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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。
The young leaves of Dendrobium nobile Lindl. Seedlings were used as experimental materials. The viability of protoplasts was used as evaluation index. The orthogonal design was used to study the effects of cellulase concentration, pectinase concentration , The optimum conditions of the four key factors of isozyme concentration and enzymolysis time were selected. The results showed that when cellulase was 9.5 mg / mL, pectinase 5.0 mg / mL, isozyme 6.0 mg / mL, enzyme Under the combined conditions of 3.0 h solution time, the viability of protoplasts was the highest (94.42%). On this basis, protoplast fusion using high calcium and pH combined with PEG4000 induction method was used to study the protoplast fusion with high activity of Chishui and Ruili Dendrobium nobile. The fusion rate of heterokaryon was used as evaluation index, and response surface methodology Optimum design of the protoplast fusion process of PEG concentration, CaCl 2 concentration, pH and fusion time four factors for the best combination screening, found that when the concentration of PEG4000 38.41%, CaCl 2 concentration of 0.44 mol / L, pH 9.26, fusion time For the combination of 28.87 min, the highest rate of heterokaryon fusion was 0.924%. At the same time, response surface analysis also found that PEG concentration and CaCl2 concentration, PEG concentration and pH, CaCl2 concentration and fusion time together have a significant effect on the fusion rate of allo-nucleosomes. In addition, response surface analysis also found that when the pH was 9.26, the values of PEG concentration, CaCl 2 concentration and fusion time within a certain range did not significantly affect the rate of heterokaryon fusion, the values were maintained at about 0.94%. The research improved the technology system of protoplast preparation and fusion of Dendrobium nobile. It provided the experimental materials for the protection, expansion and new breed selection of Dendrobium nobile, and provided the experimental reference for the related research of other related species.