| Cloves of garlic cv. Cang-shan and Gai-liang were used as plant materials to optimize thecallus induction system, according to the callus induction rate on MS medium containingdifferent type and concentration of plant growth regulators. Meantime, the effects ofAlternaria porri crude toxin on activities of protective enzymes and leaf anatomicalcharacteristics were studied at the seedling stage of both the resistant and the susceptiblecultivar of garlic. Then the clove base of garlic was used as explant to induce diseasetolerance somaclonal variation resistant to violet leaf spot under the stress of pathogen crudetoxin using both one-step and multi-step in vitro selection method and the resistant bulbs wereobtained at last. Finally, the cellular structure of the resistant callus was observed using thecytological technology and the disease resistant of the regeneration plants were identified.The results are as follows:1. In optimizing of the callus induction system, the results showed that2,4-D played akey role on callus induction, but the concentration should be less than2mg/L. The callusinduction rate was higher in the combination of1mg/L or2mg/L2,4-D and1mg/L6-BA.The study suggested that the optimal combination of plant growth regulators was1mg/L2,4-D+1mg/L6-BA.2. The pathogenicity experiment indicated that the crude toxin had relatively greaterpathogenicity. Meantime, the seeds of welsh onion were used to test the capacity of thermalstability of the pathogen crude toxin. The result suggested the crude toxin had certainresistance to high temperature and pressure. Compared to the filter sterilization, the welshonion seeds cultured in the crude toxin treated by high temperature and pressure had highergermination rate which implyed the high temperature and pressure had damaged the crudetoxin stability. The components of curde toxin was analysed by GC-MS and the resultssuggested that the major components of curde toxin were organic acids.3. The effects of Alternaria porri crude toxin on activities of protective enzymes, thecontent of malonaldehyde (MDA) and leaf anatomical characteristics of the resistant andsusceptible cultivar were studied at the seedling stage of garlic. The results showed that theSOD, POD, PAL activities appeared the increase dynamic changing trend after the pathogencrude toxin treatment and the leaf palisade tissue and spongy tissue became more compact in both cultivars after pathogen crude toxin treatment. This result indicated the crude toxin couldinduce the garlic plant disease resistance. The activities of SOD, POD and PAL in theresistant cultivar were higher than that in the susceptible cultivar at the peak and the MDAcontent in the resistant cultivar was lower than that in the susceptible cultivar.4. After making sure the optimum crude toxin concentration, the disease tolerancesomaclonal variation resistant to violet leaf spot was selected on MS media with differentlevel of pathogen culture crude toxin by use of one-step and multi-step in vitro selectionmethods. One step method of selection means that the regeneration plants were selecteddirectly from the calluses on MS media with30%crude toxin concentration. Multi-stepmethod means that the explants were used as the material to select the resistant bulbs underthe stress of10%,20%,30%of pathogen culture crude toxin step by step for subculture fourtimes. Compared to the control, the resistant callus had more tracheid which must be cellularstructure against the pathogen infect.5. When the resistant plantlets from bulblets grown to the stage of three leaves, the crudetoxin was inoculated to test the capacity of resistant disease in vitro and vivo method. Theresults indicated that the resistance plant leaf had certain tolerance to crude toxin. The SPASmolecular marker technique was also used to detect the variation of mutants. The resultsshowed that there were differential bands between the control and the resistant plants whichimplied that the plantlets with molecular variation were screened. |
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