【作者】 陈丽萍；

【导师】 程智慧；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2007， 硕士

【摘要】 加工番茄是新疆一大优势特色产业和出口创汇的拳头产品,选育适合产业需求的加工番茄品种一直是育种工作的目标。近年来随着基因工程技术的发展,科学研究愈来愈倾向于通过基因工程的手段改进加工番茄品种性状,培育耐贮运、高品质的加工番茄新种质,对加工番茄在储存与运输中的腐烂变质问题的解决有重要价值。本研究以加工番茄品种B04子叶盘和下胚轴切段为外植体,研究了不同种类和浓度的细胞分裂素(ZT、6-BA和KT)和生长素(IAA、IBA和NAA)组合及附加物AgNO3对外植体诱芽的影响;同时本研究利用农杆菌EHA105介导转化,对影响外植体转化的因素(预培养时间、侵染时间、农杆菌浓度、共培养时间)进行了深入探讨,将多聚半乳糖醛酸酶(PG)反义基因转入加工番茄品种B04,并获得了加工番茄品种B04的转基因植株。主要试验结果如下:1.在一定的浓度范围内,在MS基本培养基中添加不同浓度的细胞分裂素和生长素构成不同的诱芽培养基。在研究三种细胞分裂素对子叶和下胚轴愈伤组织及不定芽分化的诱导中,ZT明显优于6-BA,KT最差,确定最佳的诱芽培养基为MS+2.0 mg/L ZT+0.20 mg/L IAA,出芽率为83.6 %;在三种生长素对子叶和下胚轴愈伤组织及不定芽分化的诱导中,IAA与6-BA组合对芽的诱导效果比IBA、NAA好,出芽早,出芽也多,NAA组合不利于加工番茄品种B04芽的诱导;子叶外植体的出芽率明显高于下胚轴外植体,因此,本研究将子叶外植体确定为加工番茄品种B04遗传转化体系的受体;再生芽在MS+0.1 mg/L IAA生根培养基上能正常生根,并发育成完整的小植株。2.在再生培养基中添加一定浓度的AgNO3对加工番茄品种B04不定芽的发生率无显著促进作用。3.通过子叶外植体的卡那霉素敏感性筛选试验,确定加工番茄B04卡那霉素的最佳筛选浓度为30 mg/L;并对抗生素羧苄青霉素(Carb)对农杆菌的抑制效果进行了试验,本研究使用的Carb质量浓度为300 mg/L,此浓度足以抑制农杆菌的繁殖。4.建立了加工番茄品种B04子叶外植体的高频遗传转化体系,确定了预培养时间、侵染时间和侵染浓度、共培养时间等转化条件。其最佳预培养时间为2 d;当农杆菌的菌液浓度为OD600=0.6、侵染时间为10 min时转化率最高;其最佳的共培养时间为2 d。通过农杆菌介导法将PG反义基因导入加工番茄品种B04,并且获得了PG抗性植株。5.对获得的41株抗卡那霉素转基因植株进行PCR检测,结果表明:其中9株为阳性植株,其转化率为22 %,初步证实了部分加工番茄B04植株中已导入PG反义基因。更多还原

【Abstract】 The processing tomatoes production was the main induction in Xinjiang province. Choosing the best and suitable processing tomato for production was the target for breeding workers. With the development of the technique of Genetic engineering, people more and more trended towards improving the characters, breeding new varieties for storing and transporting easily, and breeding the higher quality for processing tomato, in this way, the problems of rot during the storing and transporting were well resolved. Cotyledons and hypocotyls of BO4 processing tomato were used as explants to study different combinations of cytokinin (ZT, 6-BA or KT) with auxin (IAA, IBA or NAA) and silver nitrate on bud induction; meanwhile, Agrobacterium was used for transformation, different factors of the transformation were studied(pre-cultural time,invading time,comcentration of bacteria, co-cultural time). And then transgenic plantlets with polygalacturonase gene of processing tomato B04 were obtained, and the activity of polygalacturonase was decreased. PG antisense genes were transformed into processing tomato B04.The main progress was as follows:1. The asexual systems of B04 processing tomato were established in the study. Effects of different three cytokinins on the regeneration of cotyledons and hypocotyls were compared. ZT was really better than 6-BA, KT was the most worst, the best inducting medium was MS+2.0 mg/L ZT+0.20 mg/L IAA, the induction ratio was 83.6 %;Effects of different three cytokinins on the regeneration of cotyledons and hypocotyls were compared. IAA was better than IBA, NAA was bad to the induction; The induction ratio of cotyledons explants was higher than hypocotyls explants, and the cotyledon explants was the receptor in the study of the construction of transformation system for B04 processing tomato; The regenerated buds could develop normally after roots were induced on MS+IAA0.1 mg/L, and finally could form the normal plants.2. Different concentration of silver nitrate in regeneration medium did not improve the budding induction for BO4 processing tomato.3. The inducting buds of explants based on the study of sensitive concentration of kanamycin for BO4 processing tomato was 30 mg/L; then studied the effect on the Carb to restrain the bacteria,and the concentration was 300 mg/L.4. Established the transformation system for processing tomato B04, the time of pre-culturing, the time and the bacteria concentration for invading, the time of the co-culture for explants and the bacteria were made. The suit pre-cultural time for processing tomato was 48 hours; OD600=0.6 was the fitful bacteria concentration for invading; and the time for invading was 10 minutes; the explants and the bacteria had been best co-cultured for 48 hours. Transgenic processing tomato plants were obtained.5. By PCR analysis, 9 plants were positive in 41 plants, and the rate of transformation was 22 %, the results proved that the foreign genes had been integrated into B04 processing tomato.更多还原