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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.
Stylosanthes guianensis cv. Ryan No.5 was used as material to systematically study the stanza transformation system with different selectable marker genes NptII, hpt and bar. The study found that Stylosanthes is the most suitable explant for gene transformation The seedlings were hypocotyls.The best callus formation and callus differentiation medium was MS + NAA 1.0mg / L + 6B-A4.0m gL / + 3% sucrose, pH 6. The most suitable explants for rooting The medium was MS + N AA0 mg / L + 3% sucrose pH 6. The selection pressure of Glufosinate was 0.15 mg / L, the selective pressure of Kanam ycin was 20 mg / L, and the selective pressure of Hygromycin was 15 mg / L. In the system, 30% callus was kanamycin resistant, 5% callus was Hygromycin resistant, Fifty percent of the calluses have Glufosinate resistance.