【作者】 董宁光；

【导师】 尹伟伦； 裴东；

【作者基本信息】 北京林业大学 ， 森林培育， 2012， 博士

【摘要】 不定根发生既是植物器官分化的重要理论问题,又关系到无性繁殖和完整植株再生等重大实践问题。成功诱导不定根对木本植物最大遗传增益的获得具有重要的实践意义。本研究以741杨(Populus alba(P.davidiana×P.simonii)×P.tomentosa)试管嫩茎为试材,对其不定根发生过程中的组织细胞学、相关酶活性变化、IAA(吲哚乙酸)和ABA(脱落酸)的免疫化学定位进行了分析,着重研究了741杨嫩茎不定根发生过程中内源IAA的产生、运输和积累,特别是亚细胞的分布特点。主要结果如下：1.采用石蜡切片技术结合番红-固绿染色,从组织学角度观察了741杨嫩茎不定根发生的过程,结果表明：741杨嫩茎的不定根原基起源于维管形成层细胞,其发生过程为：形成层细胞分裂分化形成根原基,根原基内细胞继续分裂分化形成不定根。2.在石蜡切片的基础上,通过PAS并结合萘酚黄S染色研究了741杨嫩茎不定根发生过程中淀粉粒和蛋白质的动态变化,结果发现：在生根诱导前,嫩茎基部的皮层、韧皮部和髓含有大量淀粉粒,诱导6d后,仅在嫩茎基部皮层中有少量淀粉粒分布,淀粉粒的转化可能为不定根的发生提供了能量和物质基础。在不定根诱导前,嫩茎基部蛋白质的量很少,随着不定根的诱导,蛋白质含量明显增多,且主要分布在维管形成层、韧皮部、根原基和根顶端分生组织这些分裂旺盛的部位,蛋白质合成增加与不定根发生密切相关。3.741杨嫩茎不定根发生过程中IAAO(吲哚乙酸氧化酶)、PPO(多酚氧化酶)和POD(过氧化物酶)的活性变化结果显示,IAAO活性在不定根的诱导期(0-6d),逐渐升高,在第6天,达到最大值,随后,IAAO活性逐渐下降。PPO活性在0-4d逐渐升高,4-6d,PPO活性基本保持恒定,在6-10d,PPO活性逐渐降低。POD活性在诱导的前两天逐渐升高,随后,POD活性逐渐下降。此结果说明,不定根的诱导与IAAO和PPO的活性呈正相关,而POD活性在741杨嫩茎不定根发生过程中的变化没有规律可循。4.运用免疫胶体金组织化学定位技术,结合IAA含量的HPLC-MS测定,研究了741杨嫩茎不定根发生过程中IAA在组织水平的变化规律,结果表明,在不定根发生的整个过程茎尖和叶片始终都有IAA分布,茎尖中的IAA主要分布在幼叶、叶原基和分生组织,叶片中的IAA主要分布在叶肉细胞,而叶柄、茎的中部和茎的基部中的IAA分布呈规律性变化,在不定根诱导前,叶柄、茎的中部和茎的基部几乎没有IAA分布,诱导6d后,在叶柄、茎的中部和茎的基部的维管组织也有明显的IAA积累,尤以嫩茎基部的IAA信号强烈,诱导8d后,根原基出现,叶柄、茎的中部的IAA信号开始减弱,而根原基中出现了强烈的IAA信号,诱导10d后,嫩茎基部出现不定根,此时叶柄和茎的中部的IAA消失了,而根中的IAA主要分布在维管组织和根尖。对嫩茎施加生长素极性运输抑制剂TIBA后,抑制了不定根的发生,也抑制了IAA在嫩茎基部维管组织的积累,但是并没有影响叶片中IAA的分布。IAA含量的HPLC-MS测定结果与IAA的免疫组织化学定位结果是一致的。以上结果表明,IAA在嫩茎基部维管组织的积累启动了不定根的发生,嫩茎基部维管组织积累的IAA可能主要由叶片经维管组织运输到嫩茎基部的,而非嫩茎基部自身合成的,当不定根形成后,由叶片到根的生长素极性运输减弱了,根中的IAA可能是由其自身合成的。茎尖的分生组织、叶原基和幼叶中的IAA可能由叶片提供。5.运用免疫胶体金电镜技术研究了不定根发生过程中IAA的亚细胞定位,结果表明,在叶片中,IAA主要分布在叶肉细胞的叶绿体中,随着不定根的诱导,有更多的IAA积累在了叶肉细胞的叶绿体中,说明叶绿体可能是IAA合成位点或者是储藏位点；在韧皮部的伴胞中,IAA主要分布在内质网和细胞膜,在细胞壁附近的细胞质中也有少量的IAA分布,在韧皮部的筛管中,细胞膜上有明显的IAA信号。在木质部的次生壁中,没有发现IAA,说明维管束中木质部可能不参与IAA的极性运输；在维管形成层细胞,IAA主要分布在细胞质、细胞膜、内质网和细胞核中,当形成层细胞分化为根原基后,IAA只出现在了根原基的细胞核,这个结果为IAA在不定根发生过程中的亚细胞作用位点提供了重要的线索,同时也暗示IAA对不定根起始和根原基发育这两个过程的调控机制是不同的。6.运用免疫胶体金组织化学定位技术,结合ABA含量的HPLC-MS测定,分析了741杨嫩茎不定根发生过程中ABA的组织学定位,发现不定根诱导前,ABA在嫩茎基部主要分布在皮层、维管束以及髓中,在根原基起始期(6d),ABA主要集中在维管束的韧皮部,在根原基形成期(8d),强烈的ABA信号分布在根原基。诱导10d后,不定根出现,此时ABA主要分布在根冠中。ABA含量的HPLC-MS测定结果与IAA的免疫组织化学定位结果一致。以上结果说明ABA在不定根发生过程中起正调控作用,同时也暗示在不定根发生过程中ABA可能参与调控同化物的运输与分配。更多还原

【Abstract】 Poplar 741(Populus alba×(P. davidiana+P. simonii) XP. tomentosa) shoots were rooted in vitro when cultured on 1/2 MS medium. The histocytology, changes of related enzym activity, histochemical localization of IAA and ABA were investigated, and the generation, transportation, accumulation and subcellular action sites were studied deeply.The main results were as follows:1 The process of adventitious root formation from poplar 741 shoots was anatomically examined, using paraffin section technology cobmining safranin-fast green staining. The reslts showed that no root primordial was fonund in the poplar 741 shoot before root induction and the induced root primodial was originated from vascular cambium. The process of the adventitious root formation from poplar 741 shoot was tha cambium cells divided and differentiated to root primordial and the root primordium grew and developed to