Optimization of Transformation Protocol for Japonica Rice cv.Taichung 65 through Agrobacterium tumefaciens

Abstract

       In the present study, we have successfully developed a transformation protocol for a rice variety Taichung 65 mediated by Agrobacterium tumefaciens with high efficiency, easy manipulation and reduction in labour work. In the protocol, we have optimized all steps of transformation, standardized components of culture media and presented several manipulation experiments. Experimental results have shown that using petri dishes with high edge (15 mm) and exposing explants not only increased the rate of callus formation, but also enhanced the size and quality of calluses. Using bacterial suspension at density OD600nm = 0.1 was optimal with high GUS expression in callus (81.25%) and low contamination. During the selection phase, transformed calluses grew better in petri dishes with high edge (15 mm) and exposing explants. Checking for the presence of R4 promoter at the molecular level showed high frequency of regenerated plantlets carrying the transgenes (up to 90.24%). GUS expression in different tissues of transgenic plants indicated the high activity of promoter R4, leading to initiate transcription and synthesis of GUS enzyme. Analysis of T1 plants confirmed the stable inheritance of transgene to the next generation.