【作者】 陈海燕；

【导师】 王乔春；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2018， 硕士

【摘要】 植物病毒和类病毒保存是病毒抗原制备、疫苗生产、检疫检验等病毒相关研究和应用的基本要求。本研究以感染马铃薯卷叶病毒(Potato leafroll virus,PLRV)、马铃薯S病毒(Potato virus S,PVS)、马铃薯纺锤形块茎类病毒(Potato spindle tuber viroid,PSTVd)的马铃薯品种‘紫花白’试管苗为材料,通过茎尖超低温保存马铃薯病毒和类病毒。获得主要研究结果如下:1.以单一感染PLRV、PVS、PSTVd及复合感染PLRV+PVS的试管苗为材料,利用小滴玻璃化超低温保存茎尖。不同带毒状况的茎尖均能成活、再生植株。2.对超低温保存后的再生苗继代培养12周后,用DAS-ELISA和RT-PCR进行带毒状况检测,结果表明,在单感PLRV和复合感染PLRV+PVS的再生试管苗中能检测到PLRV,PLRV的保存率为50%;在单感PVS和复合感染PLRV+PVS的再生试管苗中能检测到PVS,保存率均为100%;在单感PSTVd的再生试管苗中能检测到PSTVd,保存率为100%。保存后的PLRV、PVS和PSTVd均能通过嫁接感染无毒砧木。保存后的PVS和PSTVd能通过摩擦接种到无毒植株上。3.对成活细胞在超低温保存后的茎尖中的组织学观察表明,超低温保存后,大部分叶原基细胞受损,成活细胞大多位于茎尖的顶端分生组织、第1-3叶原基以及部分第4叶原基细胞中。免疫化学组织法对病毒在茎尖中的定位表明,PLRV不能侵染顶端分生组织和第1-3片叶原基,能侵染第4及其它叶原基。这样,超低温保存后有部分植株仍然是带毒PLRV的。前人的研究表明,PVS和PSTVd能侵染顶端分生组织和幼嫩的叶原基,因而再生植株的带毒率会明显高于PLRV.本研究首次利用茎尖超低温保存马铃薯病毒与类病毒,为长期、高效保存病毒和类病毒提供了技术平台。更多还原

【Abstract】 Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications such as antigen preparation for virus detection by immunology-based methods,production of vaccines,genetic transformation to produce pathogen-derived resistant transgenic plants and bionanotechnology to produce nano drugs.The objective of the present study attempted to cryopreserve Potato leafroll virus(PLRV),potato S virus(PVS)and Potato spindle tuber viroid(PSTVd).The results obtained are as followings:1.Shoot tips derived from in vitro shoots of potato ‘Zihuabai’ single-infected with PLRV or PVS or PSTVd,and co-infected with PLRV and PVS were able to survive and resume shoot regrowth following cryopreservation.2.Plantlets after 12 weeks of regeneration following cryopreservation were subject to virus detection by DAS-ELISA and RT-PCR.Results showed that PLRV was readily detected And PLRV cryopreservation frequency in was about 50% in in vitro plantlets single-infected with PLRV.PVS was detected in 100% of of plantlets when shoot tips were excised from in vitro plantlets single-infected with PVS and co-infected with PLRV and PVS.PSTVd was detected by RT-PCR in 100% of plantlets regenerated from cryopreserved shoot tips single-infected with PSTVd.The levels of these three pathogens were comparable in the plantlets before and after cryopreservation.Cryopreserved LRV、PVS and PSTVd could be transmitted to the healthy rootstocks by micrografting.Cryopreserved PVS and PSTVd could also be transmitted to the healthy plantlets by mechanical inoculation.3.Histological observation showed that many cells in the upper part of the apical dome and leaf primordia 1-3,and some cells in leaf primordium 4 were able to survive following cryopreservation.Immunohistological localization showed that PLRV was not present in the apical dome and leaf primordia 1-3,but it was found in leaf primordium 4 and older tissues.Thus,a certain number of plants regenerated from cryopreservation were still the virus-infected.Existing results demonstrated PVS and PSTVd were able to infect the apical dome and the youngest leaf primordia,and therefore,plantlets regenerated from cryopreservation were still infected with them.The present study described for the first time successful cryopreservation of potato viruses and viroid in cryopreserved shoot tips.These results would provide technical platform for efficient and long-term preservation of potato viruses and viroids,and have potential application to other plants.更多还原