Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties

Abstract

Rice is a staple food whose productivity is affected by multiple environmental stresses. The use of biotechnological tools is the best possibility to develop rice varieties with increased productivity, exceptional grain quality and resistance to various stress. Several parameters in the genetic transformation (co-cultivation period, acetosyringone concentration, temperature of the co-cultivation medium, antibiotic concentration etc.) of rice varieties—Shalimar Rice 1, Jhelum and K 332 were optimized for the first time using β-glucuronidase (GUS) gene as a reporter. The GUS expression in presence of 100 μM acetosyringone at pH 5.2 was more at 22 °C as compared to 28 °C in all the rice varieties. The selection medium containing 250 mg l−1 of cefotaxime and carbenicillin prevented Agrobacterium overgrowth and the use of 50 mg l−1 hygromycin resulted in killing of the untransformed calli. The regeneration medium containing MS B5 medium supplemented with 4% sucrose, 3 g l−1 phytagel, 2.5 mg l−1 BAP, 1 mg l−1 zeatin, 0.2 mg l−1 NAA and 0.5 mg l−1 TDZ allowed efficient regeneration of the rice calli. The overall regeneration frequency of Shalimar Rice 1, Jhelum and K 332 were 64, 75 and 77% respectively. Putative transformed plants were analyzed for the presence of the transgenes through GUS histochemical analyses and southern blotting. The present protocol provides an efficient and rapid embryogenic callus induction, transformation and regeneration system, which could be conveniently used for producing genetically modified plants and might help in the transformation of other related rice genotypes.