Transformation of indica rice (Oryza sativa L.) cv. RD6 mediated by Agrobacterium tumefaciens

Abstract

High percentage of callus induction at 97% was obtained when seeds of rice (Oryza sativa L.) cv. RD6 were cultured on modified N6 medium supplemented with 3% (w/v) sucrose, 22.5 μM 2,4-D and 0.8% agar under light condition. The suitable regeneration medium was N6 medium supplemented with 3% (w/v) sucrose, 2.5 μM IAA, 18 μM BA and 0.8% agar. A test had been performed to determine the effect of antibiotics on the regeneration of rice cv. RD6. It was found that kanamycin concentration up to 150 mg l-1 and hygromycin concentration at 10 mg l-1 were effective for selection of transformants. Cefotaxime and carbenicillin concentration up to 250 mg l-1 had the highest phytotoxicity to plant regeneration. Agrobacterium-mediated gene transfer protocols for rice cv. RD6 were performed using A. tumefaciens strain LBA4404, which harbored the plasmid pBI121 containing genes for β- glucuronidase (GUS) and kanamycin resistance (nptII), and strain EHA105, which harbored plasmid pCAMBIA1301 containing genes for β-glucuronidase (GUS) and hygromycin resistance (hptII). GUS activities were found in rice calli after co-cultivation. A number of morphologically normal fertile transgenic rice plants were obtained. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transgenic plants in T0 and T1 generation. Mendelian segregation was observed in T1 progeny.

Keywords

• Agrobacterium-mediated transformation
• rice transformation
• transgenic plants