桑树原生质体分离及高效计数方法的研究  

Study on Isolation and Efficient Counting Method of Mulberry Protoplasts

作　　者：惠甜 李芳红[2] 韩淑华 张芮 梁嘉俊 吴超男 徐柯 焦锋[1] Hui Tian;Li Fanghong;Han Shuhua;Zhang Rui;Liang Jiajun;Wu Chaonan;Xu Ke;Jiao Feng(Sericulture and Silk Research Institute,Northwest A&F University,Yangling Shaanxi 712100,China;College of Animal Science and Technology,Northwest A&F University,Yangling Shaanxi 712100,China)

机构地区：[1]西北农林科技大学蚕桑丝绸研究所,陕西杨凌712100 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《蚕业科学》2023年第5期453-459,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(32372950);国家重点研发计划项目(2019YFD1000600)。

摘　　要：为探索桑叶原生质体制备和精确计数的方法,以桑树新一之濑多倍体系列的组培自生根苗为材料,通过优化酶解液配方,利用流式细胞仪进行叶片原生质体计数试验。结果表明,酶解液为5%纤维素酶-R10+5%果胶酶+20 mmol/L KCl+20 mmol/L MES+0.4 mol/L甘露醇+10 mmol/L CaCl2+5 mmol/L β-巯基乙醇+0.1%BSA(pH 5.7)时,取28.26 mm2叶片组织,在5 mL酶解液中黑暗静置3 h,耗时较短,酶解较为充分。将酶解液稀释100倍后过滤上机,可以准确高效地检测细胞数;使用移液器量取剩余滤液体积,可以准确计算出上机吸取的滤液体积;由此,可以较为准确快速地分析制备滤液中的原生质体数目。研究结果优化了解离桑树多倍体得到原生质体的条件,为快速检测原生质体数目提供了技术支持。In order to explore the method of preparation and accurate counting of mulberry leaf protoplasts,this study used tissue⁃culture self⁃rooting seedlings of mulberry Shin⁃Ichinose polyploid series as materials,optimized the enzymatic hydrolysis formula and used flow cytometry to conduct leaf protoplast counting experiments.The results showed that the enzymolysis was sufficient and took less time,when the enzymatic hydrolysis solution was 5%cellulase⁃R10+5%pecti⁃nase+20 mmol/L KCl+20 mmol/L MES+0.4 mol/L mannitol+10 mmol/L CaCl2+5 mmol/Lβ⁃mercaptoethanol+0.1%BSA(pH 5.7),and 28.26 mm2 of leaf tissue was taken and placed in the dark for 3 h in 5 mL of the enzymatic solution.Dilu⁃ting the enzymatic solution by 100 times and then filtering it on the machine,the number of cells could be detected effi⁃ciently and accurately.Using a pipette to measure the volume of the remaining filtrate,the volume of filtrate absorbed by the machine could be accurately calculated.Thus,the number of protoplasts in the prepared filtrate could be analyzed relatively quickly and accurately.The results of the study optimized the conditions for obtaining protoplasts from polyploids of mulberry trees,and provided technical support for the rapid detection of the number of protoplasts.

关 键 词：桑树 原生质体 流式细胞仪 高效计数 

分 类 号：S888.2[农业科学—特种经济动物饲养]