Studies On Agrobacterium-midiated Genetic Transformation In Brassica Campestris Subsp. Chinensis Var. Parachinensis

(E)-β-farnesene(EβF)synthase gene were transferred into Chinese Cabbage cv. Youqingsijiu (Brassica campestris L. subsp. Chinensis Makino var. parachinensis Tsen et Lee) that were made with cultured cotyledons, by combining plant tissue culture with Agrobacterium-mediated transformation.In this research, establishment of high efficiency regeneration system, determination of various parameters involved in Agrobacterium-mediated transformation, screening and characterization of transformant were systematically researched. The results indicated that:High frequency regeneration system was a prerequisite for successful genetic transformation, it was closed related with the type of explant, seedling age, hormone concentrations and other experimental conditions. Cotyledon petiole, hypocotyl and cotyledon cuts of Chinese Cabbage (cv. Youqingsijiu) were researched and cotyledon petiole was found more appropriate to explant regeneration system. Also the impact of hormones and seedling age on the regeneration system were researched. The best conditions for regeneration of Chinese Cabbage (cv. Youqingsijiu) was 1/2 NH₄⁺ MS + 3mg/L 6-BA + 1mg/L NAA + 7mg/L AgNO₃ + 2% sucrose + 0.8% agar (pH=5.8).Expression vector pBJ40 containing target gene was transferred into Agrobacterium LBA4404 by using freeze-thaw method. Screening of positive colonies were used antibiotics Str, Rif, Amp, Kan. PCR screening indicated that the target gene had been transferred into Agrobacterium LBA4404. The strain LBA4404 could be used as engineering bacteria.Using this engineering strain infected cotyledon petiole explants, five parameters were determinated in Agrobacterium-mediated transformation. We designed a appropriate protocol in which Chinese Cabbage explants were precultured 2³ days on MS medium, dipped for 8min in LBA4404 suspension (OD₆₀₀=0.5), co-cultivated with Agrobacterium for 2³ days on 1/2 NH₄⁺ MS + 3mg/L 6-BA + 1mg/L NAA + 7mg/L AgNO₃ + 2% sucrose + 0.8% agar (pH=5.8). Under conditions, transformants grew well, transformation frequency could reach to 2.67%.The Kanamycin was applied as a selectable marker at various concentrations. We setted five concentration gradient from 0.5 to 7mg/L. The evaluation of toxic damage indicated that appropriate concentrations of Kanamycin were lower than 3mg/L. Concentration of Ampicillin was also defined. 300mg/L could inhibit the growth of Agrobacterium. But it had no significant effect on adventitious shoots.Four putative transformants were obtained. After assayed by PCR analysis, all transformants were observed the predicted band.