黄瓜小孢子培养及胚胎发生机理研究

Cucumber (Cucumis sativus L.) is an important vegetable crop worldwide that was considered high economic efficiency. But it is limited in the genetic base and germplasm resources, it is necessary to utilize efficient means to obtain high-yield and quality varieties. Microspore culture is a rapid and effective method for providing high yield and quality varieties, which develope into haploid, the homozygous diploid obtained after chromosome doubling. However, microspore culture is still not successful culture at domestic and foreign. The immature culture system and the mechanism of embryogenesis is unclear, resulting in microspore culture is difficult to apply to cucumber breeding. The project is based on the laboratory in cooperation with the basic of microspore culture of cucumber studies, and the research project concerning the key factors affecting microspore embryogenesis, the regeneration technology of embryos, the identification of ploidy and the chromosome doubling technology. The purpose is to find out the factors with the relevant the cucumber microspore culture, to establish cucumber microspore culture system, to using microspore culture to get or create a group of DH cucumber lines used in cucumber germplasm innovation. Cytoskeleton restructuring and the formation of pre-prophase band (PPB) is the key factors to induce embryogenesis, we can utilize the microscopy system to observe the changes in the cytoskeleton in course of training, for instance, microtubules and microfilament and process of embryogenesis. Simultaneously, we will observe the cytoskeleton, changes in cell morphology and the progress of embryo development, which will provide the theory for microspore culture of cucumber.

黄瓜是重要的世界性蔬菜作物，经济效益高。但其遗传基础狭窄，种质资源有限，需利用高效的育种手段提供高产、优质的品种。利用小孢子培养形成单倍体，染色体加倍后获得纯合二倍体，是一条快速有效的途径。但黄瓜小孢子培养在国内外仍是未攻克的难题，培养体系不成熟，胚胎发生机理不清，导致小孢子培养技术不能应用于黄瓜育种。本项目基于合作单位实验室对黄瓜小孢子培养研究的基础，对小孢子胚胎发生的相关因素，植株再生因素，倍性鉴定及染色体加倍等进行研究，探明影响黄瓜小孢子培养的相关因素，建立黄瓜小孢子培养技术体系；利用小孢子培养技术，获得一批黄瓜DH系，用于黄瓜种质创新；细胞骨架重组并形成早前期带微管是胚诱导的关键，利用显微系统观察培养前后小孢子细胞骨架（微管和机动蛋白丝）的变化及胚胎发生过程，研究黄瓜小孢子培养中细胞骨架、细胞形态变化及胚发育过程，为黄瓜小孢子培养提供理论依据。

双单倍体植株的生产可以在短时间内从杂合子亲本中获得纯合系，在植物育种中发挥了重要作用。诱导雄核或雌核发生是黄瓜获得双单倍体的一种有效途径，但由于缺乏有效的再生技术，使其再生率较低，阻碍了该技术在黄瓜品种改良中的应用。该研究利用黄瓜优良杂交种进行小孢子和子房培养，同时进行胚胎发生机理研究。黄瓜小孢子培养属于玩拗基因型，这几年初步建立黄瓜小孢子培养技术体系，具有胚胎发生，但最终没有获得再生植株。通过本项目研究，拓展研究了子房培养诱导双单倍体，通过4个试验(一步培养、冷预处理、基因型效应和TDZ(噻苯)浓度)，探讨了一种联合有效的未授粉子房培养直接再生植株的方法。结果表明，一步培养基（MS+0.06mg/L、蔗糖3%、琼脂7%）加4℃预处理4 d，可直接诱导植株再生。基因型间再生率差异较大，分别为73.3%和10.0%。经流式细胞仪鉴定为二倍体或单倍体，并计数保卫细胞叶绿体数。通过SSR进一步鉴定二倍体植株为纯双单倍体。这些结果表明，低温预处理和一步培养基加TDZ可用于黄瓜单倍体育种。. 诱导雌核发生生产双单倍体的机制还没有得到很好的理解。本研究采用RNA测序技术对黄瓜卵巢培养过程中的转录体进行了研究。分析了不同时期胚胎发育过程中差异表达的基因(DEGS)。结合转录和细胞形态学分析，揭示了卵巢培养胚胎发生的主要特征。细胞扩增、分裂活跃，特异基因、激素信号转导、激素反应和应激诱导基因在胚胎早期表达上调。细胞继续分裂形成前胚、球状胚、心形胚、鱼雷胚和子叶胚，GO分析表明，差异