Study on rapid propagation technology of virus-free seedlings by tissue culture in‘Shine Muscat’grape

Abstract: 【Objective】‘Shine Muscat’grape, with its unique advantages of seedless, high sugar con-tent, rich rose fragrance and good flavor, surpasses the quality of other grape varieties and is welcomed by consumers. In recent years,‘Shine Muscat’grape has been widely introduced and planted in Zheji-ang, Jiangsu, Anhui, Yunnan, Guangxi and other places, showing a broad market prospect. With the de-velopment of grape industry, the demand for‘Shine Muscat’grape seedlings is growing rapidly. How-ever, in the cultivation of this variety in China, the plants generally show the symptoms of viral diseas-es, so that the yield and berry quality were seriously affected. The traditional way of grape seedling pro-duction to remove the virus is inefficient. Therefore, it is necessary to establish a rapid propagation tech-nology system for virus-free seedlings of‘Shine Muscat’grape by combining tissue culture with heat treatment method.【Methods】Based on the heat treatment method, the virus-free zone of the stem tip can expand by inactivating the virus, and the stem tip has the advantage of taking small apical meri-stems to grow a whole plant. In this paper, the tissue culture seedlings of‘Shine Muscat’grape were treated by combining heat treatment with stem tip culture. Firstly, the stem segments with axillary bud  area of new canes were used as explants, and they were disinfected by different disinfection methods. Then they were inoculated on MS medium containing different concentrations of plant growth regula-tors. After a few days, the pollution rate, survival rate, germination rate, subculture multiplication coeffi-cient, rooting rate, root length and root number of explants were calculated. The best method of disinfec-tion, disinfection time, start medium, subculture medium, rooting medium for explants was determined.Secondly, when robust tissue culture seedlings were obtained, they were heat-treated at different culture temperatures and durations. At the end of treatment, the stem tips were selected and cultured to grow in-to intact plants. Finally, the tube seedlings were sent to qualified institutions for virus detection by RT-PCR detection.【Results】After disinfection with 75% alcohol for 30 s and 0.1% mercuric chloride for 8 min, the contamination rate and browning rate of stem explants were the lowest, and the survival rate was 86%; the explants were inoculated on MS medium containing 6-BA and NAA to induce germina-tion, and the highest germination rate was on MS medium containing 6-BA 1.5 mg · L^-1 and NAA 0.2mg · L^-1, which reached 86.4%; the sterile buds were inoculated on MS medium containing 6-BA and NAA. On the medium containing 1.0 mg·L^-1 6-BA + 0.1 mg·L^-1 NAA, the effect of proliferation was the most obvious in the subculture, and the multiplication coefficient was 3.5; from subculture, the sin-gle buds with strong growth were separated and transferred onto the medium of 1/2MS with IBA and NAA for proliferation and rooting, and the effect of rooting was the best on the medium of 1/2MS with IBA 0.4 mg·L^-1 and NAA 0.2 mg·L^-1, the rooting rate was 86%, the average number of roots was 4.13,and the average root length was 3.89 cm. detoxification treatment of tissue culture seedlings by heat treatment was combined with shoot tip culture. After 18 days of heat treatment at 37 ℃, individual plants began to gradually lose their green leaves and became withered, and finally the whole plant died. The survival rate of heat treated plants was 78%, and the shoot tip survival rate was 60%. The results showed that, regenerated plants did not contain grape leaf curl virus 1 (GLRaV-1), grape leaf curl virus 3 (GLRaV-3), grape virus A (GVA), grape spot virus (GFkV), and grape fan leaf virus (GFLV).【Conclu-sion】Through the technology of plant tissue culture, the production efficiency of grape seedlings could be improved effectively. At the same time, using heat treatment combined with detoxification technology of stem tip culture, the detoxification efficiency of virus in the tissue culture seedling was also great-ly improved. Therefore, this study preliminarily established the technology system of tissue culture and detoxification and rapid reproduction of‘Shine Muscat’grape. Moreover, it can provide technical sup-port for the factory production of detoxification tissue culture seedling of ‘Shine Muscat’grape. It can also provide the market with high quality, robust, virus- free, excellent characteristics of virus- free‘Shine Muscat’tissue culture seedlings.