大蒜抗紫斑病体细胞无性系变异筛选技术研究
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摘要
以苍山蒜和改良蒜品种为试验材料，通过在MS培养基中添加不同种类和浓度的植物生长调节剂优化大蒜愈伤组织诱导体系，研究大蒜紫斑病菌粗毒素对不同抗性品种大蒜幼苗生理特性和叶片解剖结构的影响，以紫斑病菌粗毒素为筛选压力，以2个品种蒜瓣芽基部为外植体，分别采用一步选择法和多步选择法筛选大蒜抗紫斑病变异系，观察筛选过程中病菌粗毒素对大蒜愈伤组织细胞学结构的影响，最终获得抗性小鳞茎，并对抗性植株的抗病性进行鉴定。主要结果如下：
1.以2个大蒜品种鳞芽基部为外植体，设不同植物激素配比，对大蒜愈伤组织的诱导体系进行优化。结果表明，2,4-D对愈伤组织诱导起着关键作用，但其浓度不宜高于2.0mg/L。2,4-D浓度1.0mg/L或2.0mg/L、6-BA浓度1.0mg/L时愈伤组织诱导率较高，2,4-D和6-BA浓度均为1.0mg/L为愈伤组织诱导的最佳激素配比。
2.采用离体大蒜叶片检测表明，供试紫斑病菌的粗毒素具有较强的致病能力。采用大葱种子发芽法鉴定表明，紫斑病菌粗毒素具有一定的热稳定性，但高温高压对病菌粗毒素的毒性也有一定影响。利用气质联用仪对大蒜紫斑病菌粗毒素成分进行分析，结果表明粗毒素主要成分为有机酸类。
3.大蒜紫斑病菌粗毒素处理不同抗性品种的大蒜幼苗后，抗病和感病品种叶片的SOD、POD和PAL活性呈上升趋势，叶片组织结构较处理前更加紧密，表明粗毒素胁迫可以诱导大蒜植株抗病性；抗病品种叶片SOD、POD和PAL活性峰值高于感病品种，而MDA含量低于感病品种。
4.通过愈伤组织诱导法确定了筛选抗病变异系的适宜病菌粗毒素浓度后，分别采用一步选择和多步选择两种方法进行抗病变异系筛选。一步选择法的大蒜愈伤组织在高浓度（30%）病菌粗毒素的胁迫下诱导并再生获得了抗性植株；多步选择法的外植体在10%、20%和30%的病菌粗毒素浓度逐级胁迫下筛选出抗性愈伤组织，然后在无毒培养基上继代4次分化成芽，获得了抗病小鳞茎。
5.种植抗性小鳞茎待其长至3片叶时采用粗毒素离体接种和活体喷施粗毒素进行抗性鉴定，在离体接种不同浓度粗毒素处理4d后，抗病品种在30%，40%粗毒素水平上没有发病；活体喷施粗毒素7d后对照植株发病，而抗病植株没有发病，说明抗病变异系具有一定抗病性。SRAP检测发现，抗性植株和对照植株具有差异性条带，认为抗性植株可能发生了分子水平的变异。
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.
1.以2个大蒜品种鳞芽基部为外植体，设不同植物激素配比，对大蒜愈伤组织的诱导体系进行优化。结果表明，2,4-D对愈伤组织诱导起着关键作用，但其浓度不宜高于2.0mg/L。2,4-D浓度1.0mg/L或2.0mg/L、6-BA浓度1.0mg/L时愈伤组织诱导率较高，2,4-D和6-BA浓度均为1.0mg/L为愈伤组织诱导的最佳激素配比。
2.采用离体大蒜叶片检测表明，供试紫斑病菌的粗毒素具有较强的致病能力。采用大葱种子发芽法鉴定表明，紫斑病菌粗毒素具有一定的热稳定性，但高温高压对病菌粗毒素的毒性也有一定影响。利用气质联用仪对大蒜紫斑病菌粗毒素成分进行分析，结果表明粗毒素主要成分为有机酸类。
3.大蒜紫斑病菌粗毒素处理不同抗性品种的大蒜幼苗后，抗病和感病品种叶片的SOD、POD和PAL活性呈上升趋势，叶片组织结构较处理前更加紧密，表明粗毒素胁迫可以诱导大蒜植株抗病性；抗病品种叶片SOD、POD和PAL活性峰值高于感病品种，而MDA含量低于感病品种。
4.通过愈伤组织诱导法确定了筛选抗病变异系的适宜病菌粗毒素浓度后，分别采用一步选择和多步选择两种方法进行抗病变异系筛选。一步选择法的大蒜愈伤组织在高浓度（30%）病菌粗毒素的胁迫下诱导并再生获得了抗性植株；多步选择法的外植体在10%、20%和30%的病菌粗毒素浓度逐级胁迫下筛选出抗性愈伤组织，然后在无毒培养基上继代4次分化成芽，获得了抗病小鳞茎。
5.种植抗性小鳞茎待其长至3片叶时采用粗毒素离体接种和活体喷施粗毒素进行抗性鉴定，在离体接种不同浓度粗毒素处理4d后，抗病品种在30%，40%粗毒素水平上没有发病；活体喷施粗毒素7d后对照植株发病，而抗病植株没有发病，说明抗病变异系具有一定抗病性。SRAP检测发现，抗性植株和对照植株具有差异性条带，认为抗性植株可能发生了分子水平的变异。
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.
引文
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