多花黑麦草种子外植体组织培养灭菌方法研究

Study on the Sterilization Methods in Tissue Culture of Italian Ryegrass Seed Explant

针对多花黑麦草组织培养中的污染问题,以多花黑麦草成熟种子为外植体,进行组织培养,将组培过程中的污染物进行纯化培养、鉴定,并对组织培养中降低污染的措施进行研究。结果显示:青霉菌、曲霉菌、木霉菌、毛霉菌、根霉菌、肠杆菌、假单孢菌是引起多花黑麦草组织培养污染的主要微生物;效果最好的外植体灭菌方法是用洗涤剂混合液浸泡种子6 h,然后75%乙醇表面灭菌2 min,再用0.1%次氯酸钠进行1 h浸泡灭菌;培养基中添加70mg/L制霉菌素,10 mg/L氨苄西林钠和0.1%次氯酸钠,可以很好地控制污染,并且2种方法对材料分化的影响都很小。

According to the pollution problems in tissue culture of Italian ryegrass seed explants, tissue culture was carried out by this study using the mature seeds of Italian ryegrass as explants. The pollutants from tissue culture were purified and cultured and the methods of decreasing pollution were studied. The results show that Penicilliurn , Aspergillus , Trichoderrna , Mucor , Rhizopus , Enterobacter , and Pseudornonas were the main microbes harassing the tissue culture of Italian ryegrass. The best sterilization method of explants was to immerse the seeds in soapsuds for 6 hours, and then surface sterilization for 2 minutes by alcohol （concentration 75%）, and at last soak in the sodium hypochlorite （0. 1%） for 1 hour. The results also indicate that adding 70 mg/L nystatin, 20 mg/L ampicillin sodium, and 0.1% sodium hypochlorite to the culture medium could control the pollution problem effectively. The two methods above had little influence on the differentiation of materials.