【作者】 刘颖；

【导师】 杜国强；

【作者基本信息】 河北农业大学 ， 果树学， 2011， 硕士

【摘要】 继代保存多年后苹果组培苗的生长繁殖特性和遗传稳定性的研究对苹果种质离体保存适宜年限的确定具有重要意义。本试验对不同年限接种的‘金冠’、‘乔纳金’、‘嘎啦’、‘富士’茎尖组培苗分别进行了继代增殖、不定根诱导和离体叶片不定芽再生能力研究和内源激素的分析,并从形态特征、核DNA含量、细胞学特性及分子标记AFLP分析等方面评价了其遗传稳定性。主要研究结果如下:1.外植体接种初期3~4年以内,即继代次数为24或40代时,‘富士’、‘金冠’、‘乔纳金’的新梢增殖能力较低,‘嘠啦’生根率较低,‘金冠’、‘富士’、‘嘠啦’离体叶片再生率较低,随着培养时间延长,继代次数增加,以上品种的继代增殖效率、生根能力及离体叶片再生能力均稳定在较高水平。长期继代保存的苹果品种茎尖组培苗(‘富士’已保存25年,‘金冠’、‘乔纳金’19年,‘嘠啦’10年)仍保持其器官再生能力。2.随继代次数的增加,试管苗中ABA和ZR含量呈下降趋势,反映了试管苗芽分化和不定根生长。继代初期,GA含量较高。随保存时间的延长,IAA/ZR比值稳定在较高水平,使苹果组培苗保持稳定的增值和生根能力。3.‘乔纳金’、‘金冠’、‘嘎啦’不同继代次数组培苗移栽,移栽成活率均较高;移栽初期,株高和叶面积均无显著差异,叶片数有差异,代数少的组培苗叶片数少。移栽后期,株高、叶面积、叶片数均无差异。说明3个品种组培苗遗传稳定性未发生变化。4.不同继代次数组培苗在细胞核DNA含量或细胞倍性上均没有差别,遗传性保持稳定。利用流式细胞仪测定了‘乔纳金’、‘嘎啦’、‘富士’苹果组培苗细胞核DNA含量,不同倍性品种间细胞核DNA含量差异显著。‘嘎啦’、‘富士’二倍体细胞核DNA含量平均为2.27 pg,‘乔纳金’三倍体细胞核DNA含量平均为3.13 pg。5.对‘金冠’、‘乔纳金’‘嘎啦’、‘富士’4个苹果品种组培苗进行根尖染色体制片,均未观察到染色体数目、形态等变异。6.用10对引物对,对‘乔纳金’、‘金冠’、‘富士’和‘嘎啦’四个品种的组培苗DNA样品进行了AFLP分析。引物对E-AAC/M-GAC、E-ATC/M-CCG、E-CTA/M-AGG、E-TAA/M-CTG可以区分四个品种,区分率为100%,共扩增出430条清晰可辨的AFLP带,同一品种不同继代次数间DNA样品的指纹图谱未发现特异性条带。证明在所检测的范围内继代培养未对苹果茎尖组培苗碱基序列产生影响,DNA水平上未发生变异,具有高度的遗传稳定性。更多还原

【Abstract】 For figure out the optimal life span of germplasm preserved in vitro, it is necessary to understand whether its propagating ability changes or not after subcultured for years. The shoot multiplication ability, adventitious root regeneration rate from shoots, adventitious bud regeneration rate and DNA variation from leaves of‘Fuji’,‘Golden Delicious’,‘Jonagold’and‘Gala’apple in vitro shoot explants were studied. .The main results are as follows:1.The shoot multiplication ability of‘Fuji’,‘Golden Delicious’and‘Jonagold’, the adventitious root regeneration rate of‘Gala’, and the adventitious bud regeneration rate from leaves of‘Golden Delicious’,‘Fuji’, and‘Gala’during the first four years after inoculation from field, that was at the stage of subculture from 24 to 40 times. With the increasing of the subculture times, the abilities for the given cultivars could increase and keep at a higher level stably. The apple explants repeatedly subcultured in vitro for years (25 years for‘Fuji’, 19 years for‘Golden Delicious’and‘Jonagold’, and 10 years for‘Gala’) could remain their organogenesis characteristics.2. With the increasing of the subculture times, the content of ABA and ZR decreased in the seeding cultivation. The changes reported bud initiation and adventitious root. The content of GA was high in subculture early times. With the increasing of the subculture times, the ratio of IAA/ZR stabilized higher level. So the apple subcultured in vitro for years could remain their organogenesis characteristics.3. The transplant survival rate of‘Fuji’,‘Golden Delicious’,‘Jonagold’and‘Gala’apple in vitro shoot explants was higher. At the early stage after transplanting, height and leaves the areas were no significant differences. The leaf number was less of lower subculture times apple shoot explants. The morphologies in the field no differences at the later stage after transplanting. The result showed that the apple explants repeatedly subcultured in vitro for years could remain their genetic stability.4. Content of nuclear DNA for apple cultivars including 9 diploid and 8 triploid was measured by flow cytometry. The average nuclear DNA content of diploid cultivars was 2. 27 pg and that of triploid cultivars was 3. 13 pg, which indicated that variation of nuclear DNA content between diploid and triploid was significant.5. By examining their root tip chromosome numbers,‘Fuji’,‘Golden Delicious’and‘Gala’were diploid ,‘Jonagold’was triploid. Not been observed the chromosome number and morphology of variation.6.‘Jonagold’,‘Gold Delicious’and‘Fuji’were studied to AFLP analysis. 4 primers with high polymorphism and powerful distinctiveness from 10 primer combinations produced 430 fragments. The primers were E-AAC/M-GAC、E-ATC/M-CCG、E-CTA/M-AGG、E-TAA/M-CTG. There was no difference of different subculture times.更多还原