In this study, an in vitro plant regeneration system derived from 12 days old immature embryos was developed for Agrobacterium-mediated transformation of rice cv. IR64. Callus induction was carried out on N6 medium containing 2,4-D (2.0 mg/l), kinetin (0.1 mg/l) , sucrose (30 mg/l), casein hydrolysate (300 mg/l), L-proline (500 mg/l) under 2000 lux continuous light, at 32"C. For shoot induction, after 2 weeks of culture, embryogenic calli were cultured on N6 medium supplemented with BAP (2 mg/l) , a-NM (0.1 mg/l) , casein hydrolysate (2 g/l), L-proline (500 mg/l) , sucrose (30 g/l) at 26"C, 16 h light: 8 h dark. After 2 weeks of culture, 78.67 percent immature embryos induced embryogenic callus. The regeneration efficiency achieved at 31.18 percent frequency after 4 weeks of culture. In order to confirm in vitro plant regeneration system was developed that could be applied for transformation, the authors transformed gfp gene in to immature embyos of rice cv. IR64. The callus selection and regeneration of putative transgenic plants were carried as developed plant regeneration protocol. It took about 6 -8 weeks to obtain transgenic lines that could be transferred to the greenhouse. The strong expression of gfp gene was observed on hygromycin resisrant calli
shoots
stems, leaves, roots of transgenic rice lines
seeds of To, T1 seedling under UV light of microscope. The result of analysis using PCR technique and transgene inheritance revealed that transgenes were present in genome of To transgenic rice lines and transferred to Tl generation following Mendel's model. Transformation efficiency achieved at 27.5 percent frequency. In conclusion, an in vitro plant regeneration system was developed for rapid and high efficient transformation in immature embryos of cv. IR64. This system also could be used for transformation various genes in different rice cultivars.