摘要
通过农杆菌介导法对紫花苜蓿不同品种植株进行了抗旱基因转化的研究．得到了一套紫花苜蓿基因转化的优化体系。研究表明．100mg／L的卡那霉素对苜蓿愈伤组织的生长有着显著的抑制效应。250mg／L头孢霉素能够有效地抑制农杆菌菌株LBA4404的生长。紫花苜蓿供试材料被切后直接用OD600值为0．3～0．5的农杆菌LBA4404菌液侵染10～15min；培养材料共培养3d后在愈伤组织诱导培养基MS＋2mg／L2．4-D＋0．5mg／LKT＋30g／L蔗糖＋9g／L琼脂＋250mg／L Cef上诱导出愈伤组织；在体细胞胚分化培养基MS＋1．0mg／LBA＋0．3mg／LNAA＋30g／L蔗糖＋9g／L琼脂＋50mg／LKan＋250mg／LCef上促进体细胞胚的分化；分化出的体细胞胚在再生培养基上（同分化培养基．Kan为80mg／L）进一步发育成抗性转化苗；转化的无根小苗在生根培养基1／2MS＋1．0mg／LIBA＋1％蔗糖＋0．8％琼脂＋100mg／L卡那霉素上可生长出根系。
A protocol for drought-resistent gene transformation of Medicago sativa by Agrobacterium tumefaciens mediation has been developed. Several factors were studied to improve the frequency of gene transformation of M. sativa. Induction of callus from M. sativa was completely inhibited by 100 mg/L Kanamycin. The Agrobacterium strain LBA4404 could be inhibited by 250 mg/L Cef after transformation. The explants from M. sativa were directly inoculated with A. tumefaciens strain LBA4404 whose OD600 was 0.3-0. 5 for 10-15 min. Next, the inoculated explants were incubated on MS medium supplemented with 2 mg/L 2,4-D, 0.5 mg/L KT, 30 g/L sucrose and 9 g/L agar for three days, and then the explants were transferred to MS medium supplemented with 2 mg/L 2,4-D, 0.5 mg/L KT,30 g/L sucrose, 9 g/L agar and 250 mg/L Cef to induce callus and somatic embryogenesis. They were then subcultured onto MS medium supplemented with 1.0 mg/L BA,0.3 mg/L NAA, 30 g/L sucrose, 9 g/L agar, 50 mg/L Kan and 250 mg/L Cef to promote further embryo development. After subculturing several times on the above media, transgenic plantlets could be regenerated from somatic embryos. They could root on 1/2 MS medium supplemented with 1.0 mg/L IBA, 10 g/L sucrose, 8 g/L agar, and 100 mg/L Kan.
出处
《草业学报》
CSCD
2006年第5期94-102,共9页
Acta Prataculturae Sinica
基金
科技部"奶业"专项子课题---优质饲草高效生产关键技术研究与产业化开发(2002BA518A03)
科技部国家转基因植物研究与产业化专项子课题---苜蓿等牧草抗旱
耐盐碱转基因新品种培育(J00-B-001-12)资助
关键词
紫花苜蓿
基因转化
农杆菌介导法
Medicago sativa
transformation
protocol by Agrobacterium tumefaciens mediated
