丛枝菌根真菌与胡萝卜毛状根双重培养体系研究

以发根农杆菌（Agrobacterium rhizogenes）K599诱导胡萝卜产生毛状根,接种丛枝菌根（AM）真菌摩西斗管囊霉（Funneliformis mosseae）建立双重培养体系,研究不同消毒方法、超声波、低温预处理和培养基pH对摩西斗管囊霉孢子萌发的影响及其与胡萝卜毛状根建立双重培养体系的最佳条件。研究结果表明：消毒方法3孢子萌发率最高,孢子污染率最低,15 d孢子萌发率达48.88%,而污染率仅为9.98%;超声波处理能降低孢子污染率11.17%~14.53%;低温预处理可有效提高孢子的萌发率,4℃低温处理10 d和15 d效果较好,其萌发率分别为70%和65%;AM真菌孢子在水琼脂培养基萌发最佳pH为6.5,萌发率达到48.50%,pH< 5.5或pH>8.0均抑制孢子萌发,pH 5.5和pH 8.0的萌发率分别为15.43%和16.06%。消毒孢子先于水琼脂萌发后,再挑取萌发管多,菌丝较长的孢子并将菌丝生长方向正对毛状根方向进行转接,可以提高双重培养的成功率。MSR培养基为双重培养的最优培养基。胡萝卜毛状根与AM真菌双重培养可为菌根真菌繁殖及相关分子机理研究提供可行有效的途径。

A Study to Develop an in Vitro Dual Culture System with Carrot Hairy Roots and Arbuscular Mycorrhizal Fungi

Agrobacterium rhizogenes K599 was used to induce carrot hair roots. An in vitro dual culturing system with Funneliformis mosseae and carrot hairy roots was then developed, and factors including methods to obtain axenic, supersonic processing, chilling treatment and medium pH that might affect both AM fungal spores germination and dual culturing were optimized. It was found that the third axenic method resulted in the highest germination rate (48.88%) and the lowest contamination rate (9.98%) at 15 days. Supersonic processing helped reduce AM fungal spores’ contamination by 11.17% to 14.53%, while a chilling pretreatment at 4℃ for 10 days and 15 days increased the germination rate of AM fungal spores to 70% and 65%, respectively. In water agar, the highest germination rate was achieved at a pH of 6.5 (48.50%), whereas spore germination was inhibited when pH was lower than 5.5 or higher than 8.0, with germination rate decreased to 15.43% at pH 5.5 and 16.06% at pH 8.0. Furthermore, it was found that the success rate of dual culturing was enhanced when sterilized spores were first germinated on agar medium and long hyphae were then selected for inoculation with their extension direction towards the growth direction of the carrot root. The MSR medium was found to be optimal for dual culture. The successful in vitro dual culture of carrot hairy roots and arbuscula mycorrhizal fungi provides an ideal experimental system to obtain pure AM fungal spores and to conduct future studies on the physiological and molecular mechanism of carrot mycorrhiza.