三倍体‘银中杨’叶肉原生质体制备的优化  

Preparation and Optimization of Mesophyll Protoplasts of Populus alba×P.berolinensis Yinzhong

作　　者：宋少宇[1] 张俊琦[2] 王君[1] 

机构地区：[1]北京林业大学林木育种国家工程实验室,林木花卉遗传育种教育部重点实验室,生物科学与技术学院,北京100083 [2]北京林业大学公共分析测试中心,北京100083

出　　处：《西北植物学报》2015年第9期1899-1905,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31470662);高等学校全国优秀博士学位论文作者专项资金(201267)

摘　　要：以三倍体杨树品种‘银中杨’(Populus alba×P.berolinensis Yinzhong)无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明:酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3%Cellulase RS+0.5%Macerozyme R-10+0.3%Pectinse Y-23+0.6mol/L甘露醇+0.6g/L MES+1g/L BAS,酶解时间为8h,原生质体产量和活力分别为2.13×107个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高(1.06×107个/g),可满足进一步的原生质体培养等技术的要求。The technique for mesophyll protoplasts isolation and purification of a triploid poplar variety,Populus alba×P.berolinensis Yinzhong,was studied,for further genetic improvement through cell fusion and transgenic engineering.The leaves of aseptic seedling were used for mesophyll protoplast isolation and purification.The results showed that the yield and viability of the protoplasts were significantly influenced by the concentration of enzyme mixture,osmotic pressure and incubation time.Under the optimum condition for mesophyll protoplast isolation,CPW＋3% Cellulase RS＋0.5% Macerozyme R-10＋0.3% Pectinse Y-23＋0.6mol/L mannitol＋0.6g/L MES＋1g/L BAS with 8hincubation in dark,the protoplast yield could reach up to 2.13×10^7 per gram of leaves and the viability could be 80.18%.The best purification method was rising with sucrose density centrifugation.The yields were up to 1.06×10^7 protoplasts when purified with sucrose at the concentration of 40%.The protocol could meet the demands of protoplast culture-based techniques.

关 键 词：'银中杨’ 原生质体 分离 纯化 

分 类 号：Q242[生物学—细胞生物学] Q813.2