Transgenic Indica rice ( Oryza sativa L.) plants obtained by direct gene transfer to protoplasts

We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43. Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines. In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol. Protoplasts were cultured in maltose-containing medium. Hygromycin B selection was applied to 14-day-old dividing cell colonies. Resistant calli were readily obtained after 3 weeks of selection. Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity. Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay. The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts.