【作者】 余密密；

【导师】 卢欣石；

【作者基本信息】 北京林业大学 ， 森林培育， 2006， 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)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性育种开辟了一条新的途径。本实验通过农杆菌介导法对紫花苜蓿不同品种植株进行了果聚糖合成酶基因转化研究,获得了一套紫花苜蓿不同品种的遗传转化体系。主要结果如下:1基因转化所用的农杆菌菌株LBA4404对头孢霉素敏感,当头孢霉素为300mg/L时就能完全抑制细菌的生长,同时该浓度的头孢霉素对胚性愈伤组织的形成影响不大。2适合于紫花苜蓿的卡那霉素选择压是80mg/L,100mg/L的卡那霉素对愈伤组织的生长有着显著的抑制效应,选择方式采用延迟筛选。3不同基因型的转化效率不同,在实验的品种中和田苜蓿的转化效率最高。表现最好的外植体部位为下胚轴。4共培养3d的愈伤组织表现良好。但是从后期再生阶段看来不是必须。5转化中菌液浓度、侵染时间、预培养时间和共培养时间是相互关联的,表现最佳的组合是:菌液浓度为OD600 =0.3～0.5;侵染时间10～15min;预培养3d;共培养3d。6试验中共培养基采用MS+2mg/L 2,4-D + 0.5mg/L KT+30g/L蔗糖+7g/L琼脂,培养材料在MS+2mg/L 2,4-D+0.5mg/L KT+30g/蔗糖+7g/L琼脂+300mg/L Cef培养基上诱导出愈伤组织,在体细胞胚分化培养基MS +1.0mg/L BA + 0.3mg/L NAA 30g/L蔗糖+7g/L琼脂+50mg/L Kan +300mg/L Cef上促进体细胞胚的分化,分化出的体细胞在再生培养基MS +1.0mg/L BA + 0.3mg/L NAA+30g/L蔗糖+7g/L琼脂+80mg/L Kan +300mg/L cef上进一步发育,转化无根小苗在生根培养基1/2 MS+1.0mg/L IBA+1%蔗糖+0.6%琼脂+100mg/L Kan上诱导根系。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) perennial legume crop is wildly planted in the world. The development and application of gene engineering creates a new path for alfalfa resistant breeding. In this research, a protocol for SacB gene transformation of alfalfa by Agrobacterium tumefaciens mediated has been developed.The mainly conclusions are as follows,1. 300mg/L Cef could beused to inhibit the Agrobacterium strain LBA4404 after transformation availably;2. The results showed that induction of callus from alfalfa was completely restrained by 100mg/L Kanamycin, the suitable selection pressure should be 100mg/L Kanamycin;3. The different gene types showed different results. In this experiment, the hypocotyls from hetian had highest calli induction frequence.4. The hypocotyls had the highest Kanr calli induction frequence under together cultivation time for 3days, but it showed that together cultivation was not necessary during the cultibation followed.5. Agrobacterium concentration, inoculation time, advance culture time and together culture time were associated. The best combination contains Agrobacterium concentration whose OD600 was 0.3～0.5, The explants from alfalfa were inoculated for 10～15 minutes, advance culture time and together culture.6. The inoculated explants were incubated on MS medium supplemented with 2mg/L 2,4-D,0.5mg/L KT, 30g/L sugar and 7g/L agar ,then the explants were transferred into MS medium supplemented with 2mg/L 2,4-D,0.5mg/L KT, 30g/L sugar and 7g/L agar and 300mg/L Cef to induce callus and somatic embryogenesis and subcultured onto MS medium supplemented with 1.0mg/L BA,0.3mg/L NAA, 30g/L sugar, 7g/L agar,50mg/L Kan and 300mg/L Cef to promete further embryos development. Transgenetic plantlets regenerated from somatic embryos on medium mentioned above after several subcultruing. Transgenic plantlets could root on 1/2 MS medium supplemented with 1.0mg/L IBA,10g/L sugar,8g/L agar and 80mg/L Kan.更多还原