摘要
以刺槐未成熟合子胚的愈伤组织为材料,试验通过正交试验设计,对酶液质量分数(纤维素酶R-10、果胶酶Y-23、离析酶R-10)、分离时间和分离液渗透压(即甘露醇质量分数)3个影响因素分析,并采用蔗糖等密度离心法中的上浮法纯化后,得到分离最佳条件:纤维素酶R-10质量分数为2.5%,果胶酶Y-23质量分数0.6%和离析酶R-10质量分数1%,甘露醇质量分数11%,酶解时间10 h。在此条件下,原生质体产量达2.24×106个/mL,活力为92.13%,原生质体净得率为2.06×106个/mL。在此基础上,采用PEG诱导法,对原生质体的融合时间和融合剂PEG6000质量分数等相关影响因素进行了相关探讨,结果表明最佳融合条件为25%的PEG6000诱导下,融合30 min,融合率达到10.97%。
With the immature zygotic embryos callus material of Robinia pseudoacacia L.,we studied the protoplast isolation and protoplast fusion with PEG. The main three factors were the enzyme concentration,dissociation time and osmotic pressure（ the enzyme concentration includes the concentrations of cellulase R-10,pectinase Y-23 and macerozyme R-10）. By sucrose isodensity centrifugation,the best conditions to get amount protoplasts from the immature zygotic embryos callus material of Robinia pseudoacacia L. were 2.5% of cellulase R-10,0.6% enzyme of pectinase Y-23,1% of macerozyme R-10,11% of mannitol,and 10 h of hydrolysis time. Under these conditions,the yield of protoplasts was 2.24×106/mL FW,and the activity was 92.13%. Thus,the actual yield of protoplasts was 2.06×106/mL FW. In terms of fusion research,with the method of PEG6000-induced,the integration time and the concentration of PEG6000-related factors were discussed.The fusion rate was10.97% with 25% PEG6000-induced and the time lasting of 30 min.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2014年第10期28-33,37,共7页
Journal of Northeast Forestry University
基金
国家自然科学基金项目(31170629)
林业公益性行业科研专项(201104013)
关键词
刺槐
原生质体分离
PEG6000
原生质体融合
Black locust（Robinia pseudoacacia L.）
Protoplast isolation
PEG6000
Protoplast fusion
作者简介
王欢，女，1988年9月生，林木育种国家工程实验室（北京林业大学），硕士研究生。
通信作者：李云，林木育种国家工程实验室（北京林业大学），教授。E-mail：yunli63@163．com。
