An efficient in vitro regeneration and transformation system of Jatropha curcas L.

Abstract

Jatropha curcas L., a drought-tolerant oil yielding perennial plant, has received much attention as a potential source for biodiesel production. It has ability to grow in tropical or subtropical wastelands and dry lands. A serious limitation to the development of jatropha is the lack of highyield varieties. The development of efficient regeneration and transformation systems are prerequisite for a number of biotechnological interventions for the improvement of jatropha. An efficient in vitro regeneration system of jatropha has been developed from juvenile cotyledon. A two-step protocol has been standardized in which, firstly comprising growth hormones concentration is optimized and it was found that MS medium supplemented with 1.5 mgL-1 BA + 0.05 mgL-1 IBA + 0.5 mgL-1TDZ turned out to be the best treatment for shoot induction (3.82 ± 0.18 shoots/explant). Secondly, the best treatment for shoot induction was fortified with different concentrations of growth additives for further shoot proliferation. Among the different concentrations used, glutamine at 25 mgL-1 and adenine sulfate at 25 mgL-1 were found to be the most effective, resulted with maximum of 9.09 ± 0.37 shoots/explant and 93.0 % regeneration frequency. Regenerated shoots were cultured on elongation medium containing 0.5 mgL-1 BA and different concentrations of GA3. The combination of 0.5 mgL-1 BA and 0.5 mgL-1 GA3 was found to be the best for shoot elongation (2.13 ± 0.18 cm). The highest frequency of root induction was on the medium with 0.5 mgL-1 IBA (40 %). In this study, we have developed an efficient vacuum infiltration-assisted Agrobacterium–mediated transformation protocol for the jatropha plant. We examined the effect of two physical treatments, vacuum infiltration (5, 10 or 15 min) combined with shaking (10 min) or without it in order to improve transformation efficiency for jatropha using β-glucuronidase (GUS) assay. The highest mean number of GUSpositive clusters per explants (0.36 ± 0.02) was achieved when explants were exposed to 15 min of vacuum infiltration without shaking in Agrobacterium suspension. A transformation efficiency of 95 % was obtained. The procedures described here, will be useful for the mass propagation of elite cultivars and the production of transgenic plants through Agrobacteriummediated transformation.