怀黄菊间接体胚受体再生体系的建立及CmTGA1的遗传转化  

Establishment of Transgenic Acceptor by Indirect Somatic Embryogenesis Regeneration and Transformation of Cm TGA1 Gene in Chrysanthemum morifolium cv. ‘Huaihuang'

作　　者：赵喜亭[1,2,3] 蒋丽微[1] 王苗[1] 朱玉婷[1] 张文芳[1] 李明军[1,2,3] 

机构地区：[1]河南师范大学生命科学学院,新乡453007 [2]河南省高校道地中药材保育及利用工程技术研究中心,新乡453007 [3]绿色药材生物技术河南省工程实验室,新乡453007

出　　处：《植物学报》2016年第4期525-532,共8页Chinese Bulletin of Botany

基　　金：国家自然科学基金(No.31372105);中国博士后科学基金(No.2011M500457);中央高校基本科研业务费专项资金(No.2011JS076);河南省高校科技创新团队支持计划(No.15IRTSTHN020);河南省创新型科技人才队伍建设工程(No.C20130037)

摘　　要：植物受体再生及遗传转化体系的建立是对植物进行转基因操作时至关重要的一个技术环节。以菊花优良种质怀黄菊(Chrysanthemum morifolium cv.‘Huaihuang’)为试材,探讨胚状体诱导所需的最佳培养基及芽分化和根生长的最适抗生素选择压。在此基础上,将抗病基因Cm TGA1通过同源转化的方法转入怀黄菊,获得再生植株。结果表明,在附加1.5 mg·L-1IAA、0.5 mg·L-1 6-BA和1.0 mg·L-1 2,4-D的MS培养基上诱导培养15天后,再去除2,4-D进行分生培养,并进一步诱导芽再生,最终86%的供试外植体通过胚状体途径获得再生芽;在芽分化时所需潮霉素选择压为5.0 mg·L-1,生根时潮霉素选择压为4.5 mg·L-1。对转化植株进行半定量PCR(semi-quantitative PCR)和实时荧光定量PCR(real-time quantitative PCR)检测,结果表明,Cm TGA1基因成功整合到转化植株基因组中,从而建立了怀黄菊间接体细胞胚途径转基因受体再生体系。该技术的建立为通过转基因手段解决生产中存在的怀黄菊病害感染严重和种质退化等问题奠定了基础。The transgenic acceptor regeneration system and the genetic transformation system are important in the plant transgenic manipulation. This study explored the optimal medium of embryoid induction and the optimal antibiotic selection concentrations of shoot and root regeneration of Chrysanthemum morifolium cv. ＇Huaihuang＇. CmTGA1, a disease-resistant gene, was transformed into ＇Huaihuang＇ by using homeosis and regenerated new lines. Explants were cultured for 15 d on MS basal medium supplemented with 1.5 mg.L^-1 IAA, 0.5 mg.L^-1 6-BA and 1.0 mg.L^-1 2,4-D, then transferred to the meristematic medium without 2,4-D and cultured for 8-10 weeks, finally the regeneration bud was induced for 2 to 3 weeks on the developed medium. 86% of explants gained regeneration buds from embryoids. The selected concentration of hygromycin B was 5.0 mg.L^-1 for shoot regeneration and 4.5 mg.L^-1 for rooting. The integration of CmTGA1 into the genome of the transformation lines was confirmed by semi-quantitative PCR and real-time quantitative PCR. Finally, the transgenic acceptor regeneration system with indirect somatic embryogenesis was established successfully for Huaihuang, and this technology may lay the foundation for solving some production problems such as serious disease infection and germplasm degradation by genetic engineering methods.

关 键 词：怀黄菊 间接体细胞胚 再生体系 遗传转化 

分 类 号：S567.239[农业科学—中草药栽培]