| Application of virus-free propagules is the premise and basis of high-quality and efficient production of apple,and virus-free original propagules preservation is one of the important links of virus-free propagulesling breeding.Problems such as high cost and poor standardization of existing apple original propagules preservation technology in production hinder the development of apple virus-free propagulesling industry.In this study,the apple virus-free original propagules material obtained in the earlier period was used as the test material to study and optimize the technology of tissue culture medium-term preservation and establishment of virus-free foundation stock field of apple,in order to provide theoretical and technical guidance for apple virus-free original propagules preservation.The main research results are as follows:1.The medium-term preservation technology of virus-free apple propagules was establishedIn this study,tissue culture propagules of apple virus-free variety ‘Yanfu 6’ and apple virus-free rootstock ‘Qingzhen 1’ were used as experimental materials.The effects of culture temperature,sucrose concentration and mannitol concentration on the mid-term preservation of apple virus-free propagules were studied.(1)The results of temperature screening showed that the preservation effect of ‘Yanfu6’ and ‘Qingzhen 1’ at 12℃ was better than that at 25℃.Medium medium was MS medium,adding different concentrations of sucrose and mannitol combined to screen proliferation rate,shoots length,survival rate and regeneration rate.The optimal medium for medium preservation showed that 4.5% sucrose +0.5～1.0% mannitol could be stored for 9～12months.The survival rate and regeneration rate decreased with the increase of middle preservation time(1,3,5,7,9,12 months).The regeneration rate of ‘Qingzhen 1’ was 85.5%after being stored at 12℃ for 1 year,but only 66.6% after being stored at 25℃ for 9months.In conclusion,the medium preservation condition of 12℃ is better than 25℃.(2)In order to evaluate the influence of medium preservation on genetic stability of virus-free materials,the test tube propagules of ‘Qingzhen 1’(unpreserved,medium preservation and conventional preservation)were used as materials for SNPs and Indels analysis based on resequencing.The results showed that the number of SNPs and Indels were detected between the unpreserved and mid-preserved materials of ‘Qingzhen 1’(128609;12591)less than the amount between unpreserved and conventional preservation materials(157374;17137).This may be related to the reduction of the number of subgenerations by intermediate preservation.Thus,metaphase preservation reduces the rate of genetic variation.2.The morphological and physiological changes of 12 virus-free apple materials during growth and development were revealed,and the foundation stock field was establishedIn this study,12 kinds of virus-free tissue culture propagules of apple cultivars and rootstocks,such as ‘Liquan Fuji’,‘Buckeye gala’,‘Gala-3’,‘Huatian Fuji’,‘Aztec Fuji’,‘Yanfu6’,‘Yanfu 8’,‘Honeycrisp’,‘B.9’,‘Qiuzi’,‘M.26’,‘M.9-T337’,were used as experimental materials.The toxic apple variety ‘Liquan Fuji(V)’ and apple rootstock ‘B.9(V)’ were used as control materials.Sugar-free rooting and growth status of different cultivars and rootstocks of virus-free apple materials were investigated and physiological and biochemical indexes of leaves were determined.(1)The rooting results of sugar-free tissue culture showed that apple variety ‘Gala-3’had the best rooting rate(95.0%),and apple rootstock ‘B.9’ had the best rooting rate(91.2%).The root morphology of ‘Gala-3’ was better than that of ‘Aztec Fuji’.The root morphology of apple rootstock ‘M.9-T337’ was better than that of apple toxic rootstock‘B.9(V)’.(2)The results of growth investigation showed that the growth curve of virus-free apple materials showed an “S” shape.But the growth rate of different apple varieties and rootstocks was obviously different.The growth rate of apple variety ‘Gala-3’ was the fastest,and that of ‘Honeycrisp’ was the slowest.The growth rate of apple rootstock ‘M.9-T337’ was the fastest,and that of ‘B.9(V)’ with poison was the slowest.(3)The results of leaf photosynthetic index showed that the photosynthetic efficiency of the apple variety ‘Honeycrisp’ was the highest,while that of ‘Yanfu 8’ was the lowest.The photosynthetic efficiency of apple rootstock ‘M.26’ was the highest,while that of toxic‘B.9(V)’ was the lowest.The results showed that the pigment content of the leaves of the apple variety ’Bakai’ was the highest,while that of the apple variety ‘Liquan Fuji’ was the lowest.The leaf pigment content of apple rootstock ‘M.26’ was the highest,while that of toxic rootstock ‘B.9(V)’ was the lowest.(4)In the comparison between virus-free and virus-free(CK)apple varieties and rootstocks(root analysis,growth rule,leaf physiological and biochemical indexes),The indexes of virus-free varieties and rootstocks were better than those of virus-free varieties and rootstocks. |
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