水曲柳原生质体分离初探  

Study on protoplast isolation of Fraxinus mandshurica Rupr.

作　　者：王姿童 宋悦悦 刘林 薛佳丽 詹亚光[1] 齐凤慧[1] WANG Zi-tong;SONG Yue-yue;LIU Lin;XUE Jia-li;ZHAN Ya-guang;QI Feng-hui(School of Life Sciences,Northeast Forestry University,Harbin 150040,China)

机构地区：[1]东北林业大学生命科学学院,黑龙江哈尔滨150040

出　　处：《生物技术》2021年第2期183-188,共6页Biotechnology

基　　金：黑龙江省应用技术研究与开发计划项目(GA19B201);黑龙江省大学生创新创业训练计划项目(201810225341);东北林业大学大学生创新项目(DC2020031)。

摘　　要：[目的]通过优化限制因素,探索水曲柳原生质体制备的最佳方法。[方法]采用酶解法,对影响原生质体分离的影响因素,设计正交实验,通过显微镜观察计数比较原生质体的分离效果。[结果]在M7和M9的酶液组合对原生质体分离效果最好,产量分别为6.61×105个/g和5.76×105个/g,活力分别为97.57%和97.07%。在11%的渗透压中原生质体产量最高。pH 5.2时产量最高,pH值增加不利于原生质体产量增加。酶解时间对原生质体产量差异显著,虽然14 h产量最高,但6 h、8 h、12 h、14 h之间产量差异不显著。[结论]采用酶解法初步获得了水曲柳原生质体分离的最适条件,在预处理3 h,渗透压为11%,在1.5%纤维素酶+0.8%离析酶+0.4%半纤维素酶中,pH值为5.2,黑暗条件下酶解14h。[Objective]Optimizing conditions for preparation of the protoplasts from the leaves of Fraxinus mandshurica.[Method]Orthogonal experiments were designed based on factor affecting the isolation of the protoplast in enzymatic degradation,and the isolated protoplasts were counted using a microscope.[Result]Among all enzymatic solutions,M7 and M9 got the best isolation results,with 6. 61 ×105 cells/g and 5. 76 ×105 cells/g in yields and 97. 57% and 97. 07% in protoplast activity,respectively. For osmotic pressure,11% treatment got the best yield,where M7 < M9. The significant highest yields were harvested at pH 5. 2,indicating higher pH is detrimental for isolation. The degrading duration was significantly affected the yields,and the highest yields were harvested at 14 hours,although the yields were not significantly different at 6 hours,8 hours,12 hours and 14 hours.[Conclusion]A optimum condition for protoplast isolation from F. mandshurica was presented: pretreatment for 3 hours,osmotic pressure 11%,1. 5% cellulase + 0. 8% macerozyme + 0. 4% hemicellulase degrading solution,pH 5. 2,degradation for 14 hours in dark.

关 键 词：曲柳 原生质体 细胞活力 酶液组合 

分 类 号：Q943.2[生物学—植物学] S792.11[农业科学—林木遗传育种]