Research Of Apple Stock ’M26’ Viruses Elimination And Rooting Technology

Our country is one of the largest apple cultivation area in the world, but our country’s yield per unit area is low, the harm of apple virus and traditional backward cultivation mode are the main cause of this phenomenon. Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are the the most serious harm for apple, the three latent viruses always mixed infect trees, affecting the normal physiological metabolism of fruit trees and resulting in a decline in the fruit yield and quality, the growth of new shoots decrease, the fruit trees finally loss of cultivation value. The research based on PCR technology, established a multiple RT-PCR detection method. Then we used virus inhibitor, heat treatment and tip culture methods to eliminate the viruses.Grafing is the main breeding way of apple trees, ’M26’ is an apple stock which has apparent dwarf effect, the advantage of blossom and fruit yield in advance, therefore become the new style stock, but it is difficult to root, so it can be only used as interstocks. This research solved the problem of’M26’difficult to root through plant tissue culture technology and molecular biology methods, analyzed Rosaceae plant adventitious root formation marker gene ARRO-1 expression level variation in ’M26’ adventitious root formation stage, researched mechanism of’M26’difficult to root, fundamentally overcome the difficult rooting problem, then, established an efficient’two-step’rooting system of’M26’, provided a solid foundation for ’M26’ root stock application. At the same time established an efficient in vitro regeneration system of’M26’, enriched the content of woody plant tissue culture. This study made the following achievements,1. Used three apple latent viruses coat protein genes as purpose genes, nad5 as internal genes, setted up an efficient and rapid multiple RT-PCR detection system. When the template amount in PCR reaction system was 0.24 ug, the detection system can detect three kinds of purpose genes at the same time.2. The elimination of three Apple latent viruses,(1) Used ’M26’ as experiment material, combianed heat treatment with stem-tip cultivation, cultivating the material at initivative temperature of 25 ℃, rising 1℃ every day until 38℃, processing material at 38℃ for 50d, peeling 5mm stem-tips under binocular anatomical lens, and cultured the stem-tips in the regeneration medium MS+TDZ 4.0mg/L+NAA 0.5mg/L, cultivated at 25 ℃ for 40d. The virus elimination rate of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) respectively were 80%,0 and 40%. Afterwards used the first stem-tips as test materials, peeling stem-tips again, the three viruses elimination rate increased to 100%,20% and 80%.(2) The virus elimination of virus inhibitor, added 20mg/L ribavirin to subcultivation medium and processing material for 60d, three apple latent viruses all can be eliminated.3. Used whole leaves as explant, first, cultured the explants with optimal regeneration medium MS+TDZ 4.0mg/L+NAA 0.5mg/L in the dark for 15d, and in the light for 25d, later shifted to subcultivation medium MS+6-BA 0.5mg/L+IBA 0.1mg/L+GA30.2mg/L for further cultivation, adventitious bud regeneration rate and proliferation rate respectively were 98% and 1.58. For apple stock ’M26’ two-steps rooting, inducted the buds(about 2cm) in the dark for 4d with 1/2 MS+IBA 1.5mg/L for root primordium formation, secondary, rooting with 1/2MS medium for 20d, adventitious roots induction rate, average root number and average root length respectively were 100%,6.1 and 4.56, seedling transplanting survival rate was 100%.