adventitioud root.2 Changes of starch grains and proteins during adventitious root formation from poplar 741 shoots was investigated, using PAS and naphthol yellow staining methods. Results showed lots of starch grains were distributed in cortex, phloem and pith before root induction. After six days of induction, the satrch grains disappeared, suggesting than the starch grains provided energy and substances. Little proteins was found before root induction and proteins appeared as root induction progressed. The proteins were mainly located in the regions where cells divided vigorously, such as vascular cambium, root primordial and root apical meristem, suggesting expression of genes is active during adventitious root formation.3 Changes in IAAO, PPO and POD activity during adventitious root formation from poplar 741 shoot were analysed. During the induction phase (0-6 days) the IAAO activity increased, reached maximum on day 6 and then declined subsquently. PPO activity increased during 0-4 days, and was stable during 4-6 days, then decreased the following day. POD activity enhanced during the first two days and declined subsquently. The results suggest IAAO and PPO activitis were closely associated with adventitious root induction. Adventitious root formation was not correlated with POD activity.4 The spatial distribution of endogenous indole-3-acetic acid (IAA) and its dynamic changes during the adventitious root formation from poplar 741 shoots, using an immunohistochemical approach and HPLC-MS technology. The results showed the IAA signal in the shoot apex and lamina was always strong during adventitious root formation. And in shoot apex, the IAA was mainly located in the shoot apical meristem, leaf primordial and young leaf, in leaf, IAA was mainly located in mesophyll. However, the IAA in the petiole, middle and basal regions of the shoot presented regular changes. Before root induction, little IAA signal was found in the petiole, middle and basal regions of the shoot. After six days of root induction, obvious IAA signal was accumulated in the vascular bundles of the petiole, middle and basal regions of the shoots, especially in the vascular bundle of the shoots. After eight days of root induction, the cells of the vascular cambium developed into visible root primordia, which bore a strong IAA signal, and IAA signal in petiole and middle region of the shoot decreased. After ten days of root induction, the root primordia developed into adventitious roots, the markedly IAA signal was detected in root tip and root vascular cylinder, and little IAA signal was found in the petiole and middle region of the shoot.. Application of TIBA on poplar 741 shoot inhibited the adventitious root formation and the accumulation of IAA in the vascular bundle of the basal region of the shoot, but not affect the distribution of IAA in lamina. The content of IAA detected by HPLC-MS was consistent with the immunohistochemical results. The above results suggest accumulation of IAA in the vascular bundle of basal region of the shoot initiate the adventitious root occurrence, and differentiation and development of root primordial need IAA. IAA accumulated in the vascular bundle of the badal region of the shoot results from its polar transportation from mesophyll of the laminas, rather than by in site IAA generation. After adventitious root formed, the IAA transportation from lamina to the basal region of the shoot decreased, suggesting the IAA in the adventious root was sythesized by itself. Application of TIBA also affected the IAA distribution in shoot apical, namely the IAA signal in apical meristem, leaf primordial and young leaf was reduced, suggesting the IAA in this tissues was supplied by leaf.5 Subcellular localization of IAA during adventitious root formation from poplar 741 shoot studied by immuno-gold electro microscopic technology revealed organelle-specific distribution. In mesophyll, Particles representing immunostaining of IAA were clearly present in chloroplast starch grains on day 0 of induction. As the root induction progressed, stronger immunostaining of IAA was observed, still restricted to the chloroplast starch grains, suggesting the chloroplast starch grains was the site of IAA synthesis or store. In companion cells of the phloem, IAA gold particles were maily distributed in endoplasmic reticulum and plasma membrane. In sieve elements of the phloem, the most intens IAA signal was observed in the plasma membrane. In the secondary cell walls of the xylem, no gold particles was labelled. Thses results suggested that IAA polar transport from lamina to the basal region of the shoot during rooting was mediated by phloem. In cmbium cells, IAA gold particles were distributed most abundantly in the plasma membrane, endoplasmic reticulum, nucleus and partly in the cytoplasm. In root primordium cells, IAA gold particles were mainly distributed in nucleus. Thesw results provide important clues for the actiong sites of IAA during adventitious root formation and also suggest different IAA regulation mechanism exist in different cell duing adventitious root induction.6 The immunohistolocalization of ABA were investigated during adventitious root formation from poplar 741 shoot, using using an immunohistochemical approach and HPLC-MS technology. The results showed ABA was mainly distributed in the cortex, vascular bundle and pith before root inducation. After six days of induction, ABA was mainly concentrated in the phloem. After eight days of root induction, the cells of the vascular cambium developed into visible root primordia, which bore a strong ABA signal. After ten days of root induction, the root primordia developed into adventitious roots, the markedly IAA signal was detected in root cap. The content of ABA detected by HPLC-MS was consistent with the immunohistochemical results. These results suggest ABA positively regulates the adventitious root formation. And the localization of ABA in phloem suggests ABA may involved in the assimilates distribution during adventitious root formation.更多还原