Central University of Bihar, India
Nitish Kumar is a plant biologist and has been working on Plant Tissue Culture, Molecular markers Development and Transgenic Technology. Recently he has started working on Microbial Biotechnology focusing on Bioremediation. He obtained Masters Degree in Agricultural Biotechnology from Himachal Pradesh Agricultural University, Palampur and PhD Degree in Botany from Bhavnagar University and worked at CSIR-Central Salt & Chemical Research Institute, Bhavnagar as a JRF/SRF during his PhD tenure.
Jatrophacurcas 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 4404harbouringthe 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 OD600=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 µM6-benzyl aminopurine (BA), and 5.5 µMα-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 µMindole-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.
Keywords: Agrobacterium tumefaciens, Genetic transformation, Jatrophacurcas, Leaf explants