农杆菌敏感小麦基因型筛选与转化条件的优化

Screening of Wheat Genotype Sensitive to Agrobacterium tumefaciens Infection and Optimizing Agrobacterium-mediated Transformation Conditions

以13个基因型小麦的幼胚愈伤和新春9号幼胚为受体材料,利用农杆菌菌株C 58C1（含有GUS外源基因）进行侵染,通过组织化学染色的方法统计GUS基因瞬时表达率,以筛选对农杆菌敏感的小麦基因型及适宜的菌液浓度和侵染时间,优化农杆菌转化的条件,为逐步建立小麦农杆菌的转化体系奠定基础。结果显示,不同基因型小麦GUS瞬时表达率在0-95.9%之间,其中新春9号、小偃328和济麦19的GUS基因瞬时表达率均在90%以上;对新鲜幼胚和预培养7d的幼胚愈伤组织分别侵染30min和60min,前者GUS阳性率均为0,后者分别为92.0%和95.9%,说明预培养可以显著提高受体材料对农杆菌的敏感性,提高转化效率;在不同菌液浓度（OD600=0.5、1.0、1.5）侵染时,GUS基因瞬时表达率分别为93.4%、94.0%和87.6%,不同侵染时间（15,30,60min）下,GUS基因瞬时表达率分别为94.6%、92.0%和95.9%。综合考虑认为,适宜的小麦农杆菌转化条件为幼胚经过4~7d预培养,在菌液浓度OD600=1.0时进行15min的侵染。

In this study immature embryo callus from 13 wheat genotypes and Xinchun 9as explants were infected by Agrobacterium strain of C 58C1 harboring pPTN 290 plasmid with GUSas report gene,and the infected explants were tested by X-Gluc staining for GUSgene transient expression for screen out the sensitive genotypes to the Agrobacteriumand the suitable Agrobacteriumconcentration of infection suspension and infected duration,in order to optimization wheat Agrobacterium-mediated transformation condition and to providing reference for establish wheat Agrobacterium mediated transformation system.The result showed that GUSgene transient expression frequency of 13 wheat genotypes was from 0to 95.9%,genotypes of Xinchun 9,Xiaoyan 328 and Jimai 19 were sensitive to the Agrobacterium infection which GUS gene transient expression rate was above 90%.The fresh immature embryo could＇t be infected by Agrobacterium,and when the immature embryo precultured7 days,the GUS gene transient expression frequency could up to 92% and 95.9% respectively with infected 30 mins and 60 mins.This suggest that preculture could signicantly improve explant sensitivity for Agrobacterium and improve the transformation efficiency.With different concentration（OD600=0.5,1.0,1.5）of Agrobacteriuminfection suspension infected the callus,the GUS gene transient expression rate was 93.4%,94.0% and 87.6% respectively,and with the different infected duration（15min、30min、60min）,the rate could up to 94.6%,92.0% and 95.9%.The optimal conditions for wheat Agrobacterium-mediatedtransformation were obtained for immatue embryos for precultured 4to 7days,followed by immersing in Agrobacterium suspension with OD600=1.0infection for 15 min.