【作者】 于娅；

【导师】 马峙英； 李付广；

【作者基本信息】 河北农业大学 ， 作物遗传育种， 2003， 硕士

【副题名】以茎尖分生组织和胚性愈伤组织为轰击受体

【摘要】 棉花是世界主要的经济作物之一，但是近几年受到虫害，尤其是棉铃虫的危害，其产量与品质均受到严重影响。广泛使用化学杀虫剂控制害虫，不但破坏生态平衡，污染环境，而且害虫还逐渐对其产生了抗耐性。植物基因工程研究的深入，为控制农业害虫开辟了一条新途径。我们将不同抗虫机理的双价抗虫基因(Bt+CpTI)导入陆地棉品种，旨在培育双价转基因抗虫棉，拓宽棉花的抗虫谱，同时建立基因枪遗传转化棉花的技术体系，为棉花的现代分子育种增添新的内容和新的种质材料。 本试验以陆地棉栽培品种“中棉所19”、“中棉所24”、“中棉所27”的茎尖分生组织，以“中棉所24”的胚性愈伤组织为材料，建立起了比较完善的基因枪转化体系。并把抗虫双价基因Bt+CpTI表达载体导入了棉花，获得了抗卡那霉素的转基因棉花新材料。主要结果如下： 1 以棉花茎尖分生组织为轰击受体的技术体系建立 取棉花种仁接于MSB培养基中，取苗龄为40～60小时的茎尖，将茎尖制备成(生长点+2叶原基)的状态，将制备好的茎尖分生组织竖直放于培养皿中。轰击压力为1300psi，距离为9cm，真空度为27～28inHg，轰击后接入加有活性炭1g／L的MSB培养基中过渡培养4～7天，至组织长成为肉眼可见的明显长大的绿芽点时，将其转移到筛选培养基MSB+KM(0，45，60，80，85～100mg／L)上进行筛选，获得抗性植株。三个品种茎尖的平均转化率为2.94％。 2 以胚性愈伤组织为轰击受体的技术体系的初步建立 取7日苗龄的下胚轴，接入MSB+0.01mg／L IAA+0.01mg／L KT+0.01mg／L 2，4-D以诱导愈伤组织，进而转入合适培养基中诱导出胚性愈伤组织。选取颜色新鲜、颗粒状的胚性愈伤组织，平铺于培养皿中心直径3cm处。渗透处理以甘露醇为渗透剂，浓度为0.3mol／L，前处理时间为2～4hr，后处理时间为16hr为最佳。轰击参数：飞行距离9cm，真空度27～28inHg，气压1300psi。过渡培养后接于培养基MSB＋KM（25，50mg／L）中筛选抗性胚性愈伤组织转化子，然后再诱导分化至获得j可生抗性植株。3转基因植株的鉴定 抗性转化植株经PCR检测，证明目的基因Bt杀虫基因及 CPTI基因均导入了棉花基冈组中。更多还原

【Abstract】 In this paper, Biolistics transformation systems were established with apical meristems of "CCRI 19", "CCRI 24","CCRI 27" and the embryogenic callus cells of "CCRI 24". Thought these systems the bivalent gene of Bt+CpT I were introduced into cotton, and the transgenic plants resistant to Kanamycin were obtained. The main results were as follow:1 , The establishment of Biolistics tranformation systems for cotton on apical meristemsThe kernels of cotton were incubated into the MSB medium. When the seedings were 40~60 hours age, they were dissected and remained the state of meristem and 2 leaf primordia. These meristems were placed straightly into the plates with some MSB medium and bombarded by gold powders with the Helium pressure of 1300 psi, the bombardment distance of 9cm, and the vacuum pressure of about 27~28 inches Hg. Subsequently the bombarded meristems were cultured into MSB medium with activated charcoal 1.0g/L for 4~7 days and then were transferred to the selective medium MSB+KM 45, 60, 80, 85-100mg/L. Finally the regenerated plants resistant to kanamycin were obtained with the transformation rate amounting to 2.94% . 2, The etablishment of Biolistics tranformation systems for cotton on embryogenic callus cellsThe hypocotyls of 7-day-old seedlings were incubated into MSB+0.01mg/L IAA+0.01 mg/L KT+0.01 mg/L 2,4-D to induce the callus issues, then the callus issues were transferred to another medium for inducing embryogenic callus issues. By the osmotic treatment (mannitol, the concentration of 0.3 mg/L, pre-treatment 2-4 hours, post-treatment 16 hours), the embryogenic callus which were fresh and the shape of grain were placed into the districts of diameter of 3cm in the plates. Thebombardment parameters: the Helium pressure of 1300 psi, the bombardment distance of 9cm, and the vacuum pressure of about 27~28 inches Hg. By transition culture, the embryogenic callus were transferred to selective medium to select transformation and the plants resistant to kanamycin were obtained. 3 , Confirmation of transgenic plantsThe PCR assay of kanamycin-resistant transformants showed that the target gene had been integrated into cotton genome accompanying with Bt+CpT Igene .更多还原