SEM at the end of in vitro culture. 0.5 to 1 cm samples were taken at areas where shoot organo- genesis may be formed, and fixation was conducted in 2.5% gluteraldehyde and 0.1 M phosphate buffered saline solu- tion (pH 7.4), OsO4 (osmium tetraoxide). After dehydration at first through a graded ethanol series and then ethanol: amyl acetate series, samples were finally soaked in pure amyl acetate, dried via CO2 critical point drying. SEM anal- yses were carried out at the Research and Application Cen- ter for the Research Laboratory of Mugla Sitki Kocman University. Data collection and statistical analysis All experiments were repeated at least two times and standard errors of the means were calculated. Data were analyzed by using Statistica statistical program package. All data obtained were subjected to analysis of variance (one-way ANOVA). Factor analysis of variance was per- formed to examine the effect of the explant type, 24-epiBL pretreatment and their interaction on the investigated pa- rameters. Comparisons with P-values ≤ 0.05 were consid- ered significantly different. Results The study was performed according the effect of 24-epi- BL of NaCl stress evaluated on in vitro shoot regeneration and plantlet formation. It was found that the regeneration percentage in 24-epiBL non-treated cotyledon explants was the highest in the NaCl-free control (Fig. 1). Increasing NaCl concentrations negatively affected regeneration per- centage, ranging from 90% to 3% (from 0 to 80 mM NaCl, respectively). The appearance of buds and development of leaves from 24-epiBL treated and non-treated cotyledon ex- plants were observed after two weeks in culture media. In addition both direct and indirect shoot organogenesis oc- curred from cotyledons explants (Fig. 2). When we evalu- ated the effect of 24-epiBL treatment against NaCl stress, both 24-epiBL treatments at 20 mM NaCl, 1 µM 24-epiBL at 40 mM NaCl and 80 mM NaCl showed statistically posi- tive effects on regeneration percentage (Figs. 1 and 2); on the other hand, 24-epiBL treated cotyledon explants did not regenerate under a 100 mM NaCl condition (data not shown). Similarly, regeneration percentage decreased in 24-epi- BL non-treated the hypocotyl explants under salt stress (Fig. 3). In these explants, the generation percentage was 96% in the NaCl-free control medium and it was also de- termined that regeneration was76%, 54%, 34%, 7%, 10% from 20 mM NaCl to 100 mM NaCl. In general there was a gradual decrease for the regeneration percentage from 0 to 100 mM NaCl, but a particularly clear decrease in the re- generation percentage was observed at 80 mM and 100 mM NaCl. In the all NaCl concentrations, regeneration in hypocotyl explants was higher than in cotyledon explants. Adventive shoots with leaves were observed from control to 60 mM NaCl, but leaf formation did not occur at 80 mM and 100 mM NaCl. Direct and indirect shoot organogene- sis occurred from cotyledon explants for all treatments (Fig. 4). When the effect of 24-epiBL against NaCl stress was evaluated in this study, the increase of the regenera- tion percentage with 1 µM 24-epiBL pretreatment at 60 mM and 80 mM NaCl was statistically significant (Fig. 3). The factorial analysis showed that the explant type at 60, 80 and 100 mM NaCl concentrations had a significant ef- fect on regeneration. 24-EpiBL pretreatment, on the other hand, except with 100 mM NaCl, affected regeneration at all the NaCl concentrations (On-line Suppl. Tab. 1). Ex- plant type × 24-epiBL interaction was found to lead to sta- tistically significant differences at 20, 40, 60 and 100 mM NaCl concentrations. AMELIORATIVE EFFECTS OF 24-EPIBRASSINOLIDE ON NACL-STRESSED TOMATO ACTA BOT. CROAT. 