【作者】 谭德冠；

【导师】 张家明；

【作者基本信息】 海南大学 ， 作物遗传育种， 2011， 博士

【摘要】 巴西橡胶树属大戟科橡胶属,是多年生的异花授粉乔木,在热带地区广泛种植。其所产的天然橡胶是四大工业原料之一,也是重要的战略物资,在国民经济中占有举足轻重的地位。巴西橡胶树花药培养是其遗传改良的重要技术手段,但体胚发生频率低是制约其组培苗工厂化生产和构建遗传转化体系的主要因素。本研究主要围绕如何改良巴西橡胶树花药体胚发生来开展一系列工作。我们对影响花药培养体胚发生的内外因素进行深入研究并对其培养条件进行改良；由于游离小孢子培养具有提高胚胎发生的优点,我们尝试在橡胶树开展这方面的工作；转基因技术是遗传改良的重要技术手段,我们克隆了胚胎发生关键基因并转化橡胶树的花药愈伤,以期获得具高体胚发生能力的转基因株系；我们首次发现花药愈伤中存在乳管细胞,探讨花药愈伤乳管细胞分化与体胚发生关系,对改良体胚发生也有一定的意义。上述这些研究的开展,将对提高橡胶树花药体胚发生和改良其花药培养有着重要的意义。本研究所取得的结果如下：(1)首次系统研究了橡胶树花药愈伤的形态特征、组织细胞学区别。橡胶树花药胚性与非胚性愈伤有着明显差别,前者表现为组织疏松脆性,表面凹凸不平,有较多小球状突起,细胞中含有较大的胞核,细胞形状呈规则小圆球形,细胞排列紧密,细胞体积较小；后者存在两种类型,第一种表现为结构紧密,整块组织硬实,表面较平滑,细胞形态不规则,胞质稀少,部分细胞含有极小的胞核,细胞体积大,第二种表现为组织松软、水渍状,细胞不含有胞核,胞质极稀薄并液泡化,细胞形状不规则,细胞排列较疏松,胞间间隙较大,细胞体积较大。同时发现胚性愈伤发生频率与其体胚发生频率呈极显著相关；胚性愈伤在培养后期细胞合成依然保持活跃,细胞分裂依然旺盛,细胞分化力高。首次对橡胶树花药胚性愈伤胚胎发生的生理生化机制进行深入研究。同一品种的胚性愈伤在整个培养过程中淀粉和蛋白质含量均高于非胚性愈伤,在培养后期开始积累淀粉,可能是为体胚发生作准备,为新的发育阶段提供物质和能量。胚性愈伤高含量的蛋白质可能是其体胎发生的关键因素。胚性愈伤中类胡萝卜素含量较高,表明其具有较强的抗氧化能力,使其细胞不易褐化,降低了组织块的褐化程度。高体胚发生频率的品种其胚性愈伤细胞代谢较活跃,分化力较强,从而消耗更多的碳源,同时这种碳源的消耗可能是促进其向胚胎发生的信号。胚性愈伤CAT、SOD的含量与体胚发生频率呈极显著相关,CAT、SOD可能是胚性愈伤体胚发生的基础。(2)对橡胶树花药体胚进行系统分类,共分9种类型；首次发现橡胶树花药体胚发生过程中产生双体胚现象,该类胚能正常发育成植株。对橡胶树花药体胚发生的培养条件进行改良。暗培养诱导胚状体2个月后再转移至500Lux弱光照条件下培养10-15d,然后再转移至出苗培养基培养,能够显著提高植株再生率；稀土具有提高橡胶树花药体胚发生率的效果,诱导胚胎发生培养基中以添加1mgL-’稀土培养效果最佳。(3)对橡胶树游离小孢子培养体系进行初步研究。小孢子的提取采用两种不同的方法进行,结果发现以间接研磨法为佳,虽然工作效率低,但污染率低、杂质少、培养效果好。对小孢子进行不同的预处理试验,发现用Starvation Medium B预处理液预处理小孢子2d有利于小孢子分化。比较两种不同的碳源培养效果,发现麦芽糖培养效果好。对诱导培养基中的pH值、组分进行研究,发现诱导培养基pH值为6.6时,以改良N6为基本培养基添加外源激素2,4-D 0.5 mgL-1、KT 0.5mgL-1诱导效果好,小孢子分化率达8.33%。巴西橡胶树游离小孢子分化依赖基因型,只有海垦2的小孢子具有分化力。小孢子的分化以B途径为主,初步获得小孢子分化的细胞团和微愈伤。同时以橡胶树小孢子为受体进行电激转化试验,瞬时表达结果证明外源基因已导入部分小孢子基因组中。(4)以橡胶树品种海垦2花药愈伤为受体,采用农杆菌介导法将胚胎发生基因BabyBoom转化橡胶树。结果表明：卡那霉素选择压以50mgL-1为最佳；共培养温度对转化效率影响显著,以20℃共培养效果较26℃好；侵染培养35-40d的愈伤转化效率最高,达100%。获得了抗性愈伤和转基因胚状体,经GUS组织化学法和PCR检测,表明外源基因已整合至它们的基因组中,为下一步转基因植株的获得奠定了基础。(5)通过组织化学法和免疫组织化学法首次证明巴西橡胶树花药愈伤中存在乳管细胞,虽然花药愈伤中存在少数的延伸形态的乳管细胞,但大部分表现为非延伸形态,不同于植物中现有的乳管细胞形态。这些非延伸形态的乳管细胞可能是有节乳管早期的原始特征。乳管细胞在花药愈伤中的分化依赖基因型,同时与体胚发生负相关。R107、RRIM600、热研8-79、热研7-33-97较海垦2有着更多的乳管细胞,但体胚发生率均显著低于海垦2。乳管细胞簇在PR107、RRIM600、热研8-79、热研7-33-97的愈伤中存在,但未能在海垦2花药中发现。这表明愈伤组织中的乳管细胞妨碍其体胚发生。如果能通过改良培养条件(激素调控等)来减少愈伤组织中乳管细胞数量,以期提高其体胚发生力,则有可能克服花药培养难题,这可能提供了一个改良橡胶树组织培养的连接点。更多还原

【Abstract】 Hevea brasiliensis belongs to Euphobiaceae. It is a perennial cross pollination tree and is widely planted in tropical areas. Natural rubber produced by Hevea is one of the most elemental industrial raw materials and strategic goods, which plays a key role in national economy. Anther culture is one of the most important biotechnological means to improve Hevea genetic property. However, its low embryogenic frequency prevents the large scale applications of juvenile-type clones and genetic transformation. In the present paper, a series of work was carried out to improve somatic embryogenesis of anther culture in Hevea. The internal and external factors influencing somatic embryogenesis were investigated and cultural conditions were improved. Based on the advantage of increasing the embryogenic frequency, the technique of isolated microspore culture was performed in Hevea in this paper. Transgenic technique is one of the most important approaches to crop genetic improvement and