发根农杆菌K599对菊花活体转化及其高效再生  

Hairy Roots Induced by Agrobacterium rhizogenes K599 in Chrysanthemum in Vivo and Plant Regeneration from Hairy Roots

作　　者：向太和[1] 王琳[1] 蒋欢[1] 田璟鸾[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《园艺学报》2011年第7期1365-1370,共6页Acta Horticulturae Sinica

基　　金：浙江省自然科学基金项目(Y3080184);杭州市科技创新项目(20070232H07;20091133B07)

摘　　要：发根农杆菌K599侵染菊花无菌苗刻伤的叶片形成转基因不定根,生根频率为88.0%;不定根经过诱导培养形成愈伤组织,并再生完整植株,愈伤组织诱导率和分化率分别为75.0%和63.3%。诱导的不定根和再生植株经过PCR鉴定含有K599 Ri质粒中的rolC基因,qRT-PCR检测显示rolC基因在再生转基因植株中实现了正常表达。再生植株表现出矮缩、多毛状根特征,并能正常开花。建立的利用活体材料直接作为发根农杆菌K599侵染的受体诱导不定根的遗传转化体系,能克服假阳性不定根的出现;同时,利用不定根繁殖过程中顶端生长点区域无菌的特点,通过截取不定根顶端靠近生长点区域进行继代培养,结合使用头孢霉素杀菌,能达到有效抑制和杀灭农杆菌的目的,在随后的不定根诱导愈伤及分化过程中无须再使用抗生素杀菌,提高了转基因再生效率,为菊花的矮化育种和目标基因的转移提供了良好的试验体系。The transgenic hairy roots were formed on cut leaves of chrysanthemum plantlet by Agrobacterium rhizogenes K599 with frequencies of 88.0%.The calli were induced from hairy roots and plantlets were regenerated from calli at rates of 75.0% and 63.3% respectively.The transgenic hairy roots and plantlet were confirmed by PCR analysis with primers from K599 Ri plasmid rolC gene.Quantitative RT-PCR（qRT-PCR）analysis showed that rolC gene was normally expressed in the regenerated transgenic plants.Regenerated plants harbored a character of dwarf,more hairy roots and flowering normally.Organism in vivo directly as a receptor for infection by Agrobacterium rhizogenes K599,it overcame the emergence of false-positive transgenic hairy roots.In addition,using the process of hairy roots propagation characteristics of the top growing point region sterile and the interception of adventitious roots near the growing point to the top for subculture,combined with cefotaxime sterilization,it can inhibit and kill the Agrobacterium effectively.In the subsequent callus induction and redifferentiation from hairy roots,it is no antibiotics added to medium for killing Agrobacterium.It genetically enhanced efficiency of plant regeneration.The transgenic hairy roots were induced by Agrobacterium rhizogenes K599 in chrysanthemum in vivo and plant regenerated from hairy roots highly efficiently in the study.It is useful to dwarf breeding and the target gene transformation of chrysanthemum.

关 键 词：菊花 发根农杆菌 不定根 诱导 再生 

分 类 号：S682.11[农业科学—观赏园艺]