Establishment Of Tissue Culture System And Transformation Of Jatropha Curcas

Jatropha curcas, a member of Euphorbiaceae family has evoked considerable interest as a potential biofuel plant because of its high oil content (40%) in seeds.An efficient and new regeneration system from mature cotyledons of Jatropha curcas was developed in this research. Frequency of callus induction was 100% when cultured the cotyledon on MS medium supplemented with BAP and CaCl2 after four weeks. Plant regeneration (93.47%) with the maximum number of plantlets (15/explant) was achieved on MS medium supplemented with BAP (2.0 mg/l) + CaCl2 (6 mM) + Gelzan (2.0g/l). The best rooting (90%) with no callus formation was observed on half-strength MS medium with 2.0% sucrose and supplemented with 0.12 mg/l IBA and 0.12 mg/l NAA after 4 weeks. The above-mentioned method can also be applicable to other explants such as leaf (6-8 shoots), petiole (6-8 shoots), leaf vein (2-3 shoots) and a new cultivar (multiflorous Jatropha, 2-3 shoots). Furthermore, callus developed rapidly into shoots following the transfer of callus to the medium containing 0.01 mg/l GA3 and number of shoots per explant increased to approximately 21.Callus was induced from leaf, petiole, stem and cotyledon of one month seeding cultured on CD medium (MS + 2.0 mg/l 2,4-D) and CDK medium (MS + 2.0 mg/l 2,4-D + 0.1 mg/l KN) for about one year, then the callus (0.1 g fresh wt) were transferred to 150 ml Erlenmeyer flasks containing 20 ml of MS liquid medium supplemented with 2.0 mg/l 2,4-D, 3% (w/v) sucrose. Suspension cells could be established successfully from leaf and cotyledon callus cultured on CD medium and stem and cotyledon callus cultured on CDK medium. Cotyledon callus cultured on CDK medium is the best result. Then, basal medium, inoculum (minimum) density, concentration of sucrose and plant growth regulator was optimized. The best suspension medium is as follow: B5 salts + B5 viatamins + 2,4-D (2.0 mg/l) + KN (0.2 mg/l) + sucrose (30 g/l), pH = 4.8-5.8.A number of parameters were studied to optimize transient gus expression efficiency in the Agrobacterium-mediated transformation using cotyledons. The highest transient expression efficiency (92.36%) was achieved using cotyledon disc explants infected with Agrobacterium EHA105 for 20 min, followed by co-cultivation for 6 days in a co-cultivation medium pH 5.4 at 26℃. The highest frequency of transient expression (46.00%) was observed when cotyledons were bombarded at 1350 psi with a target distance 9 cm in genetic transformation of cotyledons by particle bombardment.In vitro regeneration of Jatropha curcas offer a powerful tool for germplasm conservation, mass-multiplication of fine cultivars. Cell suspension cultures offer an in vitro system that can be served as a tool for various studies such as mass propagation, protoplast isolation, gene transfer of Jatropha curcas. The optimization of Agrobacterium-mediated and particle bombardment transient expression can evaluate factors that influence the stable transformation.