was also used in this paper. An expression vector harboring a key embryogenic gene was constructed and a transformation system was studied by Agrobaterium mediation. We found laticifer cells exist in callus cultures derived Hevea anthers for the first time. The correlation between laticifer cell differentiation and somatic embryogenesis was investigated, which may provide a hint for the improvement of somatic embryogenesis. The main results of this research were as follows:(1) The morphology and the histology of anther calli of Hevea were systemtically studied for the first time, and significant difference between embryogenic callus (EC) and non-embryogenic callus (NEC) were observed. EC was loose, friable, and unsmooth with many small spherical protuberances on its surface. EC cells were small, globular, and tightly attached to each other, and had a big nucleus. NEC included two different types, NECⅠand NECⅡ. The NECⅠwas compact, hard, and smooth, while the NECⅡwas soft, water-soaking. Histologically, both NEC cells were large and irregular in shape, and had thin cytoplasm. However, all of the NECⅡcells had no nucleus and some of NECⅠcells had an extremely small nucleus. Furthermore, the NECⅡcells were vacuolizated and had wider extracelluar gap than the NECⅠones. EC induction rate and its embryogenic rate were significantly correlated. The differentiation ability and the growth rate of the EC were still strong in the late cultural stages.Physiological and biochemical mechanism of embryogenesis of EC of the rubber tree was investigated for the first time. Starch content of EC was higher than that of NEC in all cultural stages, and EC cells accumulated more starch in its late cultural stages, which may provide material basis for further embryoid development. The high protein content may be a key factor of embryogenesis of EC. The high carotenoid content of EC indicated its cells have a strong antioxidative capacity preventing its cells brown and death. EC cells of the cultivars having high embryogenic ability had stronger differentiating ability and metabolized more animato compared to those having low embryogenic ability, which makes the former consume more carbon source. Therefore, the depletion of carbon source may activate a signal of embryoid development. The high content of CAT and SOD in EC of different cultivars and their embryogenic ability were significantly correlated, suggesting that CAT and SOD were the basis of embryogenesis.(2) Nine types of somatic embryos derived from anther calli were systematically classified, and double-embryoids were reported for the first time and they can develop into normal plantlets.Cultural conditions of somatic embryogenesis were improved. Plantlet regeneration was significantly improved by incubating the embryoids under weak light of 500 Lux for 10 to 15d after cultured for 2 months in the dark. The embryogenesis rate was significantly improved by additon of 1 mgL-1 rare earth in the induction media.