Establishment Of Virus-free Rapid Propagation Eystem And Optimization Of Detection System For New Apple Varieties

Virus disease has become one of the prominent issues that the apple industry urgently needs to solve and it spreads mainly through grafting.At present,Chinese apple industry is undergoing a large-scale replacement,and this transformation can be achieved quickly and efficiently by grafting.Therefore,the production of virus-free seedlings is of great significance to the sustainable development of the apple industry.The test aims to use virus detection,virus removal,tissue culture and rapid propagation and other technologies to produce virus-free seedlings of four apple new varieties that are'Envy','Modi','Zhongqiuwang','Yuhuazaofu'.The main findings obtained are as follows:1.Based on apple new varieties that are'Envy','Modi','Zhongqiuwang','Yuhuazaofu'as test materials,the sterile explants were established.Using a conventional sterilization for shoots borning in sandy winter dormant branches,the shoots were inoculated on medium with 2.0 g PVP·L^-1.The sterile explants of four varieties were successfully obtained.2.In the test of tissue culture and rapid propagation for the four varieties?'Envy','Modi','Zhongqiuwang','Yuhuazaofu'?,the appropriate concentration ratios of 6-BA/NAA in subculture were 1.1/0.05 mg·L^-1,1.0/0.05 mg·L^-1,1.1/0.05 mg·L^-1,and 1.0/0.05 mg·L^-1(MS was the basic medium with 2.0 g·L^-1 PVA);the appropriate concentration ratios of IAA/IBA in rooting culture were 0.5/1.0 mg·L^-1,0.75/0.5 mg·L^-1,0.5/1.0 mg·L^-1,0.5/1.0mg·L^-1?1/2 MS was the basic medium?;The suitable transplanting substrates for rooted seedlings were mosses,and the transplanting survival rates were 92.4%,88.3%,91.3%and94.5%.3.Virus of the tissue culture seedlings for each species were detected by two-step single RT-PCR before detoxification treatment.Among them,'Envy','Modi'and'Zhongqiuwang'carried ASPV,ASGV,ACLSV.'Yuhuazaofu'only carried ASGV,ACLSV.4.Stem tips of a size within the range of 0.05-0.2 mm were used from tissue culture seedlings that had been heat-treated by 4/5/6 weeks after two weeks of subculture.The detoxification methods were heat treatment+shoot tip culture and heat treatment+ultra-low temperature+shoot tip culture.The experimental results showed that with the prolongation of treatment time,the survival rate of the stem tips of two kinds of virus-free treatments of all varieties basically declined,and the detoxification rates basically showed an upward trend.The survival rate of stem tips treated with ultra-low temperature was much lower than that of stem tips treated only with heat treatment,and the detoxification rate was higher than heat treatment.5.The original two-step single detection system was optimized as a one-step multiplex detection system.The optimized reaction system:Prime Script 1 Step Enzyme Mix 1.0 ul;2×1 Step Buffer?Dye Plus?12.5?l;upstream and downstream Primer?10?M?of each virus1.0 ul;Template RNA 1.0 ul;RNase Free ddH₂O 4.5 ul;The reaction procedure:reverse transcription 50°C 20 min;pre-denaturation 94°C 2 min;entering 30 cycles?denaturation94°C 30 s,annealing 53-56°C 30 s,extension 72°C 30 s?,unlike previous studies,no final extension is required.