钩藤再生体系优化与遗传转化条件探究  

Research on Optimization of Uncaria rhynchophylla Regeneration System and Genetic Transformation Conditions

作　　者：贾娜 王晓红[1] 李林 于晓松 刘智 张明生[1] JIA Na;WANG Xiaohong;LI Lin;YU Xiaosong;LIU Zhi;ZHANG Mingsheng(College of Life Sciences,Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院,山地植物资源保护与种质创新教育部重点实验室,贵阳550025

出　　处：《种子》2022年第4期36-43,共8页Seed

基　　金：国家重点研发计划课题(2016 YFC 0502604);贵州省科技计划重大专项课题(黔科合平台人才[2017]5411-06);国家喀斯特石漠化防治工程技术研究中心建设项目(2012 FU 125 X 13);贵州省高层次创新型人才培养计划项目(黔科合人才[2015]4031号);贵州省中药材现代产业技术体系建设项目(GZCYTX-02)。

摘　　要：为探究钩藤(Uncaria rhynchophylla(Miq.)Miq.ex Havil.)组织培养与遗传转化适宜条件,以钩藤种子萌发的无菌苗叶片为外植体,筛选、优化钩藤再生体系建立条件。通过设置不同预培养时间、农杆菌浸染浓度、浸染时间、共培养时间、共培养基中AS浓度、抗生素浓度等,探究钩藤遗传转化的适宜条件。结果表明,钩藤愈伤组织诱导的适宜培养基为:WPM+3 mg/L 2,4-D+2 mg/L 6-BA+0.5 mg/L NAA+30 mg/L蔗糖+避光培养;不定芽诱导培养基为:WPM+2 mg/L 6-BA+0.5 mg/L NAA;最适遗传转化因子组合为:预培养2 d,共培养基中添加AS的浓度为120μmol/L,OD_(600)=0.8,浸染时间10 min,共培养时间2 d。经PCR检测证明,GUS基因可顺利转入钩藤基因组中并作为其遗传转化的指示基因。In order to explore the suitable conditions for tissue culture and genetic transformation of Uncaria rhynchophylla and to screen the explants from sterile seedling leaves germinated from Uncaria rhynchophylla seeds,and to optimize the conditions for establishing a regeneration system of Uncaria rhynchophylla.By setting different pre-culture time,Agrobacterium infection concentration,diffusion time,co-cultivation time,AS concentration in co-culture medium,antibiotic concentration,etc.,the optimal conditions for genetic transformation of Uncaria rhynchophylla were explored.The results showed that the suitable medium for callus induction of Uncaria rhynchophylla was WPM+3.0 mg/L 2,4-D+2.0 mg/L 6-BA+0.5 mg/L NAA+30 g/L sucrose+Culture in dark;adventitious bud induction medium:WPM+2 mg/L 6-BA+0.5 mg/L NAA;optimal combination of genetic transformation factors:pre-culture for 2 d,and the concentration of AS added in the co-culture medium was 120μmol/L,OD_(600)=0.8,immersion time 10min,co-culture time 2 days.The PCR test proved that the GUS gene could be successfully transferred into the genome of Uncaria rhynchophylla and used as an indicator gene for its genetic transformation.

关 键 词：钩藤 愈伤组织 再生体系 遗传转化 

分 类 号：S567.239[农业科学—中草药栽培]