小麦遗传转化中潮霉素筛选体系的建立及应用  

Establishment and Application of the Hygromycin Selection System in Wheat Transformation

作　　者：杨桥[1] 蔺自敏[1] 侯详文 姚明镜[1] 

机构地区：[1]华中农业大学植物科学技术学院,湖北武汉430070

出　　处：《麦类作物学报》2014年第3期304-310,共7页Journal of Triticeae Crops

基　　金：农业部转基因生物新品种培育重大专项(2011ZX08010-001)

摘　　要：为建立小麦遗传转化中高效的潮霉素筛选体系,以小麦品种郑麦9023的幼胚愈伤组织为材料,探讨了潮霉素浓度、处理时间、恢复培养与否对愈伤组织分化和再生的影响,并对基因枪转化的郑麦9023、邯6172、龙麦30、龙麦31的愈伤组织进行了筛选。结果表明,对小麦幼胚愈伤组织基因枪遗传转化后的筛选以50mg·L-1潮霉素处理30d为宜;恢复培养不能提高筛选阶段愈伤组织的存活率和分化率,但能提高转化后期的成苗率。经过筛选发现郑麦9023抗性愈伤和T0代抗性植株根系及花器官中均有GUS基因的表达;4个小麦品种抗性植株中潮霉素磷酸转移酶基因(hpt)和候选基因PCR阳性率分别为78.95%和73.68%以上;龙麦30、龙麦31的T1代植株的候选基因PCR阳性率分别在33.33%和73.33%以上,T2代阳性率分别在71.43%和75.00%以上。Effective selection system is essential to high efficiency genetic transformation. In this stud- y, the effects of different hygromycin concentrations, screening time and convalescence or not on differ- entiation and regeneration of immature embryo callus of Zhengmai 9023 were investigated,and callus of Zhengmai 9023, Han 6172,Longmai 30,Longmai 31 transformed by particle bombardment were se- lected using hygromycin. The results showed that 50 mg· L-1 of hygromycin applied for 30 d was suitable for screening after transformation; convalescence could not improve the survival rate and dif- ferentiation rate of callus. However,it could promote plantlet establishment for transformational cal- lus. GUS expressions were found in Zhengmai 9023 resistant tissues,roots and flower of To genera- tion. In addition, the expressions of hygromycin phosphotransferase gene （hpt） and candidate gene were confirmed by PCR in the To resistant plants of four cultivars,positive ratio were up to 78.95% and 73.68%, respectively. For Longmai 30 and Longmai 31 ,the positive ratio of candidate gene in T1 generation were higher than 33.33% and 73. 33% ,respectively; T2 generation were over 71. 43% and 75.00% ,respectively. Results above proved that this established hygromycin selection system for im- mature embryo callus of wheat was efficient and feasible.

关 键 词：小麦 幼胚愈伤组织 潮霉素 遗传转化 

分 类 号：S512.1[农业科学—作物学] S330