Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)

Abstract

Polyhydroxybutyrate is a component of bioplas cs that is synthesized under the control of enzymes encoded by pha mul genes. The genes are naturally present in Ralstonia eutropha. However, the produc on of bioplas cs in bacteria is inefficient because the bacterial biomass is rela vely small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objec ves of this study were to transform the ssue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The gene c transforma on of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon ssue of J. curcas in a suspension of the bacterium for 30 min, followed by cocul va on for 3 d on Murashige and Skoog (MS) medium. The ssue was then placed on a selec on medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addi on of 20 mg/L kanamycin. The results showed that 12.35% of the ssue survived and regenerated into a shoot a er 1-2 months. Molecular analysis of the transformed ssue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respec vely.

An effective method for Agrobacterium tumefaciens -mediated transformation of Jatropha curcas L. using cotyledon explants

“…To apply the Agrobacterium -mediated genetic transformation approach to jatropha improvement more rapidly and efficiently, establishing the regeneration system of plant tissue culture is the precondition for plant gene transformation. As previously reported, the regenerated adventitious buds had been acquired smoothly from various types of explants in J. curcas , such as petioles, hypocotyls, leaves, epicotyls, and cotyledons, but the periods of obtaining regenerated plants were relatively long (90–140 days) [ 6–8 ]. A series of works have described an efficient and reproducible protocol in which the various explants were treated in high concentrations (5–120 mg/L) solutions of cytokinins (CTKs) such as 6-benzyl aminopurine (6-BA) and thidiazuron (TDZ) for short periods (5–80 min) in order to induce adventitious buds regeneration of soybean [ 9 ] and jatropha [ 10 , 11 ].…”