Detection Of Sweet Potato Viruses In Hangzhou And Cultivation Of Virus-free Healthy Seedlings

Sweet potato(Ipononea batatas Lam)is one of the main economic and food crops in China,which belongs to the genus Ipomoea.China is a main producer of sweet potato in the world.Field investigation has found that sweet potato were infected by a variety of viruses,resulting in a serious decline in yield and quality deterioration,which led to serious economic losses.The cultivation of virus-free and healthy seedlings is the main solution to improve the yield and quality of sweet potato.This study investigated and tested the occurrence of viral diseases of sweet potato in the 7 main planting areas of sweet potato in Hangzhou,including Lin'an city,etc.In addition.the SPFMV-stripped virus-free seedlings of sweet potato were obtained though stripping stem tips combined with droplet liquefaction method on sweet potato 1042 strain,and the breeding system of virus-free seedlings was studied as well.The results of the study are as follows:1.Investigation and sampling of viral diseases in main producing areas of sweet potato in Hangzhou.Seven main sweet potato producing areas in Hangzhou,including Yuhang,Tonglu,Xiaoshan,Fuyang,Jiande,Chun'an and Lin 'an were investigated.It was found that sweet potato in the main producing areas had the symptoms of dwarfing,floral leaf,wrinkling,and protuberance of leaf vein,etc.Twenty leaves with typical symptoms of viral diseases were collected,of which two were from Yuhang,four from Tonglu.three from Xiaoshan,two from Fuyang,four from Jiande,three from Chun'an,and two from Lin'an.It was preliminarily confirmed that the sweet potato in the main production area of Hangzhou were infected with the viral diseases.2.Detection of viral disease in sweet potato.Using the serious diseased sample(Tonglu TL-4)as material,negative staining of the diseased samples with electron microscopy showed that the tissue juice contained a large number of curved linear virions at 600?900 nm,and a large number of crystals were found in the cytoplasm of the ultrathin sections.RT-PCR results showed that the 20 diseased samples of sweet potato from Hangzhou were detected to be infected with 6 viral diseases including Sweet potato feathery mottled virus(SPFMV)?Sweet potato chlorotic stunt virus(SPCSV),Sweet potato virus G(SPVG)?Sweet potato virus C(SPVC)?Sweet potato virus Y(SPVY)and Sweet potato chlorotic flecks virus(SPCFV)among which 6 viral diseases were detected in Tong lu,4 detected in Yu hang,4 detected in Jiande.3 detected in Chun'an.2 detected in Lin'an.1 detected in Xiaoshan.and 1 detected in Fuyang.3.Establishment of the detoxification system of sweet potato stem tip droplet liquefaction.An efficient and simple method of tip droplet liquefaction was established by studying the impacts of factors including pre-culture medium sucrose concentration.the components of the glass solution and the treatment time on the regeneration rate of the stem tip.The specific method is as follows:Peel about 1.0 mm top stem tip from sweet potato 1041 aseptic seedlings with SPFMV.Cultivate the 1.0mm peeled top stem tip in pre-culture medium A(0.3M sucrose +MS.pH=5.8)and pre-culture medium B(0.5M sucrose +MS.pH=5.8)respectively for 1 day.After that,the loading solution(2M glycerin + 0.4m sucrose,pH=5.8)was used for treatment for 60 min.then PVS2(MS+30%glycerin +15%glycol +15%DMSO+ 0.4 mol/L sucrose.pH 5.8)was used for treatment for 60 min,then liquid nitrogen treatment for 30 min,and then wash solution(1.2 M sucrose +MS,pH=5.8)treatment for 20 min.Finally,the stem tip was inoculated into the restoration medium A(MS + 0.5mg/L BA without NH4-)for dark culture for 3 days.Then transfer to the recovery medium B(MS + 0.5mg/L BA)for 1 week,and transfer to the regeneration medium(BM + 0.5mg/L BA+ 0.1 mg/L NAA+ 8 mg/L GA3.BM:0.01 g/L Ca(N03)2,0.2 g/L C6H8O6,0.02 g/L C4H12N2.0.002 g/L C18H32N2O10Ca?0.1 g/L C6H14N4O2+MS).The survival rate of stem tip was 81.88%and the regeneration rate was 72.06%.The results of RT-PCR and ELISA showed that the detoxification rate of droplet liquefaction method was 100%.Meanwhile,the SPFMV detoxification rate of plants with only stem tip cultured was 33.33%.4.The breeding of 1042 virus-free seedlings without SPFMV.Cut 1042 virus-free plants without SPFMV into stem segments containing 1-2 internodes.Stem segments were inoculated in rooting medium for 3-4 weeks.After that,when the plants grew to 6-8 cm,5-7 leaves and 3-5 internodes,the plants were placed at room temperature for one week and then washed.The survival rate was 100 when the plants were transplanted into an incubator(perlite:vermiculite:peat =1:1:2)with insect-resistant net.Then the 2-month transplanted seedlings were put into the base in Hainan Island for breeding.5.Growth index testing and quality analysis of virus-free seedlings.The morphological indexes of the upper and lower parts of the virus-free seedlings and the control seedlings were detected respectively.The results showed that the shape of potato block,dry weight,leaf length.leaf width,vine length,vine thickness and number of branches of the virus-free seedlings were significantly better than those of the control seedlings.It was found that detoxification treatment was beneficial to the enhancement of leaf photosynthesis and had a positive effect on leaf quality.