Agrobacterium mediated genetic transformation and regeneration in elite rice (Oryza sativa L.) cultivar BRRI dhan56
Agrobacterium-mediated genetic transformation of rice (Oryza sativa L.) cultivar BRRI dhan56 was carried out in this study. Agrobacterium tumefaciens strain LBA 4404, which harbors the plasmid pIG121 that carries the genes for ß-glucuronidase gene, served as a reporter gene in the histochemical assay and the neomycin phosphotransferase ΙΙ (NPT ΙΙ) gene for the identification of resistance to kanamycin was used for genetic transformation. Twenty days old embryogenic calli from mature embryos of highly regenerating rice cultivar BRRI dhan56 were used to co-cultivate with 0.8 to 0.9 OD600 Agrobacterium for 25 min and the cultured was continued on agar medium for this study. The transformed colonies were selected by using 50 mg/L kanamycin and 50 mg/L rifampicin and confirmed by colony PCR. The PCR positive colonies were isolated to transform by using calli of indica rice cultivar BRRI dhan56. Putative leaf and root segments from plantlets obtained from transformation experiment with the plasmid pIG121 were GUS positive. Integration of the introduced gene into the genome was demonstrated by PCR. The maximum transformation efficiency of 32% was obtained by using 500 mg/L cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium. In this study, 100 µM acetosyringone in co-cultivation medium and co-cultivation for 3 days were the optimum conditions for maximum transformation. The expression of GUS gene revealed that the calli were successfully transformed. The results of this study would be an effective tool for crop improvement and gene-function studies on the model monocot plant rice.
Key words: Agrobacterium, Oryza sativa L., acetosyringone, β-glucuronidase, cefotaxime, plasmid, phosphotransferase, rice, transformation.
Abbreviation: GUS, β-Glucuronidase; PCR, polymerase chain reaction; MS, Murashige and Skoog; 2,4-D, 2,4-dichlorophenoxyacetic acid; MCI, callus induction medium; OD, optical density; NAA, 1-naphthaleneacetic acid; BAP, 6-benzylaminopurine.