Thodsaporn Pipatpanukul, Sumontip Bunnag, Piyada Theerakulpisut, and Manit Kosittrakun

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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.