| AM fungi can establish symbionts with 80% of terrestrial plants on the earth,promote the absorption and utilization of a large number of trace elements,improve the stress resistance of plants,affect the absorption of heavy metals by plants,and improve the soil structure.AM fungi have broad prospects in agricultural production and ecology,and have great application value.Because AM fungi have the characteristics of specific symbiosis,it is impossible to achieve pure culture in vitro.Through the following research: the acquisition and propagation of tobacco aseptic seedlings;the induction,detection and optimization of hairy roots of tobacco,screening the best combination of Agrobacterium rhizogenes and tobacco varieties;the disinfection,germination,morphological and molecular biological identification of spores,screening better disinfection treatment;establishment of dual culture system,establishment,detection and optimization of dual culture system.The following results were obtained:1.75% alcohol and sodium hypochlorite were used to disinfect tobacco seeds for 1 min and 15 min respectively.The pollution rate was less than 15%,which was a good disinfection treatment.Nine combinations of 6-BA and NAA with different concentrations were used to induce callus and adventitious bud of K326.The optimum medium was MS + 1.0mg/l 6-BA + 0.1mg/l NAA.2.Hairy roots were induced from the leaves of NC82 and K326 tobacco sterile seedlings infected with A4 and C58C1,and the induction rates were different.For the same Agrobacterium rhizogenes,the induction rate of A4 to K326(28.89%)was higher than that of NC82(22.22%),while that of C58C1 to NC82(67.78%)was significantly higher than that of K326(34.44%);for the same tobacco variety,the induction rate of C58C1 to two tobacco varieties was higher than that of A4.Agrobacterium rhizogenes and tobacco varieties(C58C1 and NC82)were screened out to obtain hairy roots rapidly.The morphological and molecular biological detection of hairy roots further proved that the induced four hairy roots were Ri T-DNA transformation roots.3.The spores of Gigaspora isolated from soil were identified as: Gigaspora gigantea,Gigaspora margarita.With the increase of disinfection time,the spore contamination rate decreased,and the spore germination rate increased first and then decreased.Among them,2% chloramine T + 2 drops of 0.25% Tween 20 for 12 min,200 mg / L streptomycin + 100 mg / L gentamicin for 10 min,the spore germination rate was 42.22%,and the contamination rate was 13.33%,which was suitable for sterilization of Gigaspora margarita spores handle.The germinating spores were identified by gene detection and phylogenetic trees were established.It was further proved that the germinating spores were Gigaspora margarita.4.The double culture system of tobacco hairy root and Gigaspora margarita spore was established successfully,and the spore was produced.At the same time,it was observed in the experiment that after two weeks of co culture,the mycelium began to find the invasion point.It was found that the mycelium of the spores which had been inoculated for three days could find the hairy root quickly and invade the hairy root.The growth state and some special structures(such as auxiliary cells,fan-shaped hyphae,crisscross hyphae,D-shaped hyphae,open circular hyphae,etc.)were observed during the culture.The double culture system of AM fungi of trichomes and Gigaspora margarita in tobacco was established successfully.The optimum medium for callus induction and adventitious bud induction was selected,and a large number of sterile tobacco seedlings and hairy roots were obtained.The dual culture system of hairy roots and Gigaspora margarita was established,and the sporulation process was completed.The results provide a technical reference for the large-scale production and application of AM fungicides. |
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