摘要
以金钗石斛(Dendrobium nobile Lindl.)再生苗的幼嫩叶片为实验材料,以原生质体活力率为评价指标,用正交试验设计对原生质体制备过程中的纤维素酶浓度、果胶酶浓度、离析酶浓度和酶解时间四个关键因素进行最佳组合筛选,发现当纤维素酶为9.5 mg/m L、果胶酶为5.0 mg/m L、离析酶为6.0 mg/m L、酶解时间为3.0 h的组合条件下,制备的原生质体活力率最高,为94.42%。在此基础上,采用高钙高pH值结合PEG4000诱导法进行原生质体融合研究,以高活力的赤水和瑞丽金钗石斛原生质体为材料,以异核体融合率为评价指标,用响应面法优化设计对原生质体融合过程中的PEG浓度、CaCl_2浓度、pH和融合时间四个因素进行最佳组合筛选,发现当PEG4000浓度为38.41%、CaCl_2浓度为0.44 mol/L、pH为9.26、融合时间为28.87 min的组合条件下,异核体融合率达到最高,为0.924%。同时,响应面法分析还发现,PEG浓度和CaCl_2浓度之间、PEG浓度和pH之间、CaCl_2浓度和融合时间两个因素共同对异核体融合率有显著影响。此外,响应面分析还发现,当pH为9.26时,PEG浓度、CaCl_2浓度和融合时间的值在一定范围内变化并不会显著影响异核体融合率的值,数值均维持在0.94%左右。该研究完善了金钗石斛原生质体制备和融合的技术体系,为金钗石斛种质资源的保护、拓展和新品种选育提供了实验材料,为其他近缘物种的相关研究提供了实验参考。
To construct the protocols of protoplast isolation and cell fusion in Dendrobium nobile, the tender leaves of tissue cultured seedling were used as the material. The survival rate was applied for evaluating the qual- ity of newly harvested protoplast, four significant factors of the cellulase concentration, pectinase concentration, macerozyme concentration and enzymolysis time were screened for their optimum levels during protoplast isolation through orthogonal experiment design. It found that the survival rate could reach the highest value of 94.42% with 9.5 mg/mL, 5.0 mg/mL, 6.0 mg/mL and 3.0 h, respectively. Then the PEG 4000 was applied for inducing the cell fusion with the method of high pH and high calcium. The heterokaryon fusion rate was selected as the assess- ment criteria, the main four factors of the PEG concentration, CaCI2 concentration, pH value and fusion time were screened for their optimum levels by using the experimental design of response surface method （RSM）. Through this method, the fusion rate could reach the highest value of 0.924% with four factors of 38.41%, 0.44 mol/L, 9.26 and 28.87 rain respectively. Meanwhile, the RSM detected that the synergistic effects reached to the significant lev- el of 0.05 between the PEG concentration and the CaCI2 concentration, PEG concentration and pH, CaCI2 concen- tration and fusion time. Furthermore, RSM also identified that the heterokaryon fusion rate was maintained around 0.94% when the pH value was 9.26 and the PEG concentration, CaCI2 concentration, fusion time were varied within narrow ranges. This study improved the protocols of protoplast isolation and cell fusion in D. nobile, which is meaningful for germplasms＇ protection and development, and it also supplied a solid bases in experimental material for breeding.
作者
徐德林
储士润
潘胤池
郑明辉
钱刚
陈芳
李林
Xu Delin Chu Shirun Pan Yinchi Zheng Minghui Qian Gang Chen Fang Li Lin(Department of Cell Biology, Zunyi Medical University, Zunyi 563000, China Department of Clinical Laboratory, The Second People ＇s Hospital of Jiangyou City, Mianyang 621701, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第8期935-944,共10页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:31560079)
贵州省教育厅2014年大学生创新创业训练计划项目(批准号:201410661007)
贵州省中药现代化科技产业研究开发专项(批准号:黔科合ZY字[2013]3002号)
遵义医学院招标项目(批准号:F-551
F-611)资助的课题~~
关键词
金钗石斛
原生质体制备
原生质体融合
正交试验法
响应面法
Dendrobium nobile Lindl.
protoplast isolation
cell fusion
orthogonal design method
response surface method
