| 'Shine Muscat'(Vitis labruscana Bailey ×V.vinifera L.Shine Musact)is a diploid table grape cultivar,which was released by National Institute of Fruit Tree Science(NIFTS).This cultivar cross breeding of 'Akitsu-21'['Steuben'(V.labruscana)×'Muscat of Alexandria'(V.vinifera)]and 'Hakunan'(V.vinifera)is kelly.And it has a large of advantageous characteristics such as crispy,juicy flesh and soluble solids can reach 20%under normal cultivation management.However,'Shine Muscat' generally exhibits similar symptoms of viral diseases in various cultivations in China,including leaf rewinding,deformity,and clear spots,which have a serious impacts on fruit yield and quality.This research aimed to identify the viral pathogens causing Shine Muscat viral diseases and explore the tissue culture system and virus-free technology suitable for 'Shine Muscat'.Main results are listed as follows:1.The viroid symptoms of the 'Shine Muscat' in different orchards were mainly manifested in the leaves.The leaves exhibit different types of malformations,including vesicular shrinkage,wrinkle deformity,tower deformity,and leaf margin rewinding.After spring germination,cutting seedlings and two-year-old saplings are easier than several-year-old trees to express symptom;while the vigorous trees generally do not show symptoms before flowering while the diseased trees will show a large number of symptoms after bearing.2.RT-PCR detection and nucleotide sequencing were used to identify the pathogens in ten selected varieties.It was found that there were six kinds of viruses including Grapevine Pinot gris virus(GVPV),Grapevine leafroll-associated virus-3(GLRaV-3),Grapevine fleck virus(GFkV),Grapevine rupestris stem pitting-associated virus(GRSPaV),Grapevine virus B(GVB),Grapevine virus E(GVE).'Shine Muscat' was infected with five kinds of viruses,including GLRaV-3,GFkV,GRSPaV,GVB and GVE,and the detection rate is higher than the average detection rate.The composite infection rate of these five viruses reached 36.36%.3.Parameters such as template concentration,primer concentration and polymerase concentration were optimized.A multiplex RT-PCR detection system for rugose wood complex disease related viruses GRSPaV,GVB and GVE was established,which consisted of 10 ?l containing TaqMix 6 ?l,template cDNA 1 ?l,GRSPaV corresponding specific upstream and downstream primers 0.6 ?l each,and other primers 0.4 ?l each.And a multiplex RT-PCR detection system of related viruses GVPV,GLRaV-3,GFkV causing grape leaf malformation was established,which consisted of 10 ?l containing TaqMix 6 ?l,template cDNA 1 ?l,GLRaV-3 corresponding specific upstream and downstream primers 0.6 ?l each,and other primers 0.4 ?l each.4.A rapid propagation system for tissue culture of 'Shine Muscat' with single bud stems as explants was created.The optimum disinfectant treatment for explants was 0.1%mercuric chloride for 12 minutes with a survival rate of 51.7%.The optimum stem's initial medium was 0.5 mg/L 6-BA and 0.5 mg/L KT with a germination rate of 56.2%.The optimum subculture medium was basic medium MS supplemented 0.5 mg/L 6-BA with proliferation rate of 1.89,and the optimum rooting medium was basic medium 1/2MS supplemented 0.5 mg/L NAA.5.A micropropagation system with stem apex for tissue culture of 'Shine Muscat'was created.The optimum disinfection treatment for explants with the size of 0.1 mm was 0.1%mercuric chloride for 8 minutes with a survival rate of 64.4%.The optimal stem apex's initial culture medium was basic medium 1/2MS supplemented 0.5 mg/L 6-BA,0.5 mg/L KT and 0.2 mg/L IBA.6.Virus-free 'Shine Muscat' seedling was created by a variety of elimination techniques,including stem apex tissue culture,heat treatment,heat treatment combined with stem apex tissue culture,ultra-low temperature treatment combined with stem apex tissue culture.Heat treatment combined with stem apex tissue culture was better than other methods,in which the regeneration rate was 90%and the virus-free frequency of GLRaV-3,GFkV,GRSPaV,GVB and GVE were 100.0%,80.0%,95.0%,70.0%and 45.0%. |
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