Elimination Of Virus In Potato (Solanum Tuberosum L.) By Cryotherapy And Analysis Of Genetic Variation

Plant virus can affect plants' growing and developing,reduce the yield and quality of crops,and it is a seriouse damage to plant.Potato degeneration is over the world,and it is aroused by potato virus,tiny virus,bacterium hypostasis or other plant virus disease.There are many infringing sources to potato.Of infringing sources,Virus is an important factor,and it could makes a drop in potato production by 20%～50%ingeneral,even above 80%.Because of difficulty in handling,time consuming and low efficiency in removing virus etc,traditional methods can not deal with virus damage absolutly,and virus damage have been more serious.As one kind of new technique taking off the plant virus,Low temperature treatment has been developing over the past few years.This technique is easy to operate,and has a high efficiency in removing plant virus,so it plays an important role in the field of the preserving plant seeds' quality.The experiment makes use of the cryopreservation method,studying the condition that the cryopreservation technology removes the potato virus.And at the same time analysing the regenerations using AFLP and MSAP technology to discuss the influences of cryopreservation to materials' geneome array and DNA methylation.It is laid technological and theoretical foundation for cryopreservation technology in the application of potato virus removal.The results as follows:1.The procedures of vitrification for potato material were modified material,The results showed that:when the materials were trained at 4℃low temperature for 7 days,then pre-cultured on the MS medium with DMSO and acetamide for 5 days,loaded with 60%PVS₂ for 30 min at room temperature and dehydrated with PVS₂ for 40 min at 0℃.it had the best results,the suvival rate was 71%,the regenerated plants grew and differentiated normally.2.The genome DNA of treatments and controls were analyzed by amplified fragment lenth polymorphism(AFLP) technique.The DNA were digested by EcoRâ… and Mseâ… ,and 385 bands were scored using 6 pairs of primers combination.No variation of bands was found during the three samples,which suggested that no nucleotide sequence of the materials had altered during cryopreservation.Indicate plant in DNA level go ahead keeping the fairly good inheritance stability.3.Methylation-sensitive amplification polymorphism(MSAP) was used to analyze the DNA methylation status of the samples and controls,using MSAP technology analysed on the plant methylation before and after the cryopreservation showed that:compared with the control,the materials after cryopreservation all had varying degrees ofmethylation changes.In the 624 bands,the controls and the samples treated had 584 bands which was exactly the same type;and there was 40 changerable bands,of which 13 sites had methylation increaced,21 sites had demethylation. 4.It was using RT-PCR to detect the virus of the materials which were still suvival after cryopreservation.The results showed that cryopreservation were able to remove potato PVY and PVS virus,the removal rates were 83%and 47%separatly and it is higher than traditional stem point culture.This method is also simple and easy operated,showing cryopreservation technique removing the potato virus is feasible.