High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation

Abstract

An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol seed calli from the varietiesNipponbare andTaipei 309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 × 106 protoplasts perg fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant transcriptional regulatory signals was used as a selectable marker gene. Hygromycin-resistant colonies were selected in the presence of 95 μM hygromycin B with apparent frequencies of 2×10−4 and 5×10−4 forNipponbare andTaipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.