【作者】 李红；

【导师】 王关林；

【作者基本信息】 辽宁师范大学 ， 植物学， 2004， 硕士

【摘要】 本研究以毛白杨系品种:二倍体雌、雄株及三倍体雌、雄株和美洲黑杨的芽及叶片为外植体材料,系统的研究了杨树组织培养的影响因素,建立了五种杨树的高频再生体系;并采用农杆菌介导的基因转化法,将耐盐碱基因(OsNHX1)导入美洲黑杨基因组中,经PCR分子鉴定,表明已获得了OsNHX1基因的转化植株。具体工作如下:1. 杨树组织培养和高频再生体系的建立选用五种杨树芽、叶片进行组织培养,诱导再生,得到了白杨系外植体最适分化培养基为:MS+BA1.0(mg·L-1,单位下同)+NAA0.1+GA0.5,分化率可以达到90%以上;美洲黑杨再生培养基为:MS+BA0.5+NAA0.1+GA0.5,分化率可以达到99%以上;生根培养基:MS+NAA0.1。2. 种间基因型的差异是影响植物外植体再生能力的决定性因素;种内基因型此种效应不明显;植物基因组中调控再生能力的基因不是性染色体连锁;调控再生能力的基因不存在染色体组倍数的叠加效应。3. 在建立了美洲黑杨高频转化受体系统的基础上,进行了农杆菌介导的耐盐碱基因(OsNHX1)的转化研究;并从工程菌的活化状态、侵染时间等方面阐述了杨树基因转化中的影响因素;采用分级筛选的方法,对抗性芽进行筛选,并在筛选生根培养基中生根,得到了转化植株;以未转化植株为阴性对照,以质粒DNA为阳性对照,进行PCR检测。结果表明,转化植株扩增的条带和质粒中的目的条带一致,而未转化植株没有扩增出任何条带,说明外源目的基因已经整合到美洲黑杨基因组上。更多还原

【Abstract】 Factors affecting Pupulus tissue culture were explored systematically by culturing buds and leaves as in vitro material of binary female and male P.tomentosa as well as triploid female and male. High-frequency Regeneration System of five species of Pupulus were established;The result of PCR has showed: OsNHX1 gene was led into the genome of P.deltoides by Agrobacterium tumefaciens mediating gene transforming and salt-resisting transgenosis plant was acquired. Concrete operations are as follows:1. Tissue Culture and Establishment of High-frequency Regeneration System of Populus Selected and tissue cultured buds and leaves of five species of Populus , then induced them into regeneration. The best medium of regeneration of P.tomentosa is:MS+BA1.0(mg·L-1, the same as follow) +NAA0.1+GA0.5; Frequency of differentiation is 90% above; To P.deltoides, regeneration medium is: MS+BA0.5+NAA0.1+GA0.5;Frequency of differentiation is 99% above; Rooting-medium is: MS+ NAA0.1; 2. Difference of genotype in different species is the determinative factor which affects the regeneration ability of in vitro explants, but the effect of that in the same species is not obvious. Gene which modifies the regeneration ability of plants is not sex-chromosome-linked and has not folded effect with multiple of genome. 3. On the basis of establishment of Populus high-frequency regenerationsystem, the study of Populus heredity conversion of anti-salt gene mediatedand induced by Agrobacterium tumefaciens was carried on. Factors as optimumconversion state, infecting time etc were expounded. Resistant planet wasscreened by increasing concentration of Km step by step and roots were inducedin screening rooting-medium.With unconversed plant as negative tester andplasmid DNA as active tester, PCR was carried on. Result showed: extendedstrip in transgenosis plant agreed with target strip in plasmid while no stripextended in unconversed plant; this indicated external target gene wasintegraded into Populus genome.更多还原