ISSN 2167-0447
International Journal of Agricultural Sciences ISSN: 2167-0447 Vol. 4 (12), pp. 341-350, December, 2014. © International Scholars Journals
Full length Research paper
Studies on Agrobacterium mediated genetic transformation of rice (Oryza sativa L.) for drought tolerance using Dreb 1 gene
Ravindra Donde, K. C. Samal and G. R. Rout
Department of Agricultural Biotechnology, College of Agriculture, Orissa University of Agriculture and Technology, Bhubaneswar-751003, Orissa, India.
Accepted 12 September,2014
E-mail: [email protected]
Abstract
Improving crops that are better adapted to abiotic and biotic stresses is gaining importance for sustainable food production in the present era of rapid climate change. The present investigation was carried out to optimize genetic transformation method in rice (Oryza sativa L. cv. Lalat) employing Agrobacterium strains GV3107 harbouring binary vector pCAMBIA2300 with rd29A::DREB1 gene construct. De-husked mature seeds of rice cv. ‘Lalat’ treated with Bavistin (1%) for 10 minutes followed by 0.1% HgCl2 + Tween-20 for 6 minutes resulted better asceptic culture with the maximum survival percentage. Incorporation of phytohormone 2, 4-D (2.5 mg/l) in the modified MS medium induced profuse, white, friable, embryogenic calli. The best in vitro regeneration from calli was obtained in the modified MS medium supplemented with BAP, NAA and Kinetin. The modified MS medium supplemented with 2.0 mg/l BAP, and 0.2 mg/l NAA was able to induce the maximum multiple shoots and organogenesis. The maximum number of multiple shoots (9.75) was recorded in modified MS medium supplemented with 2.0 mg/l BAP and 0.2 mg/l NAA within 30.75 days of culture. For root induction NAA was found to be the most effective phytohormone and effective rooting was recorded on modified MS medium supplemented with 1.0 mg/l NAA and 0.2 mg/l BAP within 25 to 30 days after culture of shoots on rooting medium. Various factor influencing transformation of rice callus and multiple shoots were optimized such as co-cultivation period (3 days) and bacterial inoculum density (0.6-0.8 at 600 nm), acetosyringone concentration (200 µM) and Kanamycin sensitivity (120 mg/l). The transformation efficiency based on Kanamycin selection was found to be 8.97 % whereas the transformation frequency on the basis of the PCR was found to be 2.56%.
Key Words: Agrobacterium tumefaciens, DREB1A, genetic transformation, Oryza sativa, abiotic stress.