Standardization of Agrobacterium mediated genetic transformation in Indica rice cv BPT-5204

Abstract

The BPT-5204 genotype from Indica rice cultivar (cv) is recalcitrant and showed low transformation frequency compare to japonica rice cv. Here we have optimized the efficient transformation protocol to minimize the time scale and enhance the transformation frequency by altering the key parameters like, acetosyringone (AS) concentration, optical density of bacterial culture and co-cultivation time. Highly proliferated 21 days old Scutellum derived embryogenic calli were infected with Agrobacterium tumefaciens harbouring pCAMBIA2300-Ds-En-Bar binary vector. T-DNA contains tetrameric CaMV35S enhancer elements along with bar gene which embedded between the transposable Ds element. At 0.5 OD600nm of Agrobacterium culture and co-cultivation for 2 days on MS co-cultivation medium containing 100 μM acetosyringone proved to be optimal and attained 6.2 % of transformation efficiency. The transformed calli and regenerated plantlets were proliferated on Murashige and Skoog (MS) medium containing phosphinothricin (PPT). Polymerase chain reaction (PCR) confirmed that intact T-DNA was successfully integrated in the rice genome. This protocol can be employed to develop transgenic rice plants with gain of functional mutagenesis.