《红富士苹果离体叶片不定芽再生机理研究》由会员分享，可在线阅读，更多相关《红富士苹果离体叶片不定芽再生机理研究（63页珍藏版）》请在金锄头文库上搜索。
1、河北农业大学 硕士学位论文 红富士苹果离体叶片不定芽再生机理研究 姓名：田春英 申请学位级别：硕士 专业：果树学 指导教师：徐继忠；邵建柱 2009-06-10 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
2、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 710d 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 1821d 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
3、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
4、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
5、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
6、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/ LNAA 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
7、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。 关键词：红富士苹果；不定芽发生；生理生化变化；影响因子 Studies on Regeneration Mechanism of Adventitious Buds from Leaves in vitro of Red Fuji Apple Author: Tian Chun-ying Majo
8、y: Pomology Tutor: Xu Ji-zhong and Shao Jian-zhu 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 slic
9、e. 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
10、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, da
11、rk 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 buds The results of observe indicated: after the leaves were inoculated , started the cell differ
12、entiation after day 4, accelerated cell division for a later, formed cell mass of meristemoids after day 710, 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
13、 of generation have diversity. 2. Studies of physiology and biochemistry 2.1 Changes of endogenous hormone contents and balance The 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 l
14、ight. 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 indica
15、ted 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 Z
16、R 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 balance After 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 th
