正交设计在杨树最佳遗传转化体系的建立  

OPTIMIZATION OF AN GENETIC TRANSFORMATION SYSTEM FOR POPLAR BY USING ORTHOGONAL DESIGN

作　　者：蔡诚[1] 吴大强[1] 纵方[1] 项艳[1] 

机构地区：[1]安徽农业大学林学与园林学院,安徽合肥230036

出　　处：《核农学报》2008年第2期136-140,共5页Journal of Nuclear Agricultural Sciences

基　　金：由安徽省人才开发专项基金(2007年度);国家十一五科技支撑项目(2006BAD03A15050-4)资助

摘　　要：本试验以建立的南林95杨高频再生体系为基础,利用正交试验设计,通过GUS组织染色分析法研究了预培养时间、菌液浓度、AS浓度、侵染时间和共培养时间5个因素在4个水平上对南林95杨遗传转化的影响,并探讨了南林95杨转化中添加卡那霉素(Kanamycin)的适宜浓度。结果表明:外植体预培养7d,在含乙酰丁香酮AS200μmol/L、菌液浓度OD值为0.6的农杆菌液中侵染20min,共培养3d为最佳遗传转化体系,适宜的卡那霉素筛选浓度为50mg/L;经GUS染色检测和PCR分析表明,GUS基因已初步整合进南林95杨基因组中。Based on the established regeneration system of Nanlin 95 （ Populus deltoids Bartr. cv. Lux × P. euramericana （Dode） Guineir ev. 1-45/51 ）, through orthogonal design, an efficient transformation and screening system of Nanlin 95 mediated by Agrobateriurn was developed. The leaves from tissue culture served as excellent explants for transformation, and the factors affecting the transformation frequency, such as preculture time of explants, concentration of Agrobacterium and acetosyringone （AS）, infection time and co-infection time were discussed by GUS coloration analysis, and the concentrations of antibiotics to inhibit the growth of Agrobacterium were also discussed. The data were analyzed by software DPS, a suitable genetic transformation system was established. The results indicated that 7 days precuhuration, 20 min Agrobacterium （OD600 = 0.6） infection with 200μmol/L AS, and 3 days＇ co-infection would lead to an increase in transformation efficiency, and the medium with kanamycin 50 mg/L was found to be the most optimal for transgenic plantlets selection ; stable integration of GUS gene in these plants were confirmed by GUS coloration analysis and PCR test.

关 键 词：杨树 正交设计 遗传转化 农杆菌 

分 类 号：S792.11[农业科学—林木遗传育种]