Cryopreservation Of Potato Leafrool Virus,Potato S Virus And Potato Spindle Tuber Viroid In Shoot Tips

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.