【作者】 黄剑；

【导师】 孙吉雄；

【作者基本信息】 甘肃农业大学 ， 草业科学， 2002， 硕士

【摘要】 盐碱地在中国分布广泛，随着我国人口增加，耕地减少，盐碱地资源的开发利用有着极其重要的现实意义。紫花苜蓿（Medicago．sativa．L）是当今世界上种植面积最大，应用最广泛的多年生豆科作物。基因工程的发展和应用为苜蓿抗盐育种开辟了一条新的途径。本实验从5个品种中选择出再生能力强的中苜一号作为材料，运用两步法建立了中苜一号体细胞胚再生体系，并从中选择出了高再生率的基因型H2。在此基础上进行了农杆菌介导的甜菜碱醛脱氢酶基因的转化。此外，利用共培后GUS染色组织分析探讨了影响转化效率的各种因素，优化了转化体系。主要结果如下： 1 建立了中苜一号体胚发生再生体系。中苜一号叶片分化能力强，改良SH+2，4—D4.0mg／L+BA0.5mg／L诱导胚性愈伤组织效果好。MSO培养基对胚状体的诱导最为有效。蔗糖浓度为2％，并附加谷胱甘肽15mg／L能促进胚状体的分化。体胚在MSO培养基上萌发生根并成苗。从中苜一号200个基因型中选择出H1、H2、H3、M1、M3，5个高再生率的基因型作为转化受体。 2 建立了遗传转化体系。从5个基因型中选择出转化效率高的H2作为最终的转化受体。以GUS染色组织分析为依据建立和优化了转化体系：叶片预培养3d，利用MC液体培养基重悬菌液至浓度为OD₆₀₀=0.3～0.5时侵染5～10min，共培养3d，然后转接到含400mg／L Cef和50mg／L Kan的MC培养基上进行选择培养。经Kan90d筛选得到8株Kan抗性植株。更多还原

【Abstract】 Saline soil is widespread in China. The development of saline soil is of significance with the increasing population and decreasing arable land. Cultivated alfalfa ( Medicago. sativa.L ) , perennial legume crop,is wildly planted in the world. The development and application of gene engineering creates a new path for alfalfa salt-resistant breeding.In this research, ZhongMuYiHao .selected from five salt-resistant varieties, has regenerated via somatic embryogenesis by two steps. The genotype H2 was selected for transformation with its high regenerative capacity. On the basis of acquired regenerating system of somatic embryogenesis, the BADH gene was transformed through agrobaterium-mediated. We have studied many factors which affect transformation frequency and established and optimized transforming system by histological detection of GUS activity after co-cultivation. The main results were as following:1 The regeneration system of somatic embryogenesis has been established. The leaf explant has the highest regenerative capacity among explants of ZhongMuYiHao. The modified SH medium containing 4.0mg/L 2,4-D and 0.5mg/L BA induced embryogenic callus very well. MSO medium with sugar 2% and no hormone is the most effective medium for embryo induction. The supplement of 15mg/L glutathione promote the formation of embryo. The embryo converted into plant in die MSO medium. Five genotypes with high regenerative capacity were selected from 200 genotypes.2 The transformation system has been established. In the agrobacterium-mediated gene transformation, leaves of genotype H2 was transformed for its high transformation frequency. And with the histological detection of GUS activity, we optimized the transformation procedure. The wounded leaves pro-cultivated on MC for 5 days. A fresh liquid culture of A.tumefaciens was centrifuged and the pellet was resuspended in liquid MC medium to me ODgoo of 0.3 to 0.5.The wounded leaves were immersed in the bacterial suspension for 5 to 10 minutes and then to-cultivated on MC for 3 days. After washing the explants to remove excessive bacterium, callogenesis was induced on MC supplemented with 50mg/L Kan as the selective agent for transformed tissue and 400mg/L Cef to inhibit bacteria] growth. After 90 days selection eight regenerated Kan-resistant plants were abtained.更多还原