作物学报2018，Vol.44Issue(3)：463-470,8.DOI:10.3724/SP.J.1006.2018.00463
茶树叶片和胚根原生质体的分离及PEG诱导融合
Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant (Camellia sinensis L.O.Kuntze)
摘要
Abstract
Plant protoplasts are important materials for studies on plant cell culture,somatic cell fusion,genetics and breeding.This work established a stable and efficient method for getting a large amount of vital and intact protoplasts from young leaves and young roots of Fudingdabai tea plant.For protoplast isolation,the young leaves of tea seedlings grown in the condition of constant temperature (23℃) and dark or shading were the best materials,and the young radicle of tea plant was also the better,While the healthy young leaves of tea plants grown in tea plantation were used as materials,only a small quantity of viable protoplasts mixed with a large number of cell fragments were obtained.The optimal enzyme solution for protoplasts isolation from young leaves of tea seedlings contained 1.5％ cellulase + 0.1％ macerozyme + 0.5％ pectolyase + 0.4 mol L-1 mannitoi + 20 mmol L 1 MES,and that from radicle of tea seedlings contained 1.5％ cellulase + 0.3％ macerozyme + 0.5％ pectolyase + 0.4 mol L-1 mannitol + 20 mmol L-1 MES.Protoplasts with high yield and viability were purified when incubated in a shaker with low speed (55 r min1 and 50 r min-1 respectively) under constant temperature (23℃) for 7 h and 8 h,and then centrifuged at 15×g for 4 min.PEG-6000 was used to induce the fusion of protoplasts from young leaves and radicle of tea plants.The fusion time,the concentration of fusion agent PEG-6000 were discussed.The optimal protoplast infusion conditions were induction with 40％ PEG-6000 for 20 min,and the fusion rate was about 10％.关键词
茶树/幼嫩叶/幼嫩胚根/原生质体分离/原生质体融合Key words
Camellia sinensis/young leaf/young root/protoplast isolation/protoplast fusion引用本文复制引用
彭章,童华荣,梁国鲁,石艺琦,袁连玉..茶树叶片和胚根原生质体的分离及PEG诱导融合[J].作物学报,2018,44(3):463-470,8.基金项目
本研究由国家自然科学基金青年项目(31400583),国家级大学生创新创业训练计划项目(201610635041)和重庆市基础与前沿研究计划一般项目(cstc2014jcyjA80011)资助.This study was supported by the National Natural Science Foundation of China (31400583),National Training Program of Innovation and Entrepreneurship for Undergraduates (201610635041),and the Basic and Frontier Research Program of Chongqing (cstc2014jcyjA80011). （31400583）