76 (2), 2017 165 Fig. 1. The regeneration percentage (%) in cotyledon explants of Lycopersicon esculentum pretreated with 24-epibrassinolide (24-epiBL) in concentrations 0, 1 and 2 µM and NaCl in the range of concentrations 0–80 mM. Different letters show the statistically significant dif- ferences between 24-epiBL pretreatments, at p≤0.05. Fig. 2. The regeneration status of 24-epibrassinolide treated and non-treated cotyledon explants of Lycopersicon esculentum in MS me- dium containing 20, 40 and 80 mM NaCl after 30 days: a) 20 mM NaCl + 0 µM 24-epiBL, b) 20 mM NaCl + 1 µM 24-epiBL, c) 20 mM NaCl + 2 µM 24-epiBL, d) 40 mM NaCl + 0 µM 24-epiBL, e) 40 mM NaCl + 1 µM 24-epiBL, f) 40 mM NaCl + 2 µM 24-epiBL, g) 80 mM NaCl + 0 µM 24-epiBL, h) 80 mM NaCl + 1 µM 24-epiBL, i) 80 mM NaCl + 2 µM 24-epiBL. Bars = 1 cm. YILMAZ-GOKDOGAN E., BURUN B. 166 ACTA BOT. CROAT. 76 (2), 2017 Fig. 3. The regeneration percentage (%) in hypocotyl explants of Lycopersicon esculentum pretreated with 24-epibrassinolide (24-epiBL) in concentrations 0, 1 and 2 µM and NaCl in the range of concentrations 0–100 mM. Different letters show the statistically significant differences between 24-epiBL pretreatments, at p≤0.05. Fig. 4. The regeneration status of 24-epibrassinolide treated and non-treated hypocotyl explants of Lycopersicon esculentum in MS me- dium containing 60, 80 and 100 mM NaCl after 30 days: a) 60 mM NaCl + 0 µM 24-epiBL, b) 60 mM NaCl + 1 µM 24-epiBL, c) 60 mM NaCl + 2 µM 24-epiBL, d) 80 mM NaCl + 0 µM 24-epiBL, e) 80 mM NaCl + 1 µM 24-epiBL, f) 80 mM NaCl + 2 µM 24-epiBL, g) 100 mM NaCl + 0 µM 24-epiBL, h) 100 mM NaCl + 1 µM 24-epiBL, i) 100 mM NaCl + 2 µM 24-epiBL. Bars = 1 cm. AMELIORATIVE EFFECTS OF 24-EPIBRASSINOLIDE ON NACL-STRESSED TOMATO ACTA BOT. CROAT. 76 (2), 2017 167 Besides regeneration percentage, adventive shoot num- ber of per explant (Tab. 1), shoot length (Tab. 2) and leaf number of per shoot (Tab. 3) were also evaluated. In the 24-epiBL non-treated cotyledon explant, shoot number of per explant was very close from 0 to 40 mM NaCl, but it decreased from 60 mM to 100 mM NaCl. The highest shoot number was interestingly observed with the 2 µM 24-epiBL pretreatment at 80 mM NaCl (11.00/explant). The shoot number decreased in the 24-epiBL non-treated at 80 and 100 mM NaCl (1.00/explant) and there was no shoot for- mation with 24-epiBL treatment at 100 mM NaCl. In this study, only the effect of 2 µM 24-epiBL pretreatment against 80 mM NaCl stress was found statistically different (Tab. 1). Shoot number derived from 24-epiBL non-treated and treated hypocotyl explants on MS medium was nega- tively affected when salt stress was increased. When we evaluated the effect of 24-epiBL against NaCl stress, only 1 µM 24-epiBL treatment at 60 mM NaCl was statistically significant (Tab. 1). The explant type used was found to have a significant effect on the adventive shoot number/ex- plant at 0, 20 and 100 mM NaCl concentrations (data not shown). 