| Arbuscular mycorrhizal fungi（AM）can form mutualistic symbionts with plant roots,promote plant growth and development,improve mineral nutrition,and enhance stress resistance.It has a broad application prospect in agriculture and forestry.However,AM fungi are specific living nutrient microorganisms,and can not achieve in vitro pure culture.In this experiment,tobacco varieties NC82 and Va116 were used as host plants to obtain tobacco sterile seedlings,and Agrobacterium rhizogenes C58C1 and A4 were used to infect injured leaves to obtain tobacco Ri T-DNA transformation root（hairy root）was used to screen the best combination of Agrobacterium rhizogenes and tobacco varieties;the spores of several AM fungi were identified by morphology and molecular biology,and the spores were disinfected,germinated and screened for better disinfection treatment;the germinated spores of several AM fungi were co cultured with hairy roots of tobacco,and the symbiotic culture system was tested.The following results were obtained:1.NC82 and Va116 tobacco seeds were soaked in 75%ethanol for 60s,disinfected with 10%sodium hypochlorite for 15min,washed with sterile water for three times,inoculated in MS solid medium and cultured at 25℃at constant temperature,the germination rate was higher than 80%,and the pollution rate was less than 15%.2.When the OD600 values of Agrobacterium rhizogenes A4 and C58C1 were0.6-0.8,the infection rates of tobacco NC82 and Va116 were 23.33%and 21.43%respectively,and the infection rates of Agrobacterium rhizogenes C58C1 to NC82 and Va116 were 70.97%and 35.71%,respectively.3.The spores of glomus flavisporum,claroideoglomus etunicatum and acaulospora mellea were disinfected by three disinfection methods.The best disinfection methods for glomus flavisporum spores were as follows:1:2%（w/v）chloramine T+2 drops of 0.25%（v/v）Tween20（solution A）for 12 min,0.02%（w/v）streptomycin sulfate and 0.01%（w/v）gentamicin sulfate（solution B）for 15 min and method 3:differential sterilization（mixture of liquid A and liquid B）for 12 min;claroideoglomus The better disinfection treatment for etunicatum spores is method 1:solution A for 10 min,solution B for 15 min and method 3 for 10 min;method 3 for acaulospora mellea spores is method 3:disinfection for 10 min and method 1:solution A for 10 min and solution B for 12 min.The germination rate of these disinfection treatments is higher,and the pollution rate is less than or equal to 15%.4.The germination rate of glomus flavisporum,Claroideoglomus etunicatum and Acaulospora mellea were increased by adding exogenous additives（NaCl,SPM,GR24）in water agar medium.Compared with water agar,the germination rate of three kinds of spores in the medium added with NaCl increased by 11.69%,8.52%and 5.83%,respectively,and that of SPM in the medium increased by 15.59%,14.63%and 10.47%,respectively;The germination rate of GR24 increased by 6.51%,1.21%and 2.34%respectively.The addition of SPM to water agar medium significantly promoted the spore germination of glomus flavisporum and Claroideoglomus etunicatum,The germination of Glomus flavisporum,Claroideoglomus etunicatum and Acaulospora mellea only increased when NaCl and GR24 were added to water agar medium.5.After four months of symbiotic culture,hyphae invaded hairy roots,arbuscular structures（BAS）and a large number of hyphae;The presence of Arbuscular branches and vesicles was observed by alkali dissociation and acid fuchsin staining,and the infection rate was 17.3%.It indicated that the in vitro double culture system of Claroideoglomus etunicatum and hairy root of tobacco had been preliminarily established.In this experiment,the most suitable disinfection treatment for tobacco seeds was selected,a large number of tobacco sterile seedlings and hairy roots were obtained,and the double culture system of tobacco hairy roots and Claroideoglomus etunicatum was successfully established.The results provide technical reference for large-scale production and application of Claroideoglomus etunicatum. |
