甘蓝型油菜抗病虫双价基因转化体系的建立

以甘蓝型油菜（Brassica napus L.）品种浙双758子叶柄外植体为受体，建立农杆菌介导转化体系，研究乙酰丁香酮、羧苄青霉素浓度和潮霉素筛选浓度等对农杆菌遗传转化效果的影响。结果发现，羧苄青霉素对农杆菌的抑制效果以500mg/L最佳，且对子叶柄外植体愈伤组织诱导和分化的影响最小，愈伤组织诱导率和芽再生率分别为75.2%和65.1%。5.0mg/L的潮霉素能完全抑制未转化再生植株的生长，使其最终褐化死亡，在此潮霉素筛选浓度下，获得了32株转化再生植株。共培养时培养基中添加100μmol/L AS的芽再生率为3.9%，显著高于未添加AS的芽再生率（2.0%），说明共培养阶段添加酚类物质乙酰丁香酮有利于转化载体T-DNA的转化。部分转化再生植株经PCR和Southern杂交检测呈阳性，病虫害接种试验表明转化植株对菌核病和小菜蛾有较好的抗性。

TRANSFORMATION OF BINARY DISEASE AND INSECT RESISTANCE GENES IN Brassica napus L.

Effects of acetosyringone, carbenicillin and hygromycin concentrations on the transgenic efficiency were studied to establish the transformation systems using leaf-petiole explants of Brassica napus L. cv. Zheshuang 758. The result showed that the optimum concentration of carbenicillin (500 mg/L) for inhibition of Agrobacterium tumefacienshad little effect on the callus induction and differentiation of leaf-petiole explants. The callus induction and differentiation of leaf-petiole explants were at the ratio of75.2% and 65.1%, respectively. Hygromycin at a concentration of 5.0mg/L was enough to inhibit the growth of non-transformed regeneration plants, causing them browning to death,whereas 32 regenerated plants were obtained at such lethal concentration. The frequency of shoot regeneration on co-cultivation medium containing 100μmol/L of acetosyringone(AS,) was 3.9% significantly higher than those without AS (2.0%), indicating that addition of phenolic compounds such as AS to the medium were beneficial to the transformationof T-DNA in co-cultivation. Regenerated plants were confirmed by PCR and Southern blot hybridization, and these transgenic lines were proven to be resistant to Plutellaxylostella and Sclerotinia sclerotiorum.