半夏悬浮培养细胞分化形成人工种胚的同步化条件  

The Synchronization Conditions of Artificial Embryos Differentiated by Suspension Cells of Pinellia ternata

作　　者：李欢 刘智 刘剑东[1] 杭烨 张明生[1] Li Huan;Liu Zhi;Liu Jiandong;Hang Ye;Zhang Mingsheng(Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),College of Life Sciences,Guizhou University,Guiyang,550025)

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：《分子植物育种》2021年第7期2320-2329,共10页Molecular Plant Breeding

基　　金：贵州省科技支撑计划项目(黔科合支撑[2018]2797);贵州大学国家级重点培育项目(黔科合平台人才[2017]5788);贵州省农村产业革命中药材产业发展专项(黔财农[2019]175号)共同资助。

摘　　要：为获得同步化的人工种胚,以半夏疏松愈伤组织为试验材料,采用悬浮培养的方法,探讨温度、咖啡因、蔗糖浓度对半夏悬浮培养细胞分化形成人工种胚的同步化作用,为半夏人工种子制作提供同步化种胚。结果表明:以MS为基本培养基,添加1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH进行悬浮培养,温度为8℃,处理12h,恢复36h,同步化效果最好,细胞分裂指数为13.86%;咖啡因浓度为2.00 mmol/L,处理2 h,恢复48 h,效果较好,细胞分裂指数为9.42%;蔗糖浓度为40 g/L,处理5 d,恢复4 d,效果一般,细胞分裂指数为5.33%。分别先后继代3次于MS+1.0 mg/L IBA+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH进行膨大培养及MS+0.1 mg/L IBA+0.5 mg/L 6-BA+10 g/L sucrose+300 mg/L CH进行分化培养。温度处理组微细胞团为42.33个,种胚分化率为71.11%;咖啡因处理组微细胞团为34.33个,种胚分化率为55.56%;蔗糖处理组微细胞团为40个,种胚分化率为57.78%。因此,半夏悬浮培养细胞同步化形成人工种胚的适宜条件为:MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH,8℃处理12 h,恢复36 h,再转接进膨大及分化培养基继续培养。本研究为半夏人工种子生产提供技术参考。To obtain synchronized artificial embryos, the effect of temperature, caffeine and sucrose concentration on the differentiation of cultured cells in Pinellia ternata was studied by suspension culture method, which can provide the seed embryo for the production of artificial seeds in Pinellia ternata. In this work, 1.0 mg/L 2,4-D+0.5 mg/L6-BA+30 g/L sucrose+300 mg/L CH was added as the basic medium for suspension culture. Interestingly, the synchronization effect was the best and then cell division index was 13.86% when the temperature was set at 8 ℃ for 12 hours and then recovered for 36 h. Meanwhile, the effect was the better and the cell division index was 9.42%when the cell was treated with 2.00 mmol/L caffeine for 2 h followed by recovering for 48 h. And the effect was moderate and the cell division index was 5.33% when the cell was treated with 40 g/L sucrose for 5 d and then re covered for 4 d. Three successive subcultures were successively cultured at MS+1.0 mg/L IBA+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH for expansion culture, and MS+0.1 mg/L IBA+0.5 mg/L 6-BA+10 g/L sucrose+300 mg/L CH for differentiation culture. At a result, in the temperature-treated group, there were 42.33 cell micromasses, and the embryo differentiation rate was 71.11%. There were 34.33 cell micromasses in the caffeine processing group,and the embryo differentiation rate was 55.56%;There were 40 cell micromasses in sucrose processing group, and the embryo differentiation rate was 57.78%. Therefore, the suitable conditions for suspension culture of cells in Pinellia ternata were: MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH, treated at 8 ℃ for 12 h,recovered for 36 h, and then transferred to the expansion and differentiation medium for further culture. This study provides some technical references for the artificial seed production of Pinellia ternata.

关 键 词：半夏(Pinellia ternata(Thunb.)Breit) 悬浮培养 细胞同步化分化 细胞分裂指数 人工种胚 

分 类 号：S567.239[农业科学—中草药栽培]