【作者】 吴金平；

【导师】 侯明生； 顾玉成；

【作者基本信息】 华中农业大学 ， 植物病理学， 2005， 硕士

【摘要】 魔芋软腐病是由胡萝卜软腐欧氏杆菌胡萝卜软腐亚种(Erwinia carotovora sub sp.carotovora)引起的重要病害,此病一旦发生对魔芋生产造成严重威胁,且难以根除。由于魔芋种质资源贫乏,生产上可利用的抗病品种相对少,目前关于本病的防治,主要是通过农业技术措施和化学药剂控制,但因本病侵染来源和传播途径较广,现有的技术措施往往难以达到理想的防病效果。 随着植物离体培养技术和突变体鉴定方法的改进,利用此项技术方法筛选抗病突变体已成为植物细胞工程的重要研究领域。本研究试图利用离体培养技术筛选魔芋软腐病抗病突变体,进而获得人工组培再生植株,旨在为生产实践应用奠定基础。 本课题以魔芋顶芽、顶芽鳞片、侧芽、皮上芽苞、拟块茎鳞片为外植体,不同外植体在各自最适培养基上诱导。结果表明:拟块茎鳞片形成愈伤组织平均诱导率高达90.6%,形成的愈伤组织在分化培养基上进行不定芽诱导,魔芋拟块茎鳞片不定芽的诱导率为96.4%,繁殖系数为4.9;而其它外植体最高的不定芽诱导率为74.2%,繁殖系数为4.1。由此可见魔芋拟块茎鳞片是最佳诱导愈伤组织的外植体。 在抗病突变体的筛选过程中,本研究用3种不同的方法处理愈伤组织:(1)以不同浓度的甲基磺酸乙酯(EMS)对魔芋不同分化程度的愈伤组织进行处理。离体筛选结果表明,用浓度为0.4%的EMS处理预培养4d的材料,平均存活率为47.2%。因此,以浓度为0.4%的EMS处理预培养4d的一批愈伤组织,将处理后存活的材料扩繁,同时与未经EMS处理的材料分别用针刺法接菌,进行魔芋抗软腐病突变体的筛选;将接菌筛选存活的愈伤组织分化,获得了19株再生植株。(2)利用细菌滤液筛选魔芋软腐病抗病突变体。将愈伤组织接种在含60%的魔芋软腐病菌滤液培养基上培养1个月,转接到普通培养基上缓和培养1个月,如此反复转接8次后,存活的愈伤组织接菌,然后接到分化培养基上,获得5株再生植株。(3)直接接菌法筛选魔芋抗软腐病突变体。配制9个不同浓度的魔芋软腐菌菌悬液直接接种愈伤组织,随着时间的推移,发现愈伤组织都被迅速大量繁殖的菌体和其分泌物覆盖,接菌浓度对所处理的愈伤组织侵染之间没有多大区别。本试验接种的愈伤组织在3周后全部死亡。 对普通继代植株和普通继代过程中表型发生变异的植株,EMS诱变后接菌筛选获得的植株和细菌滤液处理获得的植株进行过氧化物酶(POD)同工酶、超氧化物歧化酶(SOD)同工酶、多酚氧化酶(PPO)同工酶的酶谱比较分析。 POD同工酶研究结果表明:细菌滤液处理和EMS诱变后接菌筛选获得的植株表现特有的3条酶带,分别是Rf0.14、Rf0.18、Rf0.23,而用EMS诱变后接菌筛选的植株更多还原

【Abstract】 Konjaku’s soft rot disease is an important disease caused by Erwinia carotovora sub sp. Carotovora. Its occurrence would make a disaster to the product of konjaku, and it’s difficult to eradicate. For the shortage of konjaku germplasm resources, the utilizable variety of resistance is relatively few. At present, to control this disease is mostly through the control of agronomic techniques and chemical treatments. But because of the various infection sources and transmission routes, the present techniques always can’t catch the ideal control effect.With the improvement in the plant vitro culture techniques and mutants identification methods, the application of those techniques above to select resistance mutants had become a important study area of plant cell engineering. In this paper, the resistance mutant of konjaku’s soft rot disease was selected by vitro culture technique, then the manual plant regenerations were gained to establish a base for the application in product.In this research, top buds, bud scales, lateral bud, budlets and scales of artificial corm of Amorphophallus are taken as explants to induce on their respectively suitable culture media. The results demonstrated that the induction rate of scales of artificial corm of Amorphophallus is 90.6% on average, the advantitions bud induction of the formed calluses were taken on the differential media. The induction rate of advantitions buds on the scales of artificial corm is 96.4%, and the reproduction in crenses is 4.9. However, the highest induction rate of other explants is 74.2%, and the reproduction in crenses is just 4.1. Therefore scales of artificial corm of Amorphophallus are the best explants material to induce.At the screening of resistance mutants, the callus were treated with three different method as follows: firstly, Amorphophallus callus of different phases were treated with different concentrations of EMS, the in vitro results showed that: the 4-day preincubation callus were treated with 0.4% EMS, and the average survival frequencies are 47.2%. The survival callus was reproduced, then mutants resistant against Amorphophallus soft rot disease were screened using needle point inoculation method from the callus with EMS treatment and without EMS treatment. In the end, the survival calluses after the inoculation selection were dealt with differentiation, and 19 regeneration plants were obtained. Secondly, bacterium filtrates were used to screen the resistant mutants. The callus were inoculated on the culture mediums which contained 60% the Amorphophallus soft rot disease pathogen filtrates for 1 month, then they were inoculated to the ordinary culture mediums for the 1-month relaxative culture. After 8-time repeats, the survivalcalluses were treated with filed inoculation and identification, 5 regenerated plants were obtained. Thirdly the direct inoculation was used to screen the resistant mutants. The calluses were directly inoculated with the different concentration suspension liquid of Erwinia carotovora sub sp. Carotovora. With time gone, the calluses were covered with a lot of quickly reproduced thalli and its secretion. The inoculation concentration had no obvious effect on the infection of the treated callus. The inoculated callus of this experiment died entirely after 3 weeks.To the common subculture plants and the plants whose phenotype variated during the common subculture, the obtained plants which were selected after EMS induction and inoculation or treated by bacterial filtrates were carried on analysis of peroxidase (POD) zymogram, superoxide dismutase (SOD) zymogram and Polyphenol Oxidase (PPO) zymogram.Results of POD isozyme showed that, the plants treated by bacterial filtrates or selected after EMS induction and inoculation had 3 isozyme bands, with the Rf value of 0.14, 0.18 and 0.23 respectively. But the plants selected after EMS induction and inoculation had 1 special isozyme band with Rf 0.51. The special isozyme bands of the obtained plants with different screening methods maybe have something with the resistance to soft rot disease.Results of SOD isozyme showed that, the plants selected after EMS induction and inoculation had 1 specific isozyme band with Rf value of 0.40. The SOD isozyme bands’ Rf value of the 4 sampled plants were at the range from 0.53 to 0.65. So the mobility rates of every isozyme band were very close. It showed that the molecular weights of every isozyme had no obvious difference.Results of PPO isozyme showed that, the new special isozyme bands of the plants treated by bacterial filtrates or selected after EMS induction and inoculation had Rf values between 0.36 and 0.61. The mobility rate of this band area had a big range. It might be the result of the changed molecular weights of the PPO isozyme from the plants, which obtained from the resistance screening.更多还原