24-EpiBL pretreatement led to significant differ- ences at 80 mM NaCl concentration while explant type × 24-epiBL interaction did not have a significant effect. The shoot length derived from 24-epiBL non-treated cotyledon explants was observed to increase from 0 mM NaCl to 80 mM NaCl. The shoot length was not measured because the regenerated shoot swere very small (< 0.5 mm) at 100 mM NaCl. Additionally, no effect of 24-epiBL pre- treatment against NaCl stress was determined on the shoot length (Tab. 2). In 24-epiBL treated and non-treated hypo- cotyl explants, the adventive shoot length was high for all the NaCl concentrations when the compared with the con- trol. When it was evaluated the effect of 24-epiBL under salt stress, the positive effect of 1 µM 24-epiBL treatment was found statistically significant only at 100 mM NaCl (Tab. 2). Also, the effects of explant type, 24-epiBL and ex- plant type × 24-epiBL interaction on the shoot length were statistically evaluated; it was found that explant type has a significant effect on the shoot length at 0, 60 and 100 mM NaCl concentrations, 24-epiBL led to significant differenc- Tab. 2. The length of shoots derived from cotyledon and hypocotyl explants of Lycopersicon esculentum upon 24-epibrassinolide (24-epi- BL) pretreatment and NaCl treatment in the range of concentrations 0–100 mM. Values are means ± standard errors. Different letters show the statistical difference between 24-epiBL treatments and the control in the same column, at P≤0.05. 24-EpiBL (µM) Length of shoots derived from cotyledon NaCl (mM) 0 20 40 60 80 100 0 4.73±0.32a 7.45±0.54a 9.89±1.05a 7.53±1.67a 6.00±0.00a 0.00±0.00a 1 4.13±0.34a 6.45±0.76a 6.00±0.73b 5.57±1.04a 1.00±0.00b 0.00±0.00a 2 4.81±0.44a 6.49±0.51a 7.27±0.94ab 1.00±0.00a 4.66±0.66a 0.00±0.00a 24-EpiBL (µM) Length of shoots derived from hypocotyl NaCl (mM) 0 20 40 60 80 100 0 3.29±0.09a 6.84±0.31a 9.07±0.72a 8.22±0.97a 10.00±2.51a 3.50±0.95b 1 3.18±0.09a 7.77±0.47a 7.10±0.79a 8.22±0.70a 8.92±1.86a 8.33±2.33a 2 3.37±0.11a 7.17±0.40a 9.47±0.73a 5.69±0.59b 5.00±0.57a 3.90±0.73b Tab. 1. The number of 30-day adventive shoots of Lycopersicon esculentum derived from cotyledon and hypocotyl explants (shoot num- ber/explant) upon 24-epibrassinolide (24-epiBL) application and NaCl in the range concentrations 0–100 mM. Values are means ± stan- dard errors. Different letters show the statistical difference between 24-epiBL treatments and the control in the same column, at P≤0.05. 24-EpiBL (µM) Shoot number/cotyledon explant NaCl (mM) 0 20 40 60 80 100 0 8.14±1.32a 9.17±1.79a 8.27±2.28 a 5.87±2.25a 1.00±0.00b 1.00±0.00a 1 7.80±1.18a 9.08±1.45a 5.54±1.29 a 5.37±1.74a 2.66±0.33b 0.00±0.00a 2 8.00±1.63a 8.82±1.49a 6.93±2.24 a 2.87±0.49a 11.00±0.57a 0.00±0.00a 24-EpiBL (µM) Shoot number/hypocotyl explant NaCl (mM) 0 20 40 60 80 100 0 20.30±2.37a 13.50±1.68a 6.12±0.95a 3.92±0.63b 4.33±1.45a 2.50±1.19a 1 24.82±2.64a 12.79±1.82a 7.29±1.52a 8.41±1.35a 3.87±1.10a 3.25±1.60a 2 22.22±2.56a 12.75±1.52a 7.81±1.38a 5.88±0.79ab 9.66±4.97a 4.00±1.15a YILMAZ-GOKDOGAN E., BURUN B. 168 ACTA BOT. CROAT. 76 (2), 2017 es at 40 and 60 mM NaCl concentrations, but explant type × 24-epiBL interaction was found to be not significantly different (On-line Suppl. Tab. 2). The leaf number per shoot derived from 24-epiBL treat- ed and non-treated cotyledon explants was very close and there was no statistically significant effect of 24-epiBL against NaCl stress (Tab. 3). The leaf number of per shoot developed from 24-epiBL non-treated hypocotyl explants, like cotyledon explants, ranged from 1.00 to 1.33. The posi- tive effect of 2 µM 24- epiBL treatment under 0 mM and 20 mM NaCl stress was statistically significant on the leaf number per shoot (Tab. 3). When the effects of explant type, 24-epiBL and explants type × 24-epiBL interaction on the leaf number of per shoot according to NaCl concentra- tions were statistically evaluated the explant type was found to have a significant effect at 0 mM and 100 mM NaCl. 24-EpiBL pretreatment, on the other hand, led to