High Concentration of Thidiazuron Stimulates Adventitious Bud Regeneration from Cotyledon Explants in Jatropha curcas

A high-frequency protocol for induction of adventitious buds and plant regeneration from cotyledon explant of Jatropha curcas is described. The cotyledon explants of J. curcas cultured directly onto medium containing Thidiazuron (TDZ) induced regeneration of only poor quality adventitious buds that had a low regeneration frequency, and subsequently the elongation of shoot-buds was difficult and unsatisfactory. However, treating the cotyledon explants with high concentrations (10-60 mg/L) of TDZ solution for short time periods (10-60 min) helped to improve the regeneration frequency and enhance the quality of the regenerated shoot-buds significantly. The best shoot-buds induction (87.45%) and number of shoot-buds (11.23) per explant were seen when in vitro explants were treated with 20 mg/L TDZ solution for 40 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium in 30 days. The elongated shoots initiated roots to become intact plantlets in rooting medium supplemented with 0.1 mg/L indole-3-butyric acid (IBA) effectively stimulated the initiation and growth of roots with the best rooting rate (44.28%). After acclimatization, these plantlets were transplanted to soil wherein normal growth was observed. Hence, an intact plantlet could usually often be gained at 60 to 70 days of culture by applying the culture method described in the present study. This protocol could be widely used for mass production of regenerative plants and the yield of transgenic plants through Agrobacterium-mediated transformation. Introduction Jatropha curcas L. is the common name for physic nut and belongs to the Euphorbiaceae family [1]. J. curcas is a type of woody plant widely distributed in the tropical and sub-tropical areas [2]. The seeds and oil from J. curcas are unfit for human and animal consumption because of the presence of toxins such as curcin, phorbol esters and saponins [3-6]. The latex from J. curcas contains alkaloids such as Jatrophine and Jatropham, which are reported to have medicinal properties and can be used for extracting pharmaceutical compounds [7-8] and insecticides [9]. However, this tree is most famous for the high oil content in its seeds (up to 60%) [10], which could be easily processed to replace the conventional fossil diesel [11]. There has been a surge of interest in J. curcas cultivation around the world since studies showed that its methyl ester could yield biodiesel [8-13]. To meet the ever increasing demand for biodiesel, feedstock and medicinal applications, it is necessary to develop toxic-free, high-yielding, and biotic and abiotic stress resistant J. curcas. However, conventional breeding is hampered by low and inconstant seed yield due to heterozygous nature of J. curcas plants and sexual incompatibilities due to deleterious gene linkages, followed by difficulty in obtaining large seed yield, fertile progenies and requires a very long time [14,15]. Biotechnological innovations are envisaged for genetic upgrading of this crop wherein transgenic production will be the most important in achieving the above parameters. 0085 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) © 2016. The authors – Published by Atlantis Press In a genetic transformation system, regeneration of adventitious buds from the genetically transformed tissues (explants) is in most cases an essential step. To apply Agrobacterium-mediated genetic transformation approach to J. curcas improvement more rapidly and efficiently, one prerequisite is to establish an efficient plant regeneration system. Previously, shoot-buds regeneration of J. curcas had been successfully obtained from cotyledon, petiole, hypocotyl, epicotyl, and leaf tissue by conventional culture methods, but those regeneration efficiency were still not satisfactory and the periods of obtaining regenerated plants were too long (90-150 days) [16-23]. In this study, we have developed an efficient and reproducible plant regeneration procedure for J. curcas by utilizing high concentration of TDZ solution to deal with the cotyledon explants. Materials and Methods Plant Materials Seeds coded M-19 of J. curcas were grown and collected from the farm in Haikou, Hainan province of China [27]. Preparation of Culture Medium and Culture Maintenance Uniform culture conditions were applied in all experiments. Basal MS formula was used for all tissue culture experiments. Media used in our experiment contained 2.5% sucrose that were adjusted to pH 5.8-6.0 with 1 mol/L NaOH, and had 0.7% agar added prior to being autoclaved at 1.4 kg cm-2 for 20 minutes. All culture treatments were kept at 25 ± 1°C under a 12 h photoperiod of 60-80 μmol m-2s-1 intensity (cool white fluorescent tubes). Preparation of Explants The seeds were surface-sterilized for 60 s with 75% (v/v) ethanol after removing the outer seed coat, immersed in 2% (v/v) sodium hypochlorite (NaClO) for 20 min and finally rinsed 5 times in sterile distilled water. The embryos were removed from the seed and incubated on hormone-free Murashige and Skoog salt (MS) medium [24]. Cotyledons were excised from 5-day-old seedlings, cut into small pieces (5×5 mm), and used as explants. Preparation of Thidiazuron (TDZ) Solution and Treating the Cotyledon Explants with TDZ Solution TDZ (Sigma-Aldrich Co., St. Louis, MO, USA) was dissolved in 1 mol/L NaOH solution. The solution was diluted with purified water to make the following concentrations: 0, 10, 20, 30 and 60 mg/L. These were adjusted with 1 mol/L HCl or 1 mol/L NaOH to obtain a pH value range of 5.8-6.0, and filter-sterilized. Cotyledon explants were soaked in glass bottles containing different concentrations (0, 10, 20, 30 and 60 mg/L) of TDZ solution for various time periods (0, 10, 20, 40 and 60 min). After treatment, the explants were briefly placed on sterile dry filter paper in petri dish to absorb excess moisture. Direct Induction of Adventitious Shoots Regeneration and Elongation of Shoots Using Cotyledon Explants of J. curcas For inducing shoot-buds regeneration, cotyledon explants were inoculated on hormone-free MS medium after treated with TDZ solution for various time periods. For comparison, cotyledon explants were also treated using conventional methods and inoculated directly on MS medium containing different concentrations of TDZ (0, 0.3, 0.6 and 1.2 mg/L) as reported previously [20-22]. The percentage of induction of shoot buds and the number of shoot buds per explant were recorded after 30 days of culture. For shoot buds elongation, the regenerated shoot buds were transferred along with the mother tissues (explants) to MS medium supplemented with 0.4 mg/L gibberellic acid (GA3), 0.5 mg/L 6-benzylaminopurine (BA), 0.2 mg/L kinetin (KT) and 0.25 mg/L indole-3-acetic acid (IAA) 0086 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) © 2016. The authors – Published by Atlantis Press (Sigma-Aldrich Co., St. Louis, MO, USA) [23,27]. The length of the elongated shoots was recorded after 15 days of culture. Rooting Culture For inducing rooting of the elongated shoots, shoots at least 1 cm in length were isolated from the mother tissues and inoculated onto fresh half-strength MS (1/2 MS) medium supplemented with different concentrations (0, 0.1, 0.3, 0.6 and 1.2 mg/L) of indole butyric acid (IBA) (Sigma-Aldrich Co., St. Louis, MO, USA), and the results were assessed on 10, 20, 30 and 40 days of culture. Evaluation of the Results and Data Analysis All experiments were set up in a completely randomized factorial design and repeated three times with 25-30 replicates per treatment. Statistical analysis of the data was carried out using SPSS 17.0 soft ware, and data in the same column followed by different letters were significantly different at p ≤ 5% level as determined by Duncan’s multiple range test. The results were expressed as means ± SD (standard deviation) of three independent experiments.