Efficient in vitro plant regeneration from shoot apices and gene transfer by particle bombardment in Jatropha curcas

Abstract

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 µM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA 3 ; 0.5 µM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5 -6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm -3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

Indirect shoot organogenesis from leaf explants of threatened medicinal plant Cleistanthus collinus

“…There is a fast rate of cell division, growth and development and the formation of plant organs when cytokinin in combination with auxin was used. This was already reported in studies on the plant Jatroba curcus (Purkayastha, 2010). Recently, TDZ was found to be a cytokinin like activity which is one of the substituted ureas.…”

Plant Tissue Culture: Current Status and Opportunities

“…Recently successful transgenic plants of Jatropha were obtained by direct DNA delivery to mature seed-derived shoot apices via particle bombardment method [68]. This technology has an important impact on the reduction of toxic substances in seeds [69] thus overcoming the obstacle of seed utilization in various industrial sector.…”

Establishment of bispyribac selection protocols for <i>Agrobacterium tumefaciens</i>- and <i>Agrobacterium rhizogenes</i>-mediated transformation of the oil seed plant <i>Jatropha curcas</i> L.

“…ere are many reports of in vitro regeneration of plants from di erent tissues of Jatropha, including leaves (Sujatha and Mukta 1996;Sujatha et al 2005;Jha et al 2007; Deore and Johnson 2008;Khurana-Kaul et al 2010), cotyledon (Khemkladngoen et al 2011b;Kumar et al 2010a;Varshney and Johnson 2010), petioles (Sujatha and Mukta 1996;Kumar and Reddy 2010;Kumar et al 2011), hypocotyls (Sujatha and Mukta 1996;Kaewpoo and Te-chato 2010;Sharma et al 2011), epicotyls (Wei et al 2004;Kaewpoo and Te-chato 2010), nodal segments ), axillary nodes (Sujatha et al 2005), shoot tips (Rajore and Batra 2005;Purkayastha et al 2010), and stems . Based on these regeneration protocols, stable transformation of Jatropha has been reported by several groups.…”