柯氏无梗囊霉双重培养体系的构建

Dual Culture of Tobacco Va116 Hairy Roots and Acaulospora koskei

利用发根农杆菌(Agrobacterium rhizogenes)C58C1诱导烟草Va116产生毛状根,建立烟草Va116毛状根-柯氏无梗囊霉(Acaulospora koskei)双重培养体系;分析不同消毒方法、培养基pH、激素对柯氏无梗囊霉萌发的影响,筛选柯氏无梗囊霉与烟草Va116毛状根建立双重培养体系的最佳条件。结果表明:发根农杆菌C58C1菌液在600nm处的吸光值为0.6~0.8、侵染时间为8~10 min,通过抑菌、除菌培养,获得烟草Va116毛状根,诱导率为53.33%。柯氏无梗囊霉孢子在体积分数75%酒精中消毒1 min, A液(质量浓度20 g/L的氯胺(T)、每0.1 L加2滴吐温20)消毒10 min,转入B液(质量浓度200 mg/L的硫酸链霉素+质量浓度100 mg/L的硫酸庆大霉素)消毒10 min,萌发率最高(为48.33%)、污染率最低(为6.67%);柯氏无梗囊霉孢子在水琼脂培养基萌发,最佳pH为6.0;在独脚金内酯(GR24)质量浓度450 pg/L、精胺(SPM)质量浓度40 mg/L时,柯氏无梗囊霉孢子萌发效果最好,萌发率分别为61.67%、58.33%。成功建立了烟草Va116毛状根-柯氏无梗囊霉双重培养体系,产生了新生孢子,使柯氏无梗囊霉完成了整个生命周期,并记录了根外菌丝和根内菌丝的形态特征、泡囊结构的形成。

Tobacco Va116 was induced to produce hairy roots by Agrobacterium rhizogenes C58 C1, and a hairy root-Acaulospora koskei dual culture system of tobacco Va116 was established. The effects of different disinfection methods, medium pH and hormones on the germination of Acaulospora koskei were analyzed, and the optimal conditions for establishing a dual culture system between Acaulospora koskei and tobacco Va116 hairy roots were screened. The absorbance of Agrobacterium rhizogenes C58 C1 at 600 nm was 0.6-0.8, and the infection time was 8-10 min. Tobacco Va116 hairy roots were obtained through antibacterial and sterilizing culture, and the induction rate was 53.33%. The germination rate of Acaulospora koskei spores were sterilized in 75% alcohol for 1 min, solution A(20 g/L chloramine T+2 drops Tween 20 per 0.1 L) was sterilized for 10 min, and then transferred to solution B(200 mg/L Streptomycin Sulfate with 100 mg/L Gentamicin Sulfate Mixture) for 10 min was the highest(48.34%) and the contamination rate was the lowest(6.67%). The spores of Acaulospora koskei were germinated in water agar medium, the optimum pH was 6.0. When the mass concentration of strigolactone(GR24) was 450 pg/L and the mass concentration of spermine(SPM) was 40 mg/L, the spore germination effect of Acaulospora koskei was the best, and the germination rates were 61.67% and 58.34%, respectively. Tobacco Va116 hairy root-Acaulospora koskei dual culture system was successfully established, new spores were produced, and Acaulospora koskei completed the entire life cycle, and the morphological characteristics of the extraradical and intraradical mycelia and vesicle structure formation were recorded.