A Novel Cytoplasmic Male Sterility Gene T1243Expressed In Transgenic Chinese Cabbage

At present, there were very few male sterile materials in hybrid breeding practice of Chinese cabbage. T1243gene from the cytoplasmic male sterile line of tumor stem mustard (B. juncea var. tumida Tsen et Lee) was transformed to Chinese cabbage in our study. Firstly, we constructed an expression plasmid which carried the cytoplasmic male sterility (CMS)-related gene T1243. Then, on the basis of establishment regeneration system of Chinese cabbage, we obtained transgenic plants which were transformed by Agrobactrium mediated method. This study enriched breeding materials of the Chinese cabbage sterile lines and established a foundation for the research on mechanism of cytoplasmic male sterility.The details were as follows:Firstly, establish regeneration system of Chinese cabbage:five varieties of Chinese cabbage widely cultivated in Shanxi were used as materials and cotyledon with petioles as explants. We studied the effects of NAA, varieties,6-BA, AgNO3on regeneration of Chinese cabbage in vitro by an orthogonal design. The results showed that Jinyu75had the highest regeneration frequency, which was76.92%in the MS medium containing2mg/L6-BA+0.3mg/L NAA+7mg/L AgNO3Secondly, construct an expression plasmid of XF003-T1243:the expression plasmid XF003and T1243gene from tumorous stem mustard were digested by BamH I and then linked together, so we constructed the plant expression vector XF003-T1243. The results of PCR amplification and enzyme digestion suggested that the fragment of gene T1243was introduced into XF003plasmid and the plant expression vector was constructed successfully. Then the recombinant plasmid was transformed into Agrobacterium strain LBA4404.Finally, optimize the transformation system of Chinese cabbage: cotyledons with petioles were used as explants, and the culture conditions of transformed plants mediated by Agrobacterium tumefaciens were optimized. The results showed that, optimum culture medium for Jinyu75was: MS+2.0mg/L6-BA+0.3mg/L NAA+7.0mg/L AgNO3+7.5mg/L Kan+200mg/L Amp. The optimum time period of pre-culturing is2d on the darkness, and the highest frequency is35.48%when explants are immersed in bacterium concentration with OD6000.1about2minutes. Results from PCR and RT-PCR showed that T1243has been integrated into Chinese cabbage successfully and it was expressed normally.