(3) A protocol of isolated microspore culture in Hevea brasiliensis has been carefully studied in the present paper. Two different isolation methods of indirectly grinding anthers and directly grinding anthers were tested and the former was optimal with lower contamination, less impurity, and better culturing effect compared to the latter although it had a low efficiency. Different pretreatments were also studied and Starvation Medium B pretreatment for two days before the microspore incubation was the optimized one. The effects of different carbon sources and pH and components in induction media were investigated. The results showed maltose as carbon source and pH6.6 and modified N6 medium supplemented with 2,4-D 0.5 mgL-1 and KT 0.5 mgL-1 in induction media had an optimal induction effect with 8.33% differentiation rate of microspores which differentiated mainly through B way. Among five cultivars only Haiken2 obtained cell mass and microcalli from microspores, suggesting the differentiating ability of isolated microspores of Hevea is genotype-dependent. An electroporation test using microspores as receptors were carried out and the results of transient expression was confirmed the exogenous gene was expressed in microspores of Hevea by GUS staining.(4) The calli of Hevea variety Haikenl were genetically transformed using Agrobacterium EHA105 habouring pBIBBM that contained theβ-glucuronidase(GUS) and Kanamycin-resistant gene and Baby Boom gene. The results showed that the Kanamycin concentration at 50 mgL-1 was the best selective pressure, and the transformation rate of the cocultivation temperature 20℃was greatly higher than that of 26℃. This research optimized the genetic transformation system of Haiken2 by Agrobacterium mediation and obtained Kanamycin resistant callus lines and transgenic embryoids, which provided the prerequisite for getting the transgenic plantlets.(5) Laticifers are highly specialized cells present in over 20 plant families. They are well defined in planta. In vitro development of laticifers was also observed in some plants, but uncertain in the callus cultures of rubber tree, one of the most economically important latex producing plants. In the present study, we provide evidence that laticifer cells present in the callus cultures of rubber tree by histochemical and immunohistochemical studies. They present in the callus mainly as separate non-elongated form, very different from the morphology of laticifer cells in planta, excluding their origin from explants, although a few elongated laticifers were observed. These laticifer cells may be the initial characteristic of articulated laticifers.The occurring frequency of laticifer cells in the callus was genotype-dependent and negatively correlated with the somatic embryogenetic ability. The PR107, RRIM600, Reyan8-79, and Reyan7-33-97 had more laticifer cells in their callus cultures and lower cmbryogenesis compared to Haiken2, and laticifer clusters were only observed in the callus of the former four cultivars, suggesting that the presence of laticifer cells in the callus inhibit somatic embryogenesis in tissue culture of rubber tree. An improving cultural condition such as regulation exogenous hormone might be undertaken to reduce laticifer cells of calli and enhance their embryogenesis, which may provide a hint of improving anther culture of Hevea.更多还原