柱花草nptII、hpt和bar基因优化转化体系的建立

The Optimal Transformation Systems of Stylosanthes guianensis by nptII,hpt,b ar as Selection Genes

以热研5号柱花草(Stylosanthes guianensis cv.Ryan No.5)为材料,系统地研究了不同选择标记基因NptII,hpt和bar的柱花草转化体系.研究发现柱花草最适合基因转化的外植体是幼苗下胚轴.最佳愈伤组织形成和愈伤组织分化培养基为M S+NAA 1.0m g/L+6B-A4.0m gL/+3%蔗糖,pH6.最适宜外植体生根培养基是MS+N AA0mg/L+3%蔗糖(pH6).Glufosinate选择压力为0.15m g/L,Kanam ycin选择压力为20mg/L,Hygrom ycin选择压力为15m g/L.在以上的优化转化系统中,柱花草幼苗下胚轴分别侵染带NptII,hpt和bar基因的农杆菌(GV 3101)后,可获得30%愈伤组织具有kanamycin抗性,5%愈伤组织具有Hygromycin抗性,50%的愈伤组织具有Glufosinate抗性.

The transformation systems of Stylosanthes guianensis cv. Ryan No. 5 by using nptlI, hpt, bar as selection genes were studied. The results showed that the hypocotyls of the young seedlings were the best expand for gene transformation in Stylosanthes guianensis cv. Ryan No. 5. The best medium of the callus formation and differentiation was MS ＋ NAA 1.0 mg/L ＋ 6-BA 4.0 mg/L. The optimal medium for root formation from callus was MS ＋ NAA 0 mg/L. In Stylosanthes guianensis cv. Ryan No. 5, the suitable concentrations for transgene selection were 0. 15 mg/L for Glufosinate; 20 mg/L for Kamamycin, 15 mg/L for Hygromycin. In the above transformation systems, when the nptlI, hpt and bar genes were transformed to hypocotyls through Agrobacterium GV3101, 30% calli were resistant to Kanamycin; 5% calli were resistant to Hygromycin; 50% calli were resistant to Glufosinate.