Agrobacterium mediated genetic transformation and regeneration in elite rice (Oryza sativa L.) cultivar BRRI dhan56

Abstract

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.