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通过农杆菌感染法将苏云金杆菌杀虫蛋白基因转移进了甘蓝的基因组,带子叶柄的子叶作为外植体与农杆菌共培养.发生在子叶柄基部的愈伤组织在含卡那霉素(Km)15～30mg/L的MS培养基上进行筛选,约5%外植体上的愈伤组织继续长大,当移到含Km和6-BA的分化培养基上时,愈伤组织分化出绿色的芽.将芽分离培养,约80%在加有Km的培养基上被诱导生了根.未转化的对照组织在筛选培养基上不能分化出正常的芽和根系,并且逐渐褐化死亡.小菜粉蝶的幼虫被饲喂以转基因植株的叶片,幼虫出现中毒症状,表现为发育受阻和死亡.约20%受试植物的DNA与苏云金杆菌杀虫蛋白基因探针杂交显阳性带.由转基因植株的种子长成的第2代甘蓝幼苗的卡那霉素抗性和植株的抗小菜粉蝶的活力均符合孟德尔单基因分离规律.
The Agrobacterium tumefaciens insecticidal protein gene was transferred into the genome of Brassica oleracea by Agrobacterium tumefaciens method and cotyledons with cotyledon petiole were co-cultured with Agrobacterium as explant.The callus occurred at the base of cotyledonary petiole in the medium containing kanamycin (Km ) On 15 ~ 30mg / L MS medium, about 5% explants on the callus continue to grow, when transferred to differentiation medium containing Km and 6-BA, callus differentiation Green shoots were isolated from buds and about 80% of them were induced to root on medium supplemented with Km.The untransformed control tissues did not differentiate into normal buds and roots on the screening medium and gradually brownned The larva of Pieris rapae was fed on the leaves of transgenic plants and the larvae showed symptoms of poisoning, which was blocked by development and died.The DNA of about 20% of tested plants hybridized with the Bacillus thuringiensis insecticidal protein gene probe positive bands, The kanamycin resistance of the second-generation cabbage seedlings grown from seed of the plants and the vigor of the plants against Pieris rapae all met Mendel’s single-gene segregation rule.