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发根农杆菌介导八宝景天遗传转化体系的建立

标题: 发根农杆菌介导八宝景天遗传转化体系的建立
英文标题: Establishment of Genetic Transformation System of Sedum Spectabile Boreau by Agrobacterium Rhizogenes
作者: 王艳
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 八宝景天,组织培养,叶片再生,发根农杆菌,遗传转化,生长变异
英文关键词: Sedum Spectabile Boreau,tissue culture,plant regeneration,agrobacterium rhizogenes,genetic transformation,Growth variation
论文级别: 硕士
学位: 学位论文
导师: 刘桂林%梁海永
专业: 园林植物与观赏园艺
提交时间: 2009
摘要: 经发根农杆菌(rhizogenes)转化的植株不仅表现出生根能力明显提高,而且通常还表现出叶片和花的形态、色素形成、节间长短、生活周期及向地性等性状的可遗传的变异,其中许多变异如植株矮化、节间缩短、花形和叶形改变等在园林植物品种改良中有很大的应用价值.本文拟通过发根农杆菌转化八宝景天(Sedum Spectabile Boreau)来获得转基因植株并且筛选优良新品种.采用八宝景天嫩茎作为外植体进行组织培养,在附加不同浓度激素的培养基上对其进行增殖、生根培养,诱导叶片愈伤组织的发生及叶片愈伤组织不定芽的分化,建立其叶片再生体系.在建立八宝景天叶片再生体系的基础上,研究影响八宝景天遗传转化的若干因素,通过遗传转化条件的优化,采用农杆菌介导法,建立了发根农杆菌遗传转化体系,获得了转基因植株.对转化再生植株进行了形态鉴定、PCR检测,并对转化植株形态及生长变异进行了初步研究,主要研究结果如下: 1.叶片外殖体高效再生系统的建立:筛选出适宜八宝景天分化培养基是不附加任何激素的MS培养基,适宜生根培养基是不附加任何激素的1/2MS培养基, 适宜诱导叶片愈伤组织发生的培养基是MS+6-BA 0.3mg.L-1+NAA 0.1mg.L-1, 适宜诱导叶片愈伤组织不定芽分化的培养基是MS+6-BA 0.3mg.L-1+NAA 0.1mg.L-1.研究了暗培养对诱导叶片不定芽的影响,结果表明,暗培养5d有利于不定芽的诱导. 2.头孢噻肟钠(CTX)对诱导八宝景天叶片不定芽的影响和对农杆菌的抑菌效果:确定适宜的CTX浓度为400 mg.L-1. 3.影响农杆菌转化效率的几个主要因素:发根农杆菌活化次数、菌液浓度、侵染时间、共培养时间等对转化频率都有一定影响,在转化过程中菌株活化3次以上,菌液浓度OD600=0.4,侵染时间10min,共培养2 d,有利于获得最高遗传转化效率. 4.转基因植株的获得:利用介导法将发根农杆菌转入八宝景天叶片中,获得了转基因植株,对转基因植株进行了PCR检测,证明发根农杆菌Ri质粒已整合到八宝景天基因组中. 5.转基因植株形态及生长变异的研究:发现与未经转化的八宝景天组培苗相比,大部分转基因植株矮化,茎分支能力增强,节间距缩短,生根系数增高,叶片颜色变黄.
英文摘要: The infancy stem segments Sedum Spectabile Boreau of were taken as explants to be cultured, through which their floral system was established in this experiment. Besides, callus of the leaves and stem segments and the infancy stem with shoot segments of Sedum Spectabile Boreau were cultured in the different medium of concentrations of hormone. In order to provide genetically improved new germplasm resources and achieve the transgenic plants of Sedum Spectabile Boreau ,genetic transformation system was established based on the system of regeneration. Several factors affected genetic transformation of Sedum Spectabile Boreau mediated by agrobacterium rhizogenes were studied. The shape characteristic, PCR detection of transgenic plants were researched, shape and Growth variation and leaf anatomic structure were also studied. The main research work and results were as follows:1.Regeneration system for Sedum Spectabile Boreau leaf explants was established,the experiment results he resulte showed that good propagation medium was MS0,good rooting medium was 1/2MS0 , the good medium of leaves callus induced was MS+6-BA 0.3mg. L-1+NAA 0.1mg. L-1. Based on this suitable medium, the effect of dark cultivation on induction of adventitious buds from leaves explant was studied. It was found that dark cultivation for 5 days would be favorable for induction of adventitious buds.2. Effects of Cefotaxime (CTX) on induction of adventitious buds from Sedum Spectabile Boreau of triploid P. tomentosa explants and inhibition of Agrobacteria were investigated. The results indicated that 400mg.L-1 was a suitable concentration to control the propagation of Agrobacteria.3.Several crucial factors influencing the transformation efficiency were studied. The results showed that: the times of activation, the time of infection, the concentration of agrobacterium rhizogenes''''s solution esc, had some effect on transformation frequency. The highest transformation frequency were infected for 10 min with the concentration of agrobacterium rhizogenes''''s solution OD600=0.4,and then co-cultivated for 2d .4. PCR identification method was used on transplant, the results indicate T-DNA of Ri plasmid had already been combined into the transplant. 5.Shape and growth variation of transgenic plantlets were also studied.The result indicated that most of transgenic plantlets with Agrobacteria exhibited dwarf plant ,increased leaf area,increased branch, shortened internodes,decreased rooting rate ,root number and growth of stem length .