Establishment Of Virus-free System Of Two Main Strawberry Viruses By Cryotherapy

China boasts the world’s greatest strawberry planting area and yield.China produced 3.042 million tons of strawberries in 2019,accounting for about 40%of global strawberry output.With the increase in strawberry consumption demand and the development of the strawberry industry,strawberry virus disease has become an important disease of strawberries.Most strawberry viruses are compound infections,and the asexual reproduction of strawberries makes the virus accumulate continuously in the plant body and pass on from generation to generation,which eventually leads to a decrease in fruit quality and yield,leading to serious economic losses.At present,there are more than 20 viruses infecting strawberries in the world,but the main viruses infecting strawberries in Shaanxi province have not been reported in detail.As for the prevention and control of strawberry virus disease,a virus-free vaccine is the most economical and effective method.The virus-free strawberry seedlings were produced by stem tip culture and heat treatment with stem tip culture.However,the method of direct shoot tip culture has the disadvantages of low virus-free rate and difficult sampling;while heat treatment plus shoot tip culture has the disadvantages of long virus-free period and high cost.A cryopreservation system is a new and efficient method to remove plant viruses.Compared with traditional methods,the cryopreservation system has the advantages of a shorter cycle,lower cost,and higher virus-free rate.However,there is no mature and stable ultra-low temperature detoxification technology system for strawberries.Therefore,this study intends to investigate the main virus species infecting strawberries in Shaanxi province,and establish the explants with virus samples of main strawberry varieties and the ultra-low temperature virus-free technology system.In this study,150 strawberry samples suspected to be infected by the virus were collected from Xi’an Fengxiang,Gaoling,Huxian,and Meixian counties of Shaanxi Province from 2019 to 2020.RNA was extracted and reverse transcription PCR（RT-PCR）was combined with LncRNA sequencing technology.RT-PCR was used to determine the species and distribution of the strawberry virus in Shaanxi province.By optimizing sucrose concentration,pre-culture time,and loading time in the strawberry virus elimination system,an ultra-low temperature system was established for the elimination of two main viruses in strawberries.The results are as follows:There are five main viruses infecting strawberries in Shaanxi Province,namely Strawberry vein band virus（SVBV）,Strawberry mottle virus（SMo V）,Strawberry milky yellow edge virus（SMYEV）,Spinach latent virus（SPLV）,and Lycopersicon esculentum nepovirus（LENV）.SPLV and LENV viruses were first identified in strawberries.The distribution and virus-carrying rate of five viruses in the main strawberry-producing areas of Shaanxi Province were detected.SMo V and SVBV were detected in seven districts and counties,SMYEV was detected in Mei county,Lintong County,and Baqiao District,SPLV and LENV were detected in the suburbs of Xianyang City and some areas of Fengxiang County.In this study,the RT-PCR detection system suitable for these five viruses was optimized to realize the rapid and accurate detection of five strawberry viruses.In the establishment of the ultralow temperature virus-free system,the stolons of healthy strawberry plants were collected,and the explants of more than ten strawberry varieties such as‘Ningyu’,‘Hongyan’,‘Zhangji’,‘Xingxiang’,and‘Snow White’were established,and the propagation and virus detection of tissue culture seedlings were carried out.‘Ningyu’is one of the common strawberry varieties,which has a high virulence rate in our established explants.Therefore,the ultra-low temperature virus-free technology was carried out with the virus-bearing plants of this variety as materials.By optimizing sucrose concentration,pre-culture time,loading time,and other conditions,an ultra-low temperature virus-free system was established.The results showed that the suitable virus elimination condition for‘Ningyu’strawberry seedlings was as follows:after 4 weeks of a subculture of tissue culture seedlings,the healthy plants were selected,and the stem tips of 1.5～2.0 mm‘Ningyu’strawberry were removed under aseptic conditions and cultured in MS+2 M glycerin+0.5 M sucrose（p H=5.8）liquid medium for 5 days at 4℃in darkness;The shoot tip was transferred into MS+2M glycerol+0.4 M sucrose（p H=5.8）loading solution for 60min.Then the shoot tips were transferred to 100%PVS₂（MS+30%glycerol+15%ethylene glycol+15%DMSO+0.4 M sucrose）at 0℃for 25min.After that,the stem tip was transferred to sterilized tin foil paper to form PVS2 droplets and quickly put into liquid nitrogen until no bubbles were generated.The shoot tips were transferred to MS+2 M glycerol+1.2 M sucrose（p H=5.8）unloading medium for 20 min and dried on filter paper.Finally,the shoot tips were transferred to 4.43g/LMS+30 g/L sucrose+8.0 g/L AGAR+0.2 mg/L6-BA regeneration medium（p H=5.8）after 3 days of medium dark treatment under normal light conditions,and the survival rate was calculated after 30 days.The survival rate of the stem tip of‘Ningyu’strawberry was78.3%,72.3%,and 78.7%,respectively.Under the above cryotherapy system,the survival rate of the‘Ningyu’strawberry stem tip was 78.7%.The virus-free seedlings were detected by RT-PCR,and the virus-free rate was 66.7%for SMYEV and 73.3%for SVBV.