significant differences only in the control. Explant type × 24-epiBL in- teraction was found to have a significant effect at 20 mM NaCl (On-line Suppl. Tab. 3). Different stages of regeneration were observed from 24-epiBL treated and non-treated hypocotyl explants by SEM at the end of 30-day in vitro culture. SEM showed that direct regeneration process started by formation of or- ganized cell groups (Fig. 5a) and the regenerated shoots with first leaf primordia formed from these cell groups (Figs. 5b and 5c). Finally the development of regenerated shoots and adventive shoots with fully formed leaf primor- Tab. 3. The number of leaf per shoot derived from cotyledon and hypocotyl explants of Lycopersicon esculentum upon 24-epibrassinolide (24-epiBL) pretreatment and NaCl treatment in the range of concentrations 0–100 mM. Values are means ± standard errors. Different let- ters show the statistical difference between 24-epiBL treatments and the control in the same column, at P≤0.05. 24-EpiBL (µM) The number of leaf per shoot derived from cotyledon NaCl (mM) 0 20 40 60 80 100 0 1.14±0.02a 1.18±0.03a 1.17±0.05a 1.23±0.12a 1.00±0.00a 0.00±0.00a 1 1.08±0.02a 1.12±0.03a 1.22±0.08a 1.36±0.13a 1.00±0.00a 0.00±0.00a 2 1.16±0.04a 1.16±0.03a 1.24±0.08a 1.00±0.00a 1.33±0.33a 0.00±0.00a 24-EpiBL (µM) The number of leaf per shoot derived from hypocotyl NaCl (mM) 0 20 40 60 80 100 0 1.01±0.004b 1.10±0.01b 1.26±0.05a 1.20±0.07a 1.33±0.33a 1.00±0.00a 1 1.01±0.004b 1.19±0.04ab 1.25±0.08a 1.37±0.08a 1.07±0.07a 1.00±0.00a 2 1.08±0.013a 1.25±0.04a 1.17±0.05a 1.38±0.09a 1.33±0.21a 1.11±0.11a Fig. 5. Different stages of adventive shoot initiation from hypocotyl explants of Lycopersicon esculentum with or without the pretreat- ment with 24-epibrassinolide under salt stress after 30 days in culture: a) 100 mM NaCl + 1 µM 24-epiBL, b) 60 mM NaCl + 1 µM 24-epiBL, c) 20 mM NaCl + 0 µM 24-epiBL, d) 60 mM NaCl + 0 µM 24-epiBL, e) 80 mM NaCl + 1 µM 24-epiBL, f) 0 mM NaCl + 0 µM 24-epiBL; circle denotes organogenic callus, and arrow denotes leaf primordia at different developmental stages. Bars = 100 µm. AMELIORATIVE EFFECTS OF 24-EPIBRASSINOLIDE ON NACL-STRESSED TOMATO ACTA BOT. CROAT. 76 (2), 2017 169 dia was observed (Figs. 5d–f). In addition to SEM, mopho- logical observations too revealed that NaCl stress did not affect the shoot organogenesis process from hypocotyl ex- plants. But NaCl stress depressed shoot initiation and de- velopment, and NaCl stress (especially 80 mM and 100 mM NaCl) inhibited leaf formation in adventive shoots (Figs. 4 and 5a–f). Discussion In this study, at first the effects of 24-epiBL pretreat- ment on shoot growth were evaluated using in vitro shoot regeneration under NaCl stress. In vitro shoot morphogen- esis on tomatoes is the most important method used for the evaluation and screening of NaCl tolerance. Increased sa- linity leads to reduction of shoot number, shoot length, fresh weight and dry weight (Mercado et al. 2000). Sup- pressed shoot growth and development because of high sa- linity are connected with water stress, specific ion toxicity and ion imbalance or induced nutritional deficiency. Fur- thermore, the highest concentration of Na+ might cause problems to membrane stability, enzyme inhibition, and cell division and elongation defects (Aly et al. 2012). Espe- cially, reduction in growth parameters such as callus fresh weight, organogenic shoot induction and leaf formation might be the result of reduced water availability in the cul- ture media with NaCl (Chamandoosti 2007). In this study, it was determined that the shoot regeneration percentage in the cotyledon and hypocotyl explants was high in salt free medium but shoot regeneration capacity of tested explants was inhibited by increasing NaCl stress. 24-EpiBL pretreat- ment used for obtaining salt tolerance was conducted on cotyledon and hypocotyl explants, and 24-epiBL against various NaCl concentrations showed ameliorative effects. Similarly Mercado et al. (2000) determined that regenera- tion from leaf disc explants was high under salt free condi- tion in tomato Pera and HF cultivars and suffered under in- creasing salt stress. Benderradji et al. (2012) reported that callus induction, shoot proliferation from callus and shoot regeneration under salt stress negatively affected in vitro embryo culture in wheat. These findings show that shoot regeneration could depend on tomato cultivar, genotype and explant type under increasing NaCl stress. The number of regenerated shoots derived from 24-epi- BL treated and non-treated cotyledon and hypocotyl ex- plants was high under salt free medium and it decreased under salt stress. The shoot number increased with 24-epi- BL treatment under salt stress. Mohamed et al. (2011) re- ported that the shoot number decreased in cotyledon and hypocotyl explants of tomato Pearl and Beril cultivars in saline conditions. In addition, Chamandoosti (2007) showed that adventive shoot number from canola hypocotyl seg- ments decreased under stress conditions. These findings show that NaCl stress adversely affects shoot organogene- sis. The shoot length from both explants increased also from 0 to 100 mM NaCl concentration in this study. How- ever, Mohamed et al. (2011) reported that the shoot length decreased under salt stress in Pearl and Beril tomato culti- vars. The results showed that NaCl stress greatly influenced the in vitro performance of two different explants of tomato M-28 cultivars and that shoot induction and development was negatively affected by increasing NaCl stress. The physiological processes such as decline in photosynthesis, change in cell turgor, metabolite accumulation, increase in reactive oxygen species and changes in antioxidative en- zyme activity to scavenging of these reactive oxygen spe- cies, disturbance of carbon and nitrogen allocation and change in ion homeostasis occurring under NaCl stress are well known; recently it has been very popular to use vari- ous biologically active substances (proline, polyamine, plant growth regulators especially brassinosteroids) to under- stand the relationships among these physiological events for obtaining salt tolerance. In this study, the positive effects of 24-epiBL treatment were firstly determined on in vitro shoot regeneration from 24-epiBL treated cotyledon and hypocotyl explants under salt stress. Additionally hypocotyl explants had better re- sults than cotyledon explants on all parameters tested such as regeneration percentage, shoot number and shoot length. SEM analysis showed that no differences relating to the morphology of the shoot organogenesis from hypocotyl ex- plants were observed by NaCl and 24-epiBL treatments. 24-epiBL pretreatments can improve shoot growth and de- velopment under NaCl stress and may play an effective role for salt tolerance in M-28 tomato hybrid cultivar. Acknowledgements This article was a part of Emel YILMAZ GOKDO- GAN’s PhD thesis. The study was supported by Mugla Sit- ki Kocman University, Scientific Research Projects Coordi- nation Unit (Mugla, Turkey, project number: 2011/17). References Aazami, M. 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