Study On Tissue Culture And Establishment Of Genetic Transformation System Of Brassica Campestris Ssp Pekinensis(Lour)olsson

In this experiment, four varieties of Brassica campestris ssp. Pekinensis (Lour) olsson, such as Tianjinqingmaye, Nanningzaoshu No.5, fifty days fast vegetable and Zhejiangzaoshu No.5 were use as material. The regeneration system of Brassica campestris ssp. Pekinensis (Lour) olsson and an Agrobacterium-mediated genetic transformation system had been set up and some Gus transgenic plant of Brassica campestris ssp. Pekinensis (Lour) olsson had been obtained successfully.Factors effecting Brassica campestris ssp. Pekinensis (Lour) olsson explant regeneration were examined, which included the varieties, sterilant, sterilization time, soak seed time, plant hormones, ethylene inhibitor AgNO3, media basic components. A highly effective regeneration system was developed as follows: Seeds were soaked in 70% ethanol for 1 minute and surface-sterilized in 0.1% aqueous mercuric chloride solution for 12-15 minutes and washed for 3～4 time with sterilized water for 20-22 hours, then inoculated on MS basic medium or half of MS medium supplemented with GA 0.2mg/L to get the seedlings. After 6-8 days, the cotyledon explants were excised from seedlings and cultured under light on modified MS medium supplemented with NAA 0.3 mg/L, 6-13A4.0 mg/L and silver nitrate 4.0 mg/L. Shoots began to regenerate from explants after 2odays and the frequency of bud regeneration varied with different varieties. In this report, Tianjinqingmaye was 31.3%; Nanningzaoshu No.5 was 32%jifty days fast vegetable was 47.9% and Zhejiangzaoshu No.5 was 48.1%. The frequency of shoot regeneration from cotyledon explants of fifty days fast vegetable and Zhejiangzaoshu No.5 is higher. So both varieties were used as the material for transgenic experiment. Moreover, this experiment, by adopting the new potent cytokinin- Thidiazuran(TDZ) in the regeneration culture of Brassica campestris ssp. Pekinensis (Lour) olsson firstly, which had raised the rate of differentiation by 169%; A good culture effect had been made.-2-This study performed Brassica campestris ssp. Pekinensis (Lour) olsson explants for genetic transformation by Agrobacterium-mediated genetic transformation. Some important factors affecting frequency of transformation including selection agent, culture condition of Agrobacterium tumefaciens, concentration of bacterial suspension, time of infection were examined. An Agrobacterium-mediated genetic transformation system of Brassica carnpestris ssp. Pekinensis (Lour) olsson Zhejiangzaoshu No.5 and fifty days fast vegetable cotyledon explants was established as follows: Agrobacteriurn tumefaciens strains were isolated and cultured in liquid medium containing three kind of antibiotics (200rpm, 28 t) for about 22 hours then transformed them to YEB medium without any antibiotic. And then, they were cultured for 3-5 hours in the same conditions. UP to 0D6000.6, Cotyledon explants were incubated in diluted 1:10, Cotyledon explants which are excised from seven-day old seedlings were put into the Agrobacteriurn turn efaciens suspension and infected for 10 minutes. Infected explants were co-cultured for 2 days and transferred to selection medium containing 500 mg/L cefotaxime. After 25 days, resistibility shoots begin to occur and the frequency of transformation can reach 2.06%.Finally, highly effective regeneration and genetic transformation system for Brassica campestris ssp. Pekinensis (Lour) olsson was discussed and made up with the suggest for further raising of the frequency of the regeneration and the transform for MT were made up. And therefore, the base of transgenic of MT had been made.