农杆菌介导小麦遗传转化影响因素的研究

Factors Affecting Wheat Transformation Mediated by Agrobacterium tumefaciens

选用河南科技学院近年来选育的小麦品种百农160的成熟胚愈伤组织和农杆菌菌株LBA4404,对农杆菌介导遗传转化中农杆菌浓度、感染时间、乙酰丁香酮浓度、头孢霉素质量浓度等几个重要影响因素进行了研究,建立小麦高效转化体系。结果表明,菌液OD600=0.6,侵染时间30 min时对愈伤组织的生存和转化最有利;浓度为200μmol/L的乙酰丁香酮可显著提高抗性愈伤再生率;随着头孢霉素浓度的升高,愈伤组织出现褐化,生长缓慢,再生率低,所以在有效抑制农杆菌的浓度范围内,尽量采取较低的质量浓度。在后期的抑菌分化筛选中,在继代培养基中添加头孢霉素的质量浓度为500 mg/L,继代扩增筛选培养2个月,每10～15 d换一次培养基;而在分化培养基中添加200 mg/L头孢霉素,即可获得再生率高的抗性愈伤。最适条件下转化频率达2.36%。

To establish highly efficient wheat transformation system,factors which influenced wheat transformation including agrobacterium concentration,infection period,and concentration of acetosyringone and cephalosporin were studied with the mature embryo callus of wheat variety Bainong 160 and agrobacterium strain LBA4404.The results showed that the transformation efficiency was clearly improved with agrobacterium density of OD600=0.6 and incubation for 30 min.Acetosyringone with the concentration of 200 μmol/L could significantly enhance the rate of resistant calluses.Considering the inhibition of cephalosporin on agrobacterium cells,it was suggested that 500 mg/L of cephalosporin should be used in the latter stage of antibacterial screening,and then 200 mg/L of cephalosporin should be used in differentiation medium to get resistant calluses of high regeneration efficiency.The transgenic frequency was 2.36% under the optimal conditions.