紫花苜蓿基因转化的影响因素分析

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,100 mg/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。250 mg/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10~15 min;培养材料共培养3 d后在愈伤组织诱导培养基MS 2 mg/L 2,4-D 0.5 mg/LKT 30 g/L蔗糖 9 g/L琼脂 250 mg/L Cef上诱导出愈伤组织;在体细胞胚分化培养基MS 1.0 mg/L BA 0.3 mg/L NAA 30 g/L蔗糖 9 g/L琼脂 50 mg/L Kan 250 mg/L Cef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80 mg/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1/2 MS 1.0 mg/L IBA 1%蔗糖 0.8%琼脂 100 mg/L卡那霉素上可生长出根系。

Analysis of factors influencing gene transformation in Medicago sativa

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 OD_(600) 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.