Preliminary investigations of Agrobacterium-mediated transformation in indica rice MR219 embryogenic callus using gusA gene

Abstract

Preliminary steps in the genetic transformation of indica rice MR219 was investigated in the plant- Agrobacterium tumefaciens interaction. Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pCAMBIA 1305.2 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Various transformation parameters influences were optimized using embryogenic calli via β- glucuronidase (GUS) as a reporter marker. Various transformation parameters were optimized including bacterial concentration, age of embryogenic callus, pre-culture period, wounding technique, cocultivation period, immersion time and dry time before co-cultivation, acetosyringone (AS) concentration, pH of co-cultivation media and temperature of the co-cultivation period. The expression of the transient gusA gene in the plant genome was preliminary confirmed by histochemical GUS assay activity (as blue spots). The results from transient gusA gene expression of calli suggested that the Agrobacterium-mediated transfer system of T-DNA in indica rice MR219 was highly efficient. Therefore, the investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of indica rice MR219 calli.

Key words: Indica rice MR219, Agrobacterium tumefaciens, GUS expression.