【作者】 谭荣荣；

【导师】 洪霓； 王国平；

【作者基本信息】 华中农业大学 ， 植物病理学， 2006， 硕士

【摘要】 苹果褪绿叶斑病毒(Apple chlortotic leaf spot virus，ACLSV)、苹果茎沟病毒(Apple stem grooving virus，ASGV)和苹果茎痘病毒(Apple stem pitting virus，ASPV)是梨树上发生普遍的3种重要的潜隐病毒，在我国梨产区主要栽培品种上的带毒株率高达86.3％以上(王国平等，1994)。使用无病毒种苗是减轻这些病毒病危害的最有效措施(王国平等，2002)。为建立有效的病毒脱除技术，本研究以同时带有这3种病毒的砂梨离体植株为材料，比较分析了不同物理和化学处理技术与茎尖培养相结合的脱病毒效果，研究结果如下： 1．比较分析了3种不同热处理方法对‘丰水’梨离体植株生长的影响，结果表明，37℃恒温热处理对植株的生长影响最大，导致大部分植株死亡及繁殖系数的明显降低，并且对‘丰水’梨离体植株的生长产生明显的抑制作用；32℃／38℃变温处理对植株生长的影响较小，处理中后期的繁殖系数和株高与未处理植株接近或高于未处理植株；34℃／42℃处理对植株生长的影响介于前两种方法之间。 2．采用PAS-ELISA和斑点杂交技术对脱病毒处理后获得的梨再生植株带病毒状况进行了评价，结果显示，3种热处理方法对梨离体植株病毒含量的影响呈相同的趋势，即在处理时间相同时，所取茎尖愈小，再生植株病毒含量愈低；所取茎尖大小相同时，处理时间越愈长，再生植株病毒含量愈低。不同热处理方法的脱病毒效果存在一定的差异，37℃恒温热处理至35d后取1mm茎尖基本可以脱除ACLSV、ASGV和ASPV；32℃／38℃变温处理至50d后取1mm茎尖仍不能脱除这3种病毒；通过提高变温处理温度至34℃／42℃和切取较小的茎尖(0.5mm和1mm)，可以显著提高脱病毒效率，处理50d后取0.5mm茎尖即可完全脱除3种病毒。此外，发现在砂梨中ACLSV相对较易脱除，在同样条件下所需热处理周期较ASPV和ASGV可缩短5d左右。 3．采用所建立的34℃／42℃变温处理方法，对除‘丰水’梨外的另3个砂梨品种进行了处理，发现不同品种的热敏感性存在差异，但所有这些品种在该条件下处理50d-60d后，从存活植株上取0.5mm茎尖再培养获得了脱病毒率100％的效果。 4．比较分析了病毒醚不同工作浓度、处理时间及不同处理方法对‘丰水’梨离体植株生长和病毒脱除效果的影响，结果表明，在MS培养基中病毒醚含量低于25μg／ml时，或梨离体植株用含25μg／ml病毒醚的溶液浸泡1d-3d后继续在含病毒醚的MS培养基中生长时，仅出现轻微的药害，药害的程度随处理时间的延长而加重。对病毒醚处理后取1cm茎尖培养再生植株的病毒检测结果表明，在病毒醚浓度为50μg／ml时，可脱除以上3种病毒。在含25μg／ml病毒醚MS培养基中培养40d-50d后，耿1mm茎尖可脱除3种病毒。在含病毒醚的MS培养基中培养前用病毒醚浸泡更多还原

【Abstract】 Apple chlortotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) were the most popular viruses in pear plants. Previous surveys indicated that about 86.3% of pear trees in China were infected by these viruses(Wang et al, 1994). The most efficient measure for the controlling of these virus diseases is planting virus-free germplasm(Wang et al, 2002). In this study, thermotherapy, chemotherapy combined with merstem-tip culture were used for the virus elimination from in vitro Pyrus pyrifolia cv. "Fengshui" . Results were illustrated as following:1. The effect of three thermotherapy measures on the growth of in vitro Pyrus pyrifolia cv. "Fengshui" were compared. Results showed that treatment under 37℃ for 25 to 35 days could seriously inhibit the growth of theses plants, and resulted in the death of most treated plants and the reducing of proliferation rate and plant height. Treatment under 32/38℃ (8h/16h) gave a much lower death rate and the proliferation rate and height of treated plants did not show much difference from un-treated control plants. The effect of treatment under 34℃/42℃ was between these two treatments.2. Viruses of in-vitro plants regenerated from shoot tips of plant treated by thermotherapy were detected by PAS-ELISA and dot -blot hybridization. Results showed that three treatments under different temperatures had similar effect on titers of there viruses in regenerated plants, and the longer treatment period was, the lower the titers of viruses were. The efficiency of virus elimination was different for various treatments. Thermotherapy under 37℃ for 35 days combined shoot tip culture in a size 1mm could eliminate tested viruses. For treatment under 32/38℃, no virus-free plants were obtained. When the treatment temperature was raised to 34℃/42℃, all plants regenerated from shoot tip in a size 0.5mm of plants treated for 50 days were virus free. Results also showed that ACLSV could be more easily eliminated than ASPV and ASGV with a five-day shorten treatment period.3. Based on above results, three pear varieties were treated under 34℃/42℃ for virus elimination. Results showed that these varieties showed different sensitivities to such a high temperature. However, a 100% efficiency of virus elimination was achieved after a 50—60 day thermotherapy period combined shoot-tip culture in a size 0.5mm.4. Ribavirin was used for the virus elimination by adding it to MS medium at different amounts and for different culturing periods, and the growth characteristics and efficiency of virus elimination were evaluated. Plants grown in MS containing ribavirin更多还原