【作者】 田春英；

【导师】 徐继忠； 邵建柱；

【作者基本信息】 河北农业大学 ， 果树学， 2009， 硕士

【摘要】 本研究以红富士苹果继代试管苗叶片为试验材料,结合石蜡切片法对叶片离体培养下不定芽的形态发生进行了较系统的解剖观察,研究了不定芽的发生途径、分化时间及起源部位等,对叶片不定芽再生过程中一系列生理生化指标进行了测定,对比分析了光照培养和黑暗培养及培养基中细胞分裂素种类对各生理生化因子的影响,旨在探讨各因子在不定芽发生过程中的作用机制,以期为叶片再生不定芽调控提供理论依据;此外,试验还对影响叶片再生的主要因素即细胞分裂素种类及配比、暗培养时间、碳源、继代苗龄等多方面进行研究,以期建立并优化高效、稳定的红富士苹果离体叶片再生体系,从而为其遗传改良奠定基础。主要研究结果如下:1红富士苹果离体叶片不定芽起源及发育的研究结果表明:不定芽的发生过程是,叶片接种4d左右为细胞启动分化阶段,随后细胞加速分裂,培养7～10d时形成分生细胞团,培养到14d时形成不定芽原基,14d以后不定芽原基开始向完整的不定芽分化,培养到18～21d时不定芽形成,21d以后不定芽开始伸长生长;研究还发现红富士苹果离体叶片不定芽的起源部位主要是叶片表皮细胞和叶片维管束周围的维管束鞘细胞,属于多起源;发生途径具有多样性。2红富士苹果离体叶片不定芽再生过程中生理生化变化2.1内源激素含量及平衡的变化叶片细胞启动分化期需要较高含量的ZR、IAA和ABA,且暗培养下内源激素含量高于光培养的,细胞旺盛分裂期需要高含量的ZR和ABA及低含量的IAA;且暗培养下内源激素含量高于光培养的。不同外源细胞分裂素对内源激素变化的影响存在显著差异,TDZ诱导的叶片内源ZR和ABA含量显著高于BA诱导的,IAA的含量显著低于BA诱导的。内源ZR/IAA与ABA/IAA的比值均呈现先升高再降低、再升高-降低的趋势,且光下培养的比值低于暗培养的,TDZ诱导的比值高于BA诱导的。GA3的结果表明,其对叶片不定芽再生有负作用。综合分析,不定芽再生需要较高含量的ZR和ABA及一定量的IAA,同时细胞启动分化期和芽原基形成阶段高比值的ZR/IAA和ABA/IAA有利于不定芽的分化。2.2内源多胺含量及平衡的变化叶片离体培养过程中,内源多胺含量峰值均出现在培养前期,Put含量高峰出现在培养3d时,而Spm和Spd的高峰出现在培养7d时。光培养下的内源多胺含量显著低于暗培养下的,TDZ诱导的叶片内源多胺含量显著高于BA诱导的。结果表明,在叶片细胞启动分化期及不定芽原基诱导初期需要高含量的内源多胺,并且在不定芽分化时期内源多胺含量也一直维持在较高水平;同时各内源多胺之间的比值也是前期处于较高水平,但是比值之间的差异不明显。可见,不定芽再生需要较高含量的内源多胺,且内源多胺的平衡对不定芽的诱导有一定的影响。2.3酶活性的变化叶片离体培养过程中,在叶片培养初期及后期较高的SOD酶活性有利于叶片不定芽的分化及发育;在后期较高的POD酶活性有利于不定芽发育;培养前期较低水平的CAT酶活性有利于不定芽的诱导,培养后期较高的CAT酶活性有利于不定芽的形成;IOD酶活性变化比较复杂,且IOD活性与IAA含量密切相关。2.4 NO含量的变化叶片离体培养以后,叶片内NO含量明显提高。首先,叶片细胞启动分化期及细胞旺盛分裂期需要维持较高水平的NO含量,才能诱导不定芽原基的产生;其次,在培养后期也需要较高含量的NO才有利于不定芽的发育。在不定芽诱导过程中,光下培养的叶片NO含量显著低于暗培养的;TDZ诱导的叶片NO含量高于BA诱导的。3红富士苹果离体叶片高效再生不定芽体系的优化结果表明:较适宜叶片再生不定芽的生长调节剂为TDZ 6.0 mg/ L+NAA 0.1 mg/L,最适宜的暗培养时间为3周,最适宜的糖为蔗糖,最佳的继代苗龄为25d左右。在上述培养条件下红富士苹果离体叶片再生率达到100%,平均叶片再生芽数达到10.6个。另外,卡那霉素能显著抑制红富士苹果叶片不定芽再生,但因固化剂不同其抑制效果不同,在6.2 g/L琼脂做固化剂时,10 mg/L卡那霉素可完全抑制叶片再生,而在5.0 g/L Polygel的培养基上,卡那霉素50 mg/L才能完全抑制叶片再生。更多还原

