一种农杆菌介导的糜子高效遗传转化方法  

An Efficient Genetic Transformation Method for Broomcorn MilletMediated by Agrobacterium tumefaciens

作　　者：夏启玉[1] 降彦苗 刘亚男 李海权 程汝宏 郭安平[1] 刘国庆 赵辉[1] XIA Qiyu;XIANG Yanmiao;LIU Yanan;LI Haiquan;CHENG Ruhong;GUO Anping;LIU Guoqing;ZHAO Hui(Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences/Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions,Sanya,Hainan 572024,China;Institute of Millet Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang,Hebei 050035,China)

机构地区：[1]中国热带农业科学院三亚研究院/中国热带农业科学院热带生物技术研究所/海南省南繁生物安全与分子育种重点实验室,海南三亚572024 [2]河北农林科学院谷子研究所,河北石家庄050035

出　　处：《热带作物学报》2025年第1期44-50,共7页Chinese Journal of Tropical Crops

基　　金：三亚崖州湾科技城科技专项(No.SCKJ-JYRC-2022-90);中央引导地方科技发展资金项目(No.226Z6301G);国家自然科学基金项目(No.32241043)。

摘　　要：糜子(Panicum miliaceum L.)是我国传统的粮食作物,截至目前,针对糜子遗传转化方法的研究仍较少,本研究旨在建立农杆菌介导的糜子高效遗传转化方法。以糜子品种冀黍5号的成熟种子为外植体,在CIM和MSD诱导培养基上分别诱导糜子的胚性愈伤,以其作为转化受体材料,用含植物表达载体的农杆菌侵染60min并共培养6d,转接至含0.025g/L潮霉素的筛选培养基上筛选出抗性愈伤,接着在含0.015g/L潮霉素的分化培养基上分化,最后在含0.015g/L潮霉素的生根培养基上生根成苗,用载体特异性引物对再生植株进行PCR检测,鉴定其是否为阳性转基因植株。根据多个批次的转化实验统计糜子的抗性再生植株的筛选效率和转化效率。结果表明:CIM和MSD诱导培养基均能诱导出糜子胚性愈伤,但CIM培养基诱导的糜子胚性愈伤效果更好;糜子胚性愈伤在被农杆菌侵染及与农杆菌共培养后,经在含潮霉素的培养基上筛选、分化和生根后获得多株抗性再生糜子植株,3次试验获得的糜子抗性再生植株的PCR鉴定阳性率为100%,平均转化效率为30%以上。本研究成功建立了农杆菌介导的糜子遗传转化方法,操作简单,转化效率高,成本低廉,且转化不受季节限制,能规模化开展,为糜子的遗传改良提供有效的技术手段。Broomcorn millet(Panicum miliaceum L.)is a traditional grain crop in China.Currently,there is still limited research on the genetic transformation methods for P.miliaceum.This study aimed to establish an efficient genetic transformation method for P.miliaceum mediated by Agrobacterium.Mature seeds of the millet variety Jishu 5 were used as the explants to induce embryogenic callus of millet on CIM and MSD induction media,respectively.They were used as the transformation receptor materials and infected with Agrobacterium containing plant expression vectors for 60 minutes and co-cultured for 6 days.The calli were then transferred to a screening medium containing 0.025 g/L of hygromycin to screen for resistant calli.Then,they differentiated on a differentiation medium containing 0.015 g/L of hygromycin.Finally,they rooted into seedlings on a rooting medium containing 0.015 g/L of hygromycin.PCR detec-tion was performed on the regenerated plants using vector specific primers to identify whether they were positive transgenic plants.Based on the multiple batches of transformation experiments,the screening efficiency and transfor-mation efficiency of resistant regenerated plants of broomcorn millet were statistically analyzed.The results showed that both CIM and MSD media could induce embryogenic callus of broomcorn millet,but CIM medium had a better effect on inducing embryogenic callus of broomcorn millet.After being infected by Agrobacterium and co-cultured with Agrobacterium,the multiple resistant regenerated plants were obtained through screening,differentiation,and rooting on a medium containing hygromycin.The PCR identification positive rate of the resistant regenerated plants obtained from three experiments was 100%,and the average transformation efficiency was over 30%.This study successfully estab-lished an Agrobacterium mediated genetic transformation method for broomcorn millet,which is simple to operate,ef-ficient,cost-effective,and not limited by seasons.It could be carried out on a large scale,providing a

关 键 词：糜子 农杆菌 遗传转化 转化效率 

分 类 号：S516[农业科学—作物学]