| Sweet potato(Ipomoea batatas(L.)Lam.)is a major food and economic crop in China with strong adaptability and widely cultivated.Sweet potatoes are mainly propagated asexually through tubers and cuttings,which can easily cause viruses to accumulate and spread to the next generation year by year.Sweet potato virus disease causes serious yield losses in sweet potatoes in China,while also causing sweet potato varieties to degenerate and quality to deteriorate.In recent years,the occurrence of sweet potato virus in the main sweet potato producing areas of northern Anhui has become increasingly severe,with the range of damage caused by Sweet Potato Latent Virus(SPLV)expanding and co-infection with other viruses causing serious synergistic symptoms,reducing sweet potato yield and quality and seriously affecting the healthy development of the sweet potato economy.At present,planting virus-free sweet potato seedlings is an effective method to control the spread and harm of sweet potato virus disease and one of the best methods to improve sweet potato yield and quality.However,there is currently no efficient method for obtaining tissue culture seedlings free of Sweet Potato Latent Virus or for detecting tissue culture seedlings for the main sweet potato varieties in Anhui(including Pushu 32,Yushu 12,Yanshu 25)that are severely affected by Sweet Potato Latent Virus.Solving the problems of low efficiency in rapid propagation of sweet potato shoot tip culture,low efficiency in virus detection and low survival rate after transplantation,and obtaining virus-free seedlings at a high rate and accurately detecting viruses before using virus-free seedlings is the key to controlling Sweet Potato Latent Virus from the source.Therefore,this study used Pushu 32,Yushu 12,and Yanshu 25 as experimental materials.On the basis of previous work,we first obtained the gene sequence by cloning the coat protein(CP)gene of Sweet Potato Latent Virus.Then we used shoot tip meristem culture to obtain tissue culture seedlings and established a Sweet Potato Latent Virus detection system using RT-PCR.Finally,we further carried out research on the propagation and breeding of virus-free seedlings.The main research results are as follows:1.In order to obtain the sequence of the coat protein gene of Sweet Potato Latent Virus,specific primers were designed based on the reported sequence of Sweet Potato Latent Virus on NCBI using a conserved sequence of the coat protein gene of Sweet Potato Latent Virus.The coat protein gene sequence of Sweet Potato Latent Virus was amplified using RT-PCR.Sequencing results showed that the obtained coat protein gene fragment of Sweet Potato Latent Virus had 97.69% homology with the reported gene sequence.2.In order to obtain more sweet potato shoot tips,water culture and soil cultivation were compared.The results showed that compared with water culture,soil cultivation had more tender shoot tip branches and thicker tender shoot tips.The obtained shoot tips were disinfected with 0.1% mercuric chloride or a combination of ethyl alcohol and 0.1%mercuric chloride for different times to observe their contamination rate and survival rate.The results showed that the best disinfection effect was achieved by treating sweet potato shoot tips with 0.1% mercuric chloride for 7 min followed by 75% ethyl alcohol for 30 s with a contamination rate of 7.78% and a survival rate of 87.78%.The best disinfected sweet potato shoot tips were then cultured in three stages: shoot tip induction,shoot tip differentiation,and rooting into seedlings.From the growth of shoot tips at each stage and the induction rate,differentiation rate,and seedling rate of shoot tips,it was found that Pushu32,Yushu 12,Yanshu 25 had their best culture medium as combination formula No.1: Shoot tip induction: MS+2 mg/L 6-BA;Seedling differentiation: MS+0.2 mg/L NAA+0.01 mg/L6-BA;Rooting into seedlings: MS;The seedling rates of Pushu 32,Yushu 12,Yanshu 25 were 76.15%,65.00%,and 73.33%,respectively.3.A total of 339 tissue culture seedlings were obtained from three sweet potato varieties using RT-PCR(based on specific primers for SPLV coat protein gene obtained from sequencing results)to determine whether there was Sweet Potato Latent Virus present among them: Pushu 32 was tested for 141 plants;Yushu 12 was tested for 81 plants;Yanshu 25 was tested for 117 plants;The results showed that Pushu 32 had a virus removal rate of 76.60%;Yushu 12 had a virus removal rate of 61.73%;Yanshu 25 had a virus removal rate of 82.05%.4.The tissue culture seedlings that were not detected by RT-PCR for Sweet Potato Latent Virus were propagated on MS medium;1/2MS medium;1/2MS+0.5 mg/L NAA medium;The plant height;leaf number;main root number;root length were analyzed;The results showed that key indicators such as plant height and leaf number were better on1/2MS medium than on MS medium or 1/2MS+0.5 mg/L NAA medium;Therefore Pushu 32;Yushu 12;Yanshu25 were suitable for 1/2MS medium.5.In order to further improve the survival rate after transplanting virus-free seedlings;before transplanting completed propagated virus-free seedlings;they were hardened off for0 d;5 d;10 d;The results showed that Pushu 32 chose10 d hardening off with a transplant survival rate of 93.33%;Yushu 12 chose10 d hardening off with a transplant survival rate of84.44%;Yanshu 25 chose10 d hardening off with a transplant survival rate of 87.78%;These results indicate that it is necessary to harden off before transplanting virus-free seedlings.In summary this study established a detection system for removing SPLV from tissue culture seedlings providing practical operational basis for production of sweet potato virusfree seedlings detection of Sweet Potato Latent Virus. |