【Abstract】 This study was conducted with leaves collected from the test-tube plantlet of Red Fuji apple as the experimental materials. Through anatomical observation, morphogenesis of adventitious buds was researched by using method of paraffin wax slice. The regeneration ways, differentiation time and orign position of adventitious buds were studied. A series quota of physiology and biochemistry were also studied during in vitro culture. The effects of light, dark and different exogenous cytokinins on the content changes of different quotas were analysised, and the effect mechanism of these quotas was discussed. Above researches will providing a theoretical foundation for regulation of adventitious buds regeneration. Furthermore experiments studied impact factors of adventitious buds regeneration. Including kinds and ratio of cytokinins, dark incubation, c-source and so on. In order to optimize and establish the high efficient regeneration system, thus laying a foundation for transformation.1. Studies of origin and growth of adventitious budsThe results of observe indicated: after the leaves were inoculated , started the cell differentiation after day 4, accelerated cell division for a later, formed cell mass of meristemoids after day 7～10, bud primodium produced after day 14. At last adventitious buds formed and growth. Adventitious bud often originated from the epidermal cells and some vascular cylinder sheath cells. The ways of generation have diversity.2. Studies of physiology and biochemistry2.1 Changes of endogenous hormone contents and balanceThe hormone (ZR, ABA and IAA) contents of leaves had the higher level during the early stage of inoculation, and the contents were higher incubation in dark than those in light. Effects of exogenous cytokinins on endgenous hormone difference obviously, the ZR and ABA contents of leaves in culture medium appended TDZ were higher than those culture medium appended BA. IAA contents were lower than those culture medium appended BA. The results of ZR/IAA and ABA/IAA indicated that incubation in dark was higher than those in light, and in culture medium appended TDZ was higher than those appended BA. The results of GA3 indicated that adventitious buds regeneration maybe restrained by GA3. A comprehensive analysis show that regeneration of adventitious buds need high ZR and ABA contents levels and some concentration of IAA contents. and meanwhile higher ratios of ZR/IAA and ABA/IAA were closely associated with regeneration of adventitious buds.2.2 Changes of endogenous polyamine contents and balanceAfter inoculation, the polyamine contents of leaves obviously increased. During the initial stage of inoculation, the concents had reached peak, and the polyamine contents were higher incubation in dark than those in light, in culture medium appended TDZ than medium appended BA. At the stage of adventitious buds differentiation, the concents was also in higher level. The ratios of endogenous polyamine were increased at the early stage, and then gradually declined. Above all obvious indicated that it is needed high concents of endogenous polyamine at the stage of adventitious buds induction and growth. And meanwhile the balance of endogenous polyamine was closely associated with adventitious buds regeneration at the early stage.2.3 Changes of enzyme activityThe higher activity of SOD was propitious to differentiation and growth of adventitious buds at the initial stage and later stage. The higher activity of POD was propitious to growth of adventitious buds at the later stage. The loeer activity of CAT was propitious to induction of adventitious buds at the early stage, and the higher activity of CAT was propitious to form of adventitious buds at the later stage. The changes of IOD activity of leaves were complex, and the changes of IOD activity were closely associated with endogenous IAA contents.2. 4 Changes of NO contentsDuring the culture of leaves in vitro, NO contents were increased obviously after inoculation. At first, it need higher contents of NO to induce bud primodium at the stage of cell started differentiation and cell division bloom; secondly, higher contents of NO were propitious to growth of adventitious buds at the later stage. And then during regeneration of adventitious buds , the NO contents of leaves was higher incubation in dark than those in light, and in culture medium appended TDZ than those in culture medium appended BA.3. High efficient system optimize of regeneration from leaves in vitro of Red Fuji appleThe results showed that the optimum combination of growth regulators were TDZ 6.0 mg/L+NAA 0.1 mg/L, the optimum c-source was sucrose, the optimum dark incubation time was 3 weeks, the optimum shoot age was about 25d. With above culture conditions, the leaf regeneration frequency of Red Fuji reached 100% and the number of buds per leaf reached 10.6. In addition adventitious buds were restrained evidently by varying concentration Kanamycin. But agar and polygel was different. Adventitious shoots could be restrained completely by only 10mg/L Kanamycin on 6.2 g/L agar, while restrained completely by 50 mg/L Kanamycin on 5.0 g/L Polyge.更多还原