Optimization Of Virus-Free System For Tissue Culture Seedlings Of New Apple Varieties(Lines)

Apple is the largest cultivated fruit tree in China,and plays an important role in the development of agricultural economy.However,apple virus disease seriously restricts the development of apples,slows down the growth of fruit trees,and reduces fruit yield and quality.At present,no effective control method has been found.Therefore,non-toxic cultivation is the future direction of fruit production.‘5D2-36’is an important superior line for our group’s latest breeding,which is expected to be further developed into new varieties;‘Qin Yue’is the latest breeding new varieties,so it is of great significance to cultivate new varieties（lines）‘Qinyue’,‘5D2-36’virus-free seedlings.Based on the optimization of the existing virus-free system,the virus-free seedlings of four cultivars（lines）including‘Honey’,‘Qin Yue’,‘5D2-36’and‘Orin’were tested by different virus-free methods,such as shoot tip culture,heat treatment,ultra-low temperature and chemical agents,in order to obtain virus-free seedlings of several cultivars（lines）.The results are as follows:1.Three varieties of aseptic explants were successfully obtained by sterilizing the tender buds of‘Honey’,‘Qin Yue’and‘5D2-36’shoots stored in sand.2.Four varieties（lines）were subcultured and rooted.The optimum formula of subculture and rooting medium was obtained:‘Qin Yue’and the optimum concentration ratio of‘Honey’subculture medium was 6-BA 1.0 mg·L^-1+IBA 0.03 mg·L^-1;the optimum concentration ratio of‘5D2-36’and‘Orin’subculture medium was 6-BA 0.8 mg·L^-1+IBA0.03 mg·L^-11 and MS+6-BA0.75mg·L^-1+NAA 0.05mg·L^-1,respectively.The optimum concentration of rooting IBA for‘Qin Yue’,‘5D2-36’and Orin was 0.5 mg·L^-1,while that for‘Honey’was 0.7 mg·L^-1.3.Four strains（strains）of virus disease were detected by RT-PCR.The virus-carrying status of tissue culture seedlings before virus-free was successfully detected.Among them,‘Qin Yue’,‘5D2-36’,‘Honey’and‘Orin’all carried ASGV and ACLSV,but did not carry ASPV.4.The detoxification test of four varieties（lines）showed that the size of the stem tip was about 1.0 mm,the regeneration rate of heat treatment was the highest,and the regeneration rate of ultra-low temperature was very low.Chemical treatment had some influence on the survival of the stem tip,but the regeneration rate was higher than that of ultra-low temperature.After heat treatment（45 days）,the detoxification rate of ACLSV was 100%.After heat treatment（35-45 days）,ultra-low temperature detoxification,the detoxification rate of four varieties of ASGV ranged from 66.7%to 100%.After heat treatment（25 days）and chemical treatment(20 ug·mL^-1),the detoxification rate ranged from 70%to 90%.The ASGV of‘Honey’varieties was more difficult to remove than that of the other three varieties.Therefore,considering the detoxification rate,the best combination of‘Qin Yue’,‘5D2-36’and‘Orin’treatment is heat treatment for 45 days（ACLSV）+ultra-low temperature（ASGV）,and the best combination of‘QinYue’,‘5D2-36’and‘Orin’treatment is heat treatment for 35 days（ACLSV）+chemical treatment for 20 ug·mL^-1（ASGV）.The best treatment combination of‘Honey’and brittle is heat treatment for 35 days（ACLSV）+chemical treatment for 20 ug·mL^-1（ASGV）.