Agrobacterium–mediated Transformation and Regeneration of Fertile Transgenic Plants of Rapeseed (Brassica napus L.)

A method for transformation of B.napus is presented in this study. The protocol is based on infection of cotyledonary petioles of 5-day-old and hypocotyl segments of 14-day-old seedlings with an Agrobacterium tumefaciens strain LBA 4404 carrying a disarmed binary vector pBI121. The T-DNA region of this binary vector contains the neomycin phosphotransferase II (NPTII) and glucuronidase (GUS) genes. After cocultivation for 48h, the explants were placed on shoot induction media containing 15 mgl kanamycine sulfate. Shoot induction was continued 4-6 weeks. After 2 weeks, the green shoots were transferred to root induction medium. After 4-8 weeks rooted plantlets were transferred to greenhouse. 16 kanamycine resistance plants from 244 cotyledonary petioles for SLM and 20 transformed plants from 151 explants for PF and 6 kanamycine resistance plant from 75 hypocotyls were obtained for both varieties. Most of them grew to maturity, produced normal flowers and set seeds. GUS assay and polymerase chain reaction (PCR) confirmed the introduction of the T-DNA into the rapeseed genome.