Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of chinese cabbage (Brassica campestris ssp. pekinensis cv. 'spring flavor')

Abstract

A procedure for the regeneration of fertile transgenic Chinese cabbage (Brassica campestris ssp. pekinensis cv. ‘Spring Flavor’) is presented in this report. The protocol is based on infection of cotyledon explants of 5-d-old seedlings with an Agrobacterium tumefaciens strain LBA4404 carrying a disarmed binary vector pTOK/BKS-1. The T-DNA region of this binary vector contains the nopaline synthase/neomycin phosphotransferase II (nptII) chimeric gene for kanamycin resistance and the cauliflower mosaic virus 35S/coat protein gene of tobacco mosaic virus L (TMV-L) chimeric gene. After co-cultivation for 48 h, the cotyledonary petioles were placed on shoot induction media containing 15 mg/L kanamycin sulfate. Shoot induction was continued for 3？4 weeks, then subcultured once and after 2 weeks the shoots were transferred to root induction medium. After 1 week 8 putatively transformed plantlets from 200 cotyledon explants were obtained and transferred to greenhouse. Six of them grew to maturity, produced normal flowers and set seeds. Polymerase chain reaction and Southern blot hybridization analyses confirmed the introduction of the T-DNA into the Chinese cabbage genome. Further, Western blot analysis using polyclonal TMV antiserum showed most of the regenerants (5 out of 6) expressed TMV coat protein gene. Stable inheritance of the inserted clone was investigated in the next generation.