【作者】 方斌；

【导师】 韦鹏霄； 吕志仁；

【作者基本信息】 广西大学 ， 作物遗传育种， 2001， 硕士

【摘要】 本试验从大白菜四个品种：天津青麻叶、南宁早熟5号、50天快菜和浙江早 熟5号入手，从材料灭菌到大白菜转基因体系的建立，系统地研究了大白菜的再 生体系和根癌农杆菌介导的遗传转化体系，成功地获得了转GUS基因的大白菜植 株，并通过了鉴定。 通过对影响大白菜外植体再生的品种、灭菌剂、灭菌时间、浸种时间、植物 激素、乙烯抑制剂、培养基基本成分等诸多因素的对比试验，已建立起四个大白 菜品种的高效再生体系：种子先经70%乙醇处理1分钟，再用0.1%升汞溶液处理 12─15 分钟，进行表面灭菌；浸种20─22小时，然后接种在MS和 1/2MS＋GA0.2mg／L培养基上，获得无菌苗；取6─8天苗龄的无菌苗切下子叶外 植体，接种在附加NAA0.3mg／L＋6－BA4.0gm／L＋TDZ0.2mg／L＋AgNO34.0mg／L的 MS培养基上，进行光照培养。20天左右子叶外植体开始分化出芽。芽分化率因 品种不同而有差异，其中：天津青麻叶为31.3%；南宁早熟5号为32.0%；50天 快菜为47.9%；浙江早熟为48.1%。子叶外植体的芽分化率以50天快菜和浙江早 熟5号为高，故此以这两个品种作为转基因的试验材料。本试验还首次将新的高 活性细胞分裂素──TDZ引入大白菜再生培养中，芽分化率最高可提高169%， 取得了良好的培养效果。 农杆菌介导的植物遗传转化方法进行大白菜外植体的遗传转化。通 过对遗传转化过程中影响转化频率的筛选剂、根癌农杆菌生长状态、菌液浓度培 养时间等重要因素的研究，建立起以浙江早熟5号和50天快菜大白菜子叶为外植 体的一套可操作的遗传转化体系：挑单菌落接种根癌农杆菌菌株于三抗液体培养 基中，经28℃下200rpm振荡培养过夜，转接于不含抗生素的YEB培养基中，在 28℃培养3─5小时，OD600为0.6左右；取菌液稀释10倍，取7天苗龄的无菌苗， 切取子叶外植体；然后将子叶外植体浸泡侵染10分钟，吸干外植体上的菌液，共 培养两天后转入含头孢噻肟钠500mg/L的培养基上，脱苗后，转入到含Km6－8mg/L 的分化培养基上筛选，25天左右开始得抗性再生芽，转化率可达2.06%。 文中对影响大白菜外植体的高效再生和遗传转化的各种因素进行了讨论，为 进一步提高大白菜的再生频率和对金属硫蛋白的导入提供了参考意见，为转金属 硫蛋白基因打下了基础。更多还原

【Abstract】 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.更多还原