| Potato is an economically important agro-industrial crop that is conventionally propagated,however;its potential transmission of viruses through seed tubers from generation to generation is a major limitation of potato yield production.In order to produce potato virus-free and sufficient amount of potato seed tubers,several approaches of in vitro methods for virus elimination have been developed.Meristem culture has been used alone or combined with techniques such as thermotherapy,electrotherapy,cryotherapy and chemotherapy as the best alternative method for treating potato infected by viruses.Recent literature has shown that to eliminate potato virus significantly depends upon the potato cultivar,antiviral agents,type of virus,the duration of heat treatment.Appropriate duration for efficiency elimination is still under investigation.Viral elimination rate can be detected through serological methods such as enzyme-linked immunosorbent assay(ELISA)and molecular biology techniques such as real time reverse transcriptase polymerase chain reaction(real time RT-PCR)that are used for pre and post elimination virus detection to evaluate the efficiency and the accuracy of virus elimination methods.The purpose of this study is to highlight virus elimination methods in potato and recommending the most effective tool for virus detection in order to ensure the production of potato plantlet free of viruses.Potato PVY infection is associated with inhibition of potato vegetative growth.The present study was to investigate the effect of chemotherapy combined with thermotherapy on vegetative growth of in vitro potato cultured plantlets.Potato vegetative growth have been studied after the combined chemotherapy and thermotherapy.Before the treatment,RT-PCR and qPCR detection were done for confirmation of PVY infection.Shoots were subjected on MS media containing the following concentrations of ribavirin(0,15,20,and 30mg/l respectively),and finally to thermotherapy at 37 to40°C with 16 h of light(high intensity of over 10,000 lux)and at 24°C with eight 8 h of darkness for three to four weeks in thermotherapy room.After each treatment,establishment of in vitro culture was done to obtain complete regenerated plantlets sufficient for further analysis and for vegetative growth evaluation.The following experimental results are obtained:(1)The potato virus PVY was detected by RT-PCR and qPCR,and the potato cultivar "Favorita" tissue culture was confirmed to be infected with PVY virus.(2)In secondary culture,the single stem node of virus-infected potato tissue culture seedlings was treated with 15mg/L and 20mg/L ribavirin for 3 weeks,and then treated at 37℃ for 1 and 2 weeks.Compared with the control group,the vegetative growth of tissue culture seedlings was not significantly different after the treatment with one week of thermotherapy.However,after treatment with ribavirin at20mg/L for 3 weeks and then at 37℃ for 2 weeks,the vegetative growth of tissue cultured seedlings was significantly improved,and the height,node number,internode length and root number of tissue cultured plantlets were significantly increased.The results showed that the effects of PVY virus on the vegetative growth of potatoes could be significantly inhibited by the combination of 20mg/L ribavirin and 37℃thermotherapy for 2 weeks.(3)It was found by qPCR that the expression of PVY virus in potato tissue culture plantlets was slightly decreased after thermotherapy and chemotherapy with ribavirin reagent at different concentrations.However,compared with the control,the expression of PVY gene was not significantly different.The results showed that the combined treatment of ribavirin and heat at different concentrations only inhibited the growth of the virus and did not eliminate the virus completely.The results of this study will provide theoretical and technical support for reducing the inhibition of potato tissue culture seeds growth by PVY virus. |
