石豆兰无菌材料的建立及离体培养初探

Establishment of Aseptic Material in Vitro and Preliminary Study on Propagation Culture in Bulbophyllum sp.

目的:获取大量石豆兰无菌材料为后期细胞工程和基因工程等相关研究奠定基础。方法:选用假鳞茎和嫩芽为外植体,采用0.1%HgCl 2结合超声表面消毒,培养基中添加抗菌素抑制内生菌,继代时逐步降低抗菌素浓度最终获得无菌材料;初步研究激素配比对鳞芽增殖的影响。结果:HgCl 2结合超声处理能降低外部菌的污染率,提高成活率,10 min时效果最好。75 mg/L制菌霉素和50 mg/L萘啶酮酸抑制内生菌效果最好,90 d统计污染率为20%,无菌材料成活率为60%。6-BA和NAA对于芽分化影响显著,增殖倍数随6-BA浓度升高先增大后降低,2水平即浓度0.4 mg/L时显著高于其它两个水平;随着NAA浓度升高,增殖倍数逐步增大,3水平即浓度为0.2 mg/L时最大且显著高于其它两个水平。鳞芽在最佳激素配比6-BA 0.60 mg/L和NAA 0.2 mg/L条件下经过60 d培养,增殖了2.90倍。结论:通过化学和物理方法并结合抗菌素处理有效抑制了石豆兰内外菌物,优化了增殖的激素配比,获得了大量生长良好的无菌材料。

Objective:A large number of aseptic materials of Bulbophyllum sp.were obtained which laid a foundation for the later research of cell engineering and gene engineering.Methods:Pseudobulb and buds used as explants,0.1%HgCl 2 combined with ultrasonic treatment were selected for surface disinfectionto,and antibiotics were added to culture medium to inhibit endophyte.Aseptic materials were finally established by stepwise reduction concentration of the antibiotics during subculture.And the effects of hormone ratio on the proliferation of squamous buds were preliminarily studied.Results:HgCl 2 combined with ultrasonic treatment could reduce the contamination rate and improve the survival rate of explants,and the best treatment time was 10 minutes.The inhibitory effect of 75 mg/L nystatin and 50 mg/L nalidinone acid was the best,and the contamination rate and survival rate of aseptic materials for 90 days culture were 20%and 60%,respectively.The effects of 6-BA and NAA on the bud differentiation were significant.Proliferation multiple increased first and then decreased with the increase of 6-BA concentration,and the proliferation multiple of level 2 i.e.0.4 mg/L was significantly higher than the other two levels.With the increase of NAA concentration,proliferation increased gradually,and the proliferation multiple of level 3 i.e.0.2 mg/L reached the maximum value,which was significantly higher than the other two levels.Under the optimum hormone ratio of 6-BA 0.60 mg/L and NAA 0.2 mg/L for 60 days,the bulblets proliferated 2.90 times.Conclusion:Exogenic and endophytic microbes were effectively inhibited through chemical and physical methods combined with antibiotic treatments,and the hormones proportions for proliferation was optimized.Thus a large number of stetile seedlings of Bulbophyllum sp.were obtained.