兰州百合原生质体的分离纯化和应用  

Protoplast Isolation,Purification and Application of Lilium davidii var.unicolor

作　　者：韩炘 吴若楠 李心[1] 杨云尧 陈敏敏[1] 张永春[1] 杨柳燕[1] HAN Xin;WU Ruonan;LI Xin;YANG Yunyao;CHEN Minmin;ZHANG Yongchun;YANG Liuyan(Forestry&Fruit Tree Research Institute,Shanghai Academy of Agricultural Sciences,Shanghai 201403)

机构地区：[1]上海市农业科学院林木果树研究所,上海201403

出　　处：《植物研究》2024年第6期936-944,共9页Bulletin of Botanical Research

基　　金：上海市“科技创新行动计划”农业科技领域项目(22N11900300);上海市中央引导地方科技发展资金项目(YDZX20223100003004);上海市专业技术服务平台能力提升项目(21DZ2292300)。

摘　　要：该研究旨在建立兰州百合(Lilium davidii var.unicolor)原生质体分离纯化体系,以促进其基因功能研究和体细胞杂交育种。通过研究材料选择、渗透压调节剂浓度、酶添加量、酶解时间和纯化条件等关键因素,确定了酶解法提取兰州百合原生质体的最优参数。结果表明:继代30 d左右的兰州百合无菌苗叶片,在0.4 mol·L^(-1)的甘露醇环境下,使用含15 g·L^(-1)的纤维素酶和5 g·L^(-1)的离析酶的酶解液酶解3 h,即能分离完整均一的兰州百合叶肉原生质体,平均产量超过4×10^(6)个·g-1,且细胞活力超过90%。在0.4 g·mL^(-1)PEG-4000介导下转染35S::GFP质粒15 min,成功获得阳性转化子。同时,在0.5 g·mL^(-1)PEG-4000与5 mmol·L^(-1)CaCl_(2)(pH=10)作用下,原生质体发生融合,形成双核细胞。该体系的建立为兰州百合体细胞杂交育种提供了高质量的细胞材料,也为其基因功能研究搭建了平台。To promote gene function research and somatic hybrid breeding,a protoplast isolation and purification system for Lilium davidii var.unicolor was established,and the optimal parameters for enzymatic hydrolysis of protoplasts were determined by investigating the key factors such as material selection,osmotic regulator concentration,enzyme dosage,enzymatic hydrolysis time,and purification conditions.The results demonstrated that the homogeneous mesophyll protoplasts were isolated from leaves sub-cultured for about 30 d and digested with the enzyme solution containing 15 g·L^(-1) cellulase and 5 g·L^(-1) segregation enzyme for 3 h in the presence of 0.4 mol·L^(-1) mannitol,and the average yield was more than 4×10^(6) cells·g-1 with the cell viability exceeding 90%.Positive transformers were obtained successfully by 35S::GFP plasmids transfected with 0.4 g·mL^(-1) PEG-4000 for 15 min.At the same time,the protoplasts fused to form binucleated cells by 0.5 g·mL^(-1) PEG-4000 and 5 mmol·L^(-1) CaCl_(2),pH=10.This system provided high-quality cell materials for L.davidii var.unicolor protoplast fusion breeding and established a platform for gene function research.

关 键 词：兰州百合 原生质体 瞬时转化 化学融合 

分 类 号：Q813.1[生物学—生物工程]