Transformation of Brassica napus with the method of floral-dip

摘要

Two double-low rapeseed cultivars westar and Zhong Shuang No.4 (Brassica napus L.)were used as recipient materials and the C-terminal fragment of CRY1, CCT1, fused to GUS (GUS-CCT1) was transferred with Agrobacterium-mediated floral-dip method. The transformation was performed at initial blossom stage. The effects of raceme position and surfactant Silwet-77 on the transformation frequency were investigated. The results showed that the best branches for dipping treatment were the top inflorescence and the uppermost two first-order branches in the raceme. Three consecutive dippings were conducted on every another day within a 7 day period. The best concentration of Silwet-77 was 0.05% (v/v) in the transformation buffer. The dipped racemes were bagged for self pollination and the seeds harvested were screened with Kanamycin in culture medium. The Kanamycin-resistant plants were further assayed by GUS reporter gene, and then by PCR test with a pair of specially designed primers based on the sequence of CCT1. The special DNA band from PCR was reclaimed, cloned and sequenced. The sequence of DNA was compared to CCT1 on NCBI internet and the homogeneity of sequence was 100%. It was certified that the foreign gene CCT1 was successfully transferred into the genome of the recipient rapeseed. The results showed that the Agrobacterium-mediated floral-dip method can be successfully applied to genetic transformation in rapeseed.