【作者】 于丽艳；

【导师】 孙仲序；

【作者基本信息】 山东农业大学 ， 果树学， 2005， 硕士

【摘要】 本试验以苹果三倍体品种“乔那金”试管苗为试材,通过对苹果离体叶片两步培养法再生的研究,建立了乔纳金苹果试管苗叶片离体再生体系,证明了愈伤组织的发生和激素种类与浓度的关系;并在此基础上研究了愈伤组织再生对不同人工诱变方法（高激素、60Co 辐射、秋水仙素）的反应、被处理愈伤组织再生能力和再生植株性状多样性、以及再生植株不同生理生化角度（外观性状、耐盐性、耐高温性、耐低温性等）的变异性,得到了优良抗性植株并建立了优良变异株系,并从分子水平鉴定再生植株变异程度的结果。主要结果如下:1.实验证明2,4-D 促进愈伤组织的形成和增殖,影响外植体脱分化和再分化的趋向。BA 影响外植体形成愈伤组织的能力,对不定芽再生具有显著作用。IAA 对愈伤组织的形成几乎没有影响,对再生不定芽的生长发育影响较大。诱导愈伤组织培养基为:MS+BA1.0mg﹒L^-1+IAA0.2mg﹒L^-1+2,4-D0.8 mg﹒L^-1,诱导率可达100%;分化不定芽培养基为MS+BA4.0 mg﹒L^-1+IAA0.2 mg﹒L^-1,最高分化率达100%。暗培养1 月左右即可得到再分化的不定芽。2.通过利用（60）^Coγ射线辐照、秋水仙素和不同激素,对乔纳金苹果叶片愈伤组织实施人工诱变,结果表明,60Coγ射线辐照和秋水仙素处理外植体再生易导致再生植株中途死亡,难以得到有效的再生植株。（60）^Coγ射线辐照较为适宜的辐照剂量为2400R 左右,秋水仙素处理以0.5%浓度处理28h 为佳。高浓度激素的处理效果优于低浓度,可以诱发高频突变。使用高浓度激素可获得高达50%的外观变异率且死亡率很小。外观变异性状多种多样,以叶片不同特点的变异居多。3.在盐浓度分别是0.2%、0.4%、0.6%的盐胁迫试验中,结果表明随着盐胁迫时间的延长,所有再生苗和对照的综合指数均下降。在不同盐浓度下,高激素再生的芽苗对盐分适应力较强;（60）^Coγ射线辐照剂量对再生苗耐盐性影响较大,生长势弱,抗性较弱,综合指数在不同盐浓度下均以3000R 辐照下降最多;运用0.3%、0.5%、0.6%浓度秋水仙素处理28h,再生苗综合指数均大于对照,而处理时间>28h,再生苗综合更多还原

【Abstract】 The C.V. Jonagold in vitro leaves of apple triploid is used as ourexperimental materials. Using two-step culture method on in vitro leaves, theperfect regeneration system for Jonagold has been established and therelationship between hormone and the regeneration of callus was proved. Onthe basis of which, The callus reaction to different mutation methods （highconcentration of hormone, （60）^Co radiation and colchicum） were studied andthe treated callus’growth and regeneration ability was determined, as well asthe shape and prosperities of regenerative plants. The mutation degree ofregenerative plants was studied from different angles （appearance,salt-tolerance, hot-tolerance and molecular level）. Furthermore, the plantswith perfect resistance had got. The main results are as follows:1.The new theory of tissue culture method by two steps on shootregeneration of leaves of apple was advanced for the first time in this paper.And an adventitious shoot regeneration system for apple cv. Jonagold hasbeen established by it, which has based on the induced mutation breeding intissue. In this experiment, the marked effect of BA to regeneration ofexplants has been proved. And it was obvious that IAA influenced thedevelopment of shoots. 2,4-D stimulated Callus formation and production,influenced the trend of regeneration of Callus or shoot. The most optimalmedium for Callus was MS+BA1.0 mg﹒L^-1+IAA0.2 mg﹒L^-1+2,4-D 0.8mg﹒L^-1, and for shoots was MS+BA4.0 mg﹒L^-1+IAA0.2 mg﹒L^-1, thehighest frequency was 100%. The regenerative shoots have gained after darkculture for one month.2.（60）^Coradial, colchicum and different kinds and concentration hormoneswere used to manage callus to mutate and to study the effect on regenerationof apple explants. Data showed that 60Coγradial and colchicum couldcaused too much death of explants and shoots to attain efficiencyregeneration. The decent radiation intensity of 60Coγwas 2400R; Theconcentration of colchicum was 0.5% and the best time was 28h. But impactof high consistency hormones was better than low one, and the mutationrates in appearance could reach 50% with a rather low death rate. Highconcentration of hormone can produce a high frequency of mutation, and the更多还原