摘要
以转双抗虫基因741杨无菌苗叶片为材料,对叶片原生质体游离、纯化方法及影响因素等进行了分析研究。结果表明,上部叶片产量和活力均高于其他叶位,在实验的浓度范围之内,Macerozyme R-10(离析酶)、Cellulase Onozuka R-10(纤维素酶)、Driselase(Sigma)(崩溃酶)对原生质体的产量和活力无显著影响。适宜转双抗虫基因741杨叶片原生质体游离与纯化的条件为无菌苗上部叶片为材料,0.7mol/L甘露醇的CPW盐溶液预质壁分离1～1.5hCPW+0.5%Macerozyme R-10+0.25%CellulaseOnozuka R-10+0.025%Driselase(Sigma)+0.5mol/L甘露醇(pH 5.7),酶解温度27℃,酶解时间10h,用"过滤-离心-漂浮法"纯化原生质体。
Protoplasts of 741 Populus [Populus alba L. × （P. davidiana Dode＋P. simonii Carr. ） × P. tomentosa Carr. ] were isolated from leaves of sterile shoot. The different factors affecting the protoplast yield and viability were discussed with different methods of purl fication.. The results showed that there were no highly significant differences among MacerozymeR-10,Cellulase Onozuka R-10 and Driselase （Sigma） in the protoplasts yield（pH 5.7）. The best enzyme solution Filtrate-Centrifugal Float for purification contained aseptic seedling upper part leaf as materials, the CPW 0.7 mol/L mannitol salt solution pre Cytoplasm wall separation 1-1.5 h,CPW salts, 0.5% MacerozymeR-10,0.25%Cellulase Onozuka R-10, 0. 025% Driselase （Sigma） and 0.5 mol/L mannitol incubated at 27℃ for 10 hours.
出处
《河北林果研究》
2012年第4期358-362,共5页
Hebei Journal of Forestry and Orchard Research
基金
河北省自然科学基金(C2008000279)
国家高技术研究发展计划"863"计划(2011AA100201)
关键词
转双抗虫基因741杨
原生质体
游离
纯化
Populus alba L. × （P. davidiana Dode＋P. sirnonii Carr. ） ）〈 P. tomentosa Carr.
protoplast
isolation
purification
