【作者】 赵芳；

【导师】 李云；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2004， 硕士

【摘要】 刺槐(Robinia pseudoacacia L．)是我国重要的多用途经济树种，在木材、抗逆性、生物量等方面都具备十分优良的特性。寒冷是限制刺槐发展的一个重要因子(Hart K H等，2000)。在我国的黄土丘陵地带，刺槐是重要的造林先锋树种，但是在晚霜和严寒危害下，常存在着干、冠分杈，枯梢等现象，严重影响着刺槐的生长。利用基因导入技术可以在短时期内达到育种的目标，本试验旨在将抗寒转录因子导入速生用材型刺槐，希望得到抗寒性较强的刺槐新品种，促进速生用材型刺槐在北方地区的大规模推广。 建立高效再生体系是进行遗传转化的基础。本试验以MS为基本培养基，以刺槐的复叶为外植体，筛选出刺槐复叶离体培养的最佳生长调节剂为NAA、BA。通过对刺槐复叶中小叶和叶轴两部分的再生试验，确定刺槐的叶轴作为本试验进行遗传转化的受体材料。再生培养基配方为MS+BA2.0mg／L+KT1.0mg／L+NAA0.5mg／L，叶轴的再生率可达到80％以上。 并与此同时进行复叶通过愈伤组织诱导阶段分化不定芽的试验，结果表明复叶在MS+BA1.0mg／L+2，4-D1.0mg／L培养基上产生的愈伤组织可以在MS+BA1.5mg／L+NAA0.5mg／L中分化不定芽，再生的植株生长健壮，表现良好。给予不同外植体(叶片、叶柄、茎段)的再生最适条件，进行不定芽的分化试验，结果表明叶轴分化的不定芽在质和量上均最优。 以刺槐叶轴为材料，进行卡那霉素和头孢霉素的敏感性试验，确定卡那霉素进行抗性芽的初选浓度为30mg／L，继代筛选提高到50mg／L；头孢霉素抑菌浓度为200mg／L。并对转化体系进行优化，确定继代20～35d的刺槐无菌苗的叶轴最适合作为遗传转化的受体材料；延迟筛选的时间为7～9d；培养方式采用前期暗培养法，在有抗性芽产生后立刻转入光照培养，以利于抗性芽的生长。通过根癌农杆菌的介导将抗寒转录因子OsDREBL导入刺槐体内，对得到的抗性植株进行PCR检测，获得2个转基因株系。更多还原

【Abstract】 Black locust (Robiniapseudoacacia L.) is a multipurpose legume tree species in our country and it exhibits many excellent characteristics such as lumber, entironmental restoration and biomass production. Frost is an important antiblastic factor to black locust. Black locust is one of the important silviculture harbingers in loess hill of our country, but it usually shows abnormal growth in frost conditions. Genetic manipulation has made it possible to reach our breeding aim. This study was to transformate cold-resistant gene into black locust silviculture and get high cold-resistant varieties. Then improve the application of black locust in nothern region in large scale.The establishment of effective regeneration system is the foundation for Agrobacterium-mediated genetic transformation. Base on MS medium, NAA and BA were selected to be the best plant growth regulators for the regeneration of the compound leaf. Via the study for the regeneration of the folioles and rachis of the compound leaf, rachis is selected for the genetic transformation. The differentiation rate of rachis can reach the level as high as more than 80%, which were cultures on MS medium containing BA2mg/L, KTlmg/L, and NAA0.5mg/L. The compound leaves were placed on MS medium with BA1.0 mg/L and 2, 4-D1.0 mg/L for callus induction, and then the callus can regenerate adventitious buds on MS mediun supplemented with 1.5mg/L BA and 0.5mg/L. The effects of different explants types of adventitious shoots were studied, indicating that rachis had the highest level of adventitious buds differentiation.The Kanamycin and Ceftomine sensitivity of adventitious shoot and adventitious root were tested. The concentration of Kanamycin was 30mg/L for adventitious buds differentiation, and subculture selection was up to 50mg/L. Cef 200mg/L was the suitable bacteriostatic agent. Through optimizing transformation system, the results suggested: in vitro-germinated plantlet shoots (20~35d) were suitable for inoculation; selection of theinoculated rachis was delayed 7~9d; dark culture in former cycle was adopted, and when the transformed plants with Km resistance were observed, they were subculture transfered to light conditions, in order to allow the normal growth of the transformed plants. The results of molecular biological assays with PCR preliminarily indicated that the OsDREBL gene had been integrated into the genome of black locust, and two transgentic plants were obtained.更多还原