农杆菌介导的小麦成熟胚转化的影响因素  

The Influencing Factors of Agrobacterium Tumefaciens Infecting Mature Embryo of Wheat(Triticum aestivum)

作　　者：宋成丽[1] 王翾[2,3] 徐虹[1] 刘迎团[1] 张改生[2,4] 王军卫[2,4] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学农学院,陕西杨凌712100 [3]西安职业技术学院生物工程系,陕西西安710077 [4]陕西省作物杂种优势研究与利用重点实验室,陕西杨凌712100

出　　处：《麦类作物学报》2012年第2期209-214,共6页Journal of Triticeae Crops

基　　金：国家自然科学基金项目(30971844;30971769);西北农林科技大学基本科研业务费项目(QN2011003);西北农林科技大学国际科技合作基金项目

摘　　要：为了研究农杆菌介导的小麦成熟胚转化的影响因素,以6个小麦品种成熟胚为外植体进行离体培养,以EHA105和LBA4404农杆菌为供体(含有pCAMBIA1305双元载体)材料,研究了诱导培养基、分化培养基、农杆菌菌株、愈伤组织诱导培养时间、农杆菌菌液浓度和侵染时间对小麦成熟胚愈伤组织诱导、分化、再生的影响。结果表明:(1)以小麦品种小偃22的成熟胚为材料,获得最佳的诱导培养基方案(MS+2mg.L-1 2,4-D+0.5mg.L-1 KT+500mg.L-1水解酪蛋白+150mg.L-1天门冬酰胺)和分化培养基方案(MS+2mg.L-1 ZT+0.5mg.L-1 KT);(2)6个小麦品种中,洛阳8176和张掖春小麦具有较强的再生能力,筛选培养后较多的愈伤组织分化出小绿点;(3)洛阳8716和张掖春小麦的成熟胚,经愈伤组织诱导培养6-7d,农杆菌侵染后愈伤组织再生能力强;(4)农杆菌侵染后愈伤组织的再生能力与农杆菌菌株的关系不明显,而与小麦品种基因型、侵染时间和菌液浓度有较明显的关系;(5)以小麦洛阳8716和张掖春小麦成熟胚愈伤组织为受体,黑暗条件下诱导培养6-7d,在24±1℃黑暗条件下共培养3d和500mg.L-1羧苄青霉素(Cb)除菌及10mg.L-1潮霉素筛选处理后,共得到分化植株23株,其中以洛阳8716为受体材料,由农杆菌LBA4404和EHA105转化得到的植株分别为11株和4株,植株分化率分别为3.57%和1.31%;以张掖春小麦为受体材料,由农杆菌LBA4404和EHA105转化得到的植株分别为5株和3株,植株分化率分别为1.57%和1.14%。Mature embryo callis of wheat,winter wheat ＇Ginmai47＇and ＇Xinong928＇,weak spring wheat ＇Zhengmai9023＇ and ＇Xiaoyan22＇,spring wheat ＇Luoyang8716＇ and ＇Zhangyechunxiaomai＇,were used receptors to be infected by A.tumefaciens strains（EHA105 and LBA4404） with the pCAMBIA1305 vector in this experiment.The results showed that：（1）the optimal induction medium was MS＋2 mg·L-1 2,4-D＋0.5 mg·L-1 KT）,and optimal differentiation medium was MS＋2 mg·L-1 ZT＋0.5 mg·L-1 KT.（2）Callus differentiation ratios from ＇Luoyang8716＇ and ＇Zhangyechunxiaomai＇ were higher than other wheat varieties.（3）With ＇Luoyang8716＇ and ＇Zhangyechunxiaomai＇,callus induction time（6~7d） was demonstrated to be optimal for A.tumefaciens infection.（4）Distinct differences plant regeneration frequency were exerted among the different wheat varieties,infection time and concentration,whereas little differences was exerted between EHA105 and LBA4404.（5）Mature embryo callus of wheat ＇Luoyang 8716＇ and ＇Zhangyechunxiaomai＇ precultured for 6~7 d in the dark,co-cultivated with A.tumefaciens in the dark at 24±1℃ for 3~4 d,then subcultured on the differentiation medium with 500 mg·L-1 carbenicillin and 10 mg·L-1 hygromycin,23 transgenic seedlings could be got.For spring wheat ＇Luoyang8716＇,there were 11 transgenic seedlings by LBA4404 and 5 transgenic seedlings by EHA105,differentiation rate is 3.57% and 1.31%,respectively;and for Zhangyechunxiaomai,there were 5 transgenic seedlings by LBA4404 and 3 transgenic seedlings by EHA105,differentiation rate is 1.57% and 1.14%,respectively.

关 键 词：小麦 成熟胚 农杆菌 分化 

分 类 号：S512.1[农业科学—作物学] S336