Establishment Of Glomus Etunicatum Symbiotic System And Infection Of Polymines To Growth

More than80%of the plants on the earth form a mutually beneficial symbiotic with a class of soil fungi--arbuscular mycorrhizas fungi (AMF). These fungi have a series of important physiological and ecological function, such as promoting nutrient uptake and improving resistance of plants. So it can be used as biological protectants, accelerant, bio-fertilizer, bio-pesticides and so on. But it is a kind of the living fungal microorganisms. This study induced carrot hairy roots according to Agrobacterium rhizogenes mediated genetic transformation. Mycorrhiza symbiotic system of Glomus etunicatum (G.e) and carrot hairy roots was established using the roots as host. The effects of polyamine on G.e spores cultrued separately in vitro and G.e and roots cultured in symbiotic system were also studied. The main results are as follows:Hairy roots from carrot which can grow rapidly on hormone-free medium were successfully induced using the three kinds of Agrobacterium tumsfeciens A4, R1600and LBA9402as test bacterium. These hairy roots were preliminarily identified as transgenetic roots by PCR technique. They can cover the entire plate after one-month cultrue and maintain the identities after subculture. Impacts of the strain type, medium type, the amount of bacterium, concentration of bacterium and time of co-culture on hairy roots transformation frequencies were studied and the optimal conditions for carrot hairy roots induction were screened:use Agrobacterium tumsfeciens A4for infection, water agar containing50mg/L Vitamin C (Vc),20u1for the amount of bacterium,0.6for OD600of bacterium and2days for co-culture time.Sterile G.e spores were inoculated on the side of fresh3-week-root, and the petri dish was incubated in the dark vertically for35d, then it was observed by fluorescence microscopemicroscope. Complex mycelium formed around the roots, and there were many dark brown daugther spores of G.e on the tip of large number of the hyphal. Subculture of symbiotic system can be easily achieved by inoculating0.5cm3medium containing new G.e spores, mycelium and infected root segments on fresh carrot hairy roots.The effects of three kinds of polyamine including putrescine, spermidine and spermine on G.e spore germination, hyphal length and hyphal branching response at different concentration levels:40、80、120、160mg/L were researched. The results reflected that SPD and SPM increased spore germination. Three kinds of PA all promoted hyphal elongation and branching, and extended the time of hyphal branching. SPM had the most significant effect, SPD secondly, and PUT worstly. Hyphal branching of G.e treated by160mg/L SPM was up to3.75times of that of control. Hyphal length and branching increased when the concentration of PA increased.The effects of spermine with concentrations of0,80,120mg/L on G.e spores and carrot hairy roots during the process of dual monoaxenic culture were determined. Mycorrhizal clonization, infection intensity, biomass of carrot and spore production were checked on day10th,15th,20th,25th,30th and day35th in the co-cultrue process. The results showed that mycorrhizal clonization, infection intensity and biomass of carrot of SPM treatments in the early period were significantly larger than control, and the number of daugther spores formed and mutured earlier. Consequently, SPM did favor to infection of G.e hyphal to host in the early period of dual monoaxenic culture and accelerated the maturity of spores.