Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

Abstract

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explants. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), β-glucuronidase (gus), and hygromycin-phosphotransferase (hpt ) genes were used for gene transfer. A number of parameters such as preculture of explants, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD 600 = 0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 μM acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 μM thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 μg ml −1 hygromycin. Selected shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 μM BA and 8.5 μM indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 μM indole-3-butyric acid (IBA), 5.7 μM IAA, 5.5 μM NAA, and 0.25 mg l −1 activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was achieved for leaf explants using this protocol.