【作者】 王亚丽；

【导师】 林位夫； 吴继林； 郝秉中；

【作者基本信息】 华南热带农业大学 ， 作物栽培学与耕作学， 2004， 硕士

【摘要】 植物组织培养技术是植物生物学和植物基因工程学的连接点，也是现代作物栽培学的一个重要研究领域。植物体外形态发生的生物学研究为改进植物组织培养技术提供重要的理论根据。 巴西橡胶树(Hevea brasiliensis)体外再生技术在20世纪70年代已经成功，但目前巴西橡胶树组织培养技术仍然比较落后，至今只有少数无性系能够实现体外形态发生，且大多数品系体外植株再生的效率非常低下，这已经成为建立高效遗传转化体系，从分子水平进行橡胶树遗传改造的重要障碍。此外，过去对体外形态发生的组织学和细胞学分析涉及也很少。本论文通过研究橡胶树体外胚胎发生的过程，为探索利用基因转化等技术改进橡胶树组织培养技术，提高橡胶树体外体细胞胚发生能力和胚状体成苗率打基础。研究结果将对植物体外形态发生的理论研究提供宝贵的资料，为利用胚性愈伤组织作为转化受体提供组织学依据。 本试验以巴西橡胶树，海垦2的花药为外植体，诱导体胚的发生，对体细胞胚发生方式及发育过程做了一系列的组织学观察，分析了橡胶树体细胞胚发生的各个过程以及畸形胚的形成原因，初步结果如下： 1 橡胶花药体细胞胚主要以间接方式发生。体细胞胚的发生经愈伤组织过程，形成胚状体。体细胞胚起源于胚性愈伤组织中含单宁的细胞包围的单个胚性母细胞，属于单细胞起源。大多数胚状体起源于胚性愈伤组织近表层的几层细胞，极少数起源于愈伤组织的内部。 2 橡胶树体胚具有双极性，体细胞胚发生早期就具有胚根和胚芽两极。单个胚性母细胞也具有极性，第一次分裂多为不等分裂，形成二个细胞，其中近愈伤组织表层的一个为顶细胞，近愈伤组织内部的一个为基细胞。以后，顶细胞纵分裂成二细胞、四细胞到多细胞原胚，发育成球形胚、心形胚、鱼雷形胚和子叶胚；基细胞横分裂，形成单列细胞组成的胚柄，华南热带农业大学硕士毕业论文并随着体胚发育，细胞构成的胚柄。胚柄细胞继续分裂，在球形胚阶段，形成明显的由多列在胚性母细胞分裂到多细胞原胚的形成过程，细胞壁加厚，与周围其它的细胞形成明显的界限，产生孤立化。其外围的球形胚时期开始有原表皮的分化，至鱼雷胚时，可以看到明显的原形成层的分化。此外，体细胞胚的发生和分裂是不同步的，往往在同一张切片上既可看到单个的胚性母细胞，又可见到二细胞、四细胞以致多细胞原胚并存的多样化原胚细胞群体。 3橡胶树体胚的发育过程，心形胚至鱼雷形胚阶段是胚胎发育的关键时期。花药很容易诱导成球形胚或心形胚，但大多数胚状体发育停留在球形胚或心形胚阶段，不能形成正常胚(如鱼雷胚等)，而成为畸形胚，只有少量发育成正常胚，最终发育成正常植株。 4畸形胚在不同发育阶段有不同的特点。在球形胚阶段，可以分为三种:①缺少胚柄;②缺少原表皮;③球形胚败育。正常的球形胚和某些畸形球形胚继续发育，在心形胚或鱼雷胚阶段又出现出畸形胚，可以分为:①无茎端生长点;②原形成层发育不正常;③外形畸形，如有的是连胚，有的呈指状，有的呈叶片状等等。畸形胚有的败育，有的能继续发育，在子叶胚阶段形成畸形的植株。畸形植株又可以分化两大类:一是根端发育正常，茎端发育不正常，成团块状、连胚状、杯状、叶片状和喇叭口状等;二是根端、茎端发育都不正常。 5调控体胚的发育提高花药培养的效率和成苗率措施的建议。可以利用物理、化学等手段如激素调控等解决体胚细胞分裂发育非同步化的问题，然后再进一步调控体胚的分化。此外，也可以利用已知的调控植物合子胚或体外体细胞胚发生的重要基因，采用基因转化技术提高橡胶树体外体细胞胚发生能力。更多还原

【Abstract】 The technology of plant tissue culture could be considered as a junction connecting the plant biology with the plant genetic engineering and also as an important research field in modern crop cultivation. The study on cytobiology of somatic embryogenesis could provide an important theoretical foundation for improving the technology of plant tissue culture.The technique of somatic embryogenesis in Hevea brasiliensis was succeeded in 1970s. But now the technique was dropped behind because that so far, only a few the clones could produce somatic embryogenesis through this technique, and the rate of the in vitro plant regeneration in most of the clones was very low. This became the main obstacle in the way to found the high genetic transformation system for reconstructing the inheritance of Hevea in molecule level. Moreover, a few of studies were involved on the histology and cytology of in vitro morphogeny in the past days. It was studied that the process of the somaticembryogenesis and analyzed that the problem existing in technique of in vitro morphogeny in Hevea to ground for that to probe into the method to improve the technology of tissue culture in Hevea with the techniques of transgene and to improve the ability of somatic embryogenesis and plant percentage of embryoid in Hevea. The result of this study also could enrich up the data for theory of the somatic embryogenesis of plant and provide histological foundation for the using of embryonic callus tissues as transformational receptor.The anthers of Hevea Brasiliensis Haiken 2 were used as explants for inducing the somatic embryoids. The histology was observed that the generating mode and development process of the somatic embryos in Hevea by microscope and analyzed every phase of somatic embryos generating and the cause of formation of abnormal embryos in Hevea. The results were shown as follow.1 Somatic embryos of anther in Hevea generates mainly in indirect mode. The somatic embryos of anther were formed through callus tissue. The somatic embryo originated from single embryonic original cell of embryonic callus tissues, which was surrounded by tannin cells, that belonged to single cell origination. Almost all somatic embryos originated from several layers cells, which near to the external callus, only a few of them originated from the internal callus.2 Somatic embryos in Hevea had bipolarity. There were the radicle polarity and the plumule polarity in differentiation of the first somatic embryogenesis. Polarity also exited in single embryonic original cell, which divided unequally into two cells, one nearing to the external callus was the apical cell, and the other nearing to the internal callus was the basal cell. After that, the apical cell divided vertically respectively to form 2-celled, 4-celled, and multicellular proembryos and developed into globular embryoid, heart-shaped embryoid, torpedo embryoid,cotyledonary embryoid. And the basal cell divided transversely to form suspensor structure, which formed into single line cells in shape and with many lines cells in shape in the period of globular embryoid. When the embryonic original cell divided to form multicellur proembryos, the cell wall of the external cell was thickened to form a clear cell boundary to separate from ambient cells that was a phenomenon of isolation The protoderm began to be differentiated in the period of globular embryoid, and the procambium differentiated clearly in period of torpedo embryoid. Moreover, developments of somatic embryoids were non-synchronous, it could be seen in the same time that single embryonic cell, 2-celled, 4-celled and multicellular proembryos existed in one cutting generally in all process.3 The key period was from heart-shaped embryoid phase to torpedo embryoid phase, during the somatic embryogenesis in Hevea. It was easily to induce to globular embryoid or heart-shaped embryoid, but most of them stayed at this state and could not develop into normal embryoid, such as torpedo embryoid, but abnormal embryoid. Only a few of them developed更多还原