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A new strategy of high concentration short duration treatment of Thidiazuron (TDZ) for enhancing adventitious buds formation from hypocotyl explants were attempted in Jatropha curcas. The best shoot buds induction (81.91%) and number of buds per explant (10.16) were obtained from hypocotyl explants treated with 20 mg/L TDZ solution for 20 min, followed by 30 days of culture on hormone-free MS medium. The elongated shoots initiated roots to become intact plantlets in rooting medium containing indole-3-butyric acid (IBA) and L-glutamine (Gln), and the results showed that the initiation and growth of roots were effectively stimulated with supplementing 16 mg/L Gln into the rooting medium, and having the best rooting rate (51.39%). After acclimatization, an intact plantlet could usually be gained at 60 days of culture by using the culture protocol described in this study. Introduction Jatropha curcas L. is a sort of woody plant and pertains to the Euphorbiaceae family [1,2]. As is well-known that the latex from J. curcas contains alkaloids have been reported to contain medical composition and can be utilized to acquire pharmaceutical adjective constituents [3,4] and pesticides [5]. Nevertheless, the best known of this tree is the high oil yielding from its seeds (40% to 60%) [6]. Widespread interest in cultivation of J. curcas all over the world, because methyl ester extracted from J. curcas is suitable for production of biodiesel [4,5,7,8]. The most major problem for J. curcas plants not being grown on a major scale is that the low and unstable seed production because of natural hybrid [9]. With the purpose of breeding excellent varieties, the method of genetic transformation of the species might be a feasible approach [10-13]. In a general way, adventitious shoot regeneration from the transformed receptors (explants) is a precondition in genetic transformation system. Moreover, it is reported that the tissue culture cycles of common regeneration protocols were quite time-consuming (usually 3-5 months) for gaining intact plant in most of the reports in J. curcas. Consequently, it is indispensable to exploit high-efficiency methods of tissue culture, which could accelerate mass production of good-quality implants materials and capacitate breed improvement by means of technique for gene engineering. It was found that the regeneration of plants from different organs as explant sources had been implemented using tissue cultures protocols in J. curcas [14-19]. However, the regeneration efficiency of hypocotyl explants was barely satisfactory in J. curcas [20-23]. 0076 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) © 2016. The authors – Published by Atlantis Press Materials and Methods Plant Materials The mature J. curcas seed coded M-19 were the material source in our study. All plants were cultivated at a farm in Haikou, China [24]. Preparation of Explants Vigorously growing in vitro hypocotyl from 5 day old aseptic seedings were cut into in vitro hypocotyl explants of about 0.5 cm in length. Well-growing hypocotyl from 12 day old seedings were cut into in vivo hypocotyl explants of about 0.5 in length. Preparation of Thidiazuron (TDZ) Solution TDZ was dissolved into 1 mol/L NaOH to produce 0.2 mg/mL TDZ solution. And the solution was diluted with distilled water to generate the concentrations as follows: 0, 10, 20, 30 and 60 mg/L. These were adjusted with 1 mol/L HCl to acquire a pH value range of 5.8-6.0, and filter-sterilized before being used for treating the hypocotyl explants. Dealing with the Hypocotyl Explants with TDZ Solution Hypocotyl explants were immersed in glass petri dishes supplemented with various concentrations (0, 10, 20, 30 and 60 mg/L) of TDZ solution for various time periods (0, 5, 20, 30 and 40 min). After treatment, the explants were transitorily put on sterile absorbent paper to soak excess amount of moisture. Preparation of Mediums and Culture Conditions Ordinary MS media was applied to overall tissue culture experiments in this study. All mediums had been included 2.5 % sucrose and 0.6% agar, and tuned to pH 5.8-6.0 with 1 mol/L NaOH before being autoclaved at 1.4 kg cm-2 for 20 minutes. Unified culture conditions were applied in all experiments. All treatments were kept at 25 ± 1°C under a 12 h photoperiod of 60-80 μ mol m-2s-1 intensity (cool white fluorescent tubes). Regeneration of Adventitious Buds In order to induce adventitious buds regeneration, hypocotyl explants were placed onto hormone-free Murashige and Skoog’s (MS) medium [25] after treated by TDZ solution for different time periods for 30 days. The explants were put on the mediums with their morphological up-side up vertically simply for comparative experiments. What’s more, hypocotyl explants were also treated with conventional approach and horizontally inoculated onto MS medium including 0, 0.1, 0.3, 0.6 and 1.2 mg/L TDZ as reported anteriority [20-23]. Elongation of Shoot-Buds The regenerated shoot-buds were transferred with the mother tissues (explants) together onto MS medium containing 0.5 mg/L BA, 0.25 mg/L indole-3-acetic acid (IAA), 0.2 mg/L kinetin (KT) and 0.4 mg/L gibberellic acid (GA3) for 15 days [24,26]. Rooting For the sake of inducing rooting of the elongated shoot-buds, shoots (at least 10 mm in length) were separated from the maternal tissues and inoculated them on MS medium contained 0.3 mg/L indole-3-butyric acid (IBA) and various concentrations (0, 2, 8, 16, 24 and 48 mg/L) of L-glutamine (Gln) and the experimental results were recorded after cultivating 10, 20, 30 and 40 days. Gln solution was filter-sterilized and supplemented into the autoclaved medium as depicted anteriorly [27]. Process of Transplant Acclimatization Regenerated plantlets were taken out from jars carefully, washed completely for wiping off 0077 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) © 2016. The authors – Published by Atlantis Press residuary mediums, and then they were migration to plastic pots with germfree sand and soil at the ratio 1:1 and carpeted with plastic wrap for 15-20 days. Finally, the complete plants were moved to a conservatory (working Humidity 70-80% and temperature 25 ± 3°C ) for further accumulation and growth. Data Analysis All experiments were established on a design of random factor method completely and repeated 3 times with 35 explants per treatment in the present study. ‘Regeneration percentage’ was computed by dividing the number of explants that regenerated shoot-buds with the amount of explants. ‘Number of buds per explant’ was computed by dividing the number of regenerated shoot-buds with the amount of hypocotyl explants that had regenerated buds. ‘Length of shoot-buds’ was computed by the average length of elongated shoots. ‘Rooting percentage’ was computed by dividing the number of shoots that had regenerated adventitious roots with the total amount of shoots used for inducing rooting. ‘Number of roots per shoot’ was computed by dividing the number of regenerated roots with the amount of shoots that had regenerated roots. ‘Average root length’ was computed by dividing length of all regenerated roots with the number of regenerated roots. Statistical analysis of the data was implemented by utilizing SPSS 17.0 soft ware. Furthermore, data in the same column followed by different letters was significantly different by Duncan’s test at p ≤ 5% level. The experimental data was shown as means ± SD (standard deviation) of 3 standalone experiments. Results Regeneration of Shoot-Buds from Hypocotyl Explants with Conventional Techniques Hypocotyl explants were inoculated on MS medium supplemented with different concentrations of TDZ as reported anteriorly [20-23]. The results indicated that the highest centage of adventitious buds induction (24.73%) and the maximum number of induced shoot-buds per explant (2.63) were investigated when 0.6 mg/L TDZ was used (Table 1 and Fig. 1A). However, improving the TDZ concentration to exceed 0.6 mg/L would not obtain better results, so 0.6 mg/L TDZ was the most suitable concentration for the induction of shoot-buds (Table 1 and Fig. 1A). Furthermore, the quantity of regenerated buds per explants was considerably small, whilst majority of the regenerated shoot-buds were extremely tiny and underdeveloped. What’s more, further elongation of shoot-buds was very difficult at 0.6 mg/L concentration (data not shown). Table 1. Results of various TDZ concentrations on the regeneration of adventitious buds from hypocotyl explants in J. curcas after 30 days of culture. TDZ concentration (mg/L) Regeneration percentage (%) Number of buds per explant 0 0d 0d 0.1 9.93±1.81c 1.19±0.12c 0.3 18.80±1.93b 1.81±0.32b 0.6 24.73±3.06a 2.63±0.21a 1.2 17.97±2.38b 2.13±0.28ab Regeneration of Shoot-Buds from Hypocotyl Explants after Being Treated with TDZ Solution Hypocotyl explants were dealt with different concentrations of TDZ solution for 20 min before being placed onto basic MS medium free of hormone. The experimental results showed clearly that the protocol of treatment with TDZ solution was more efficient than conventional means on induction of shoot-buds regeneration from hypocotyl explants. Moveover, the highest percentage of adventitious buds induction (81.91%) and the maximum number of induced shoot-buds per explant (10.16) were obtained on account of the utilization of 20 mg/L TDZ solution (Table 2 and Fig. 1B) . 0078 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) © 2016. The authors – Published by Atlantis Press When the concentrations of TDZ solution were not more than 20 mg/L, the percentage of adventitious buds induction and the number of induced shoot-buds per explant were proportional to the TDZ concentrations directly. Nevertheless, the application of higher than 20 mg/L resulted in the percentage of shoot-buds regeneration significantly decreased (Table 2). Hence,

Efficient Protocol for Introducing Regeneration of Adventitious Buds from Hypocotyl Explants in Jatropha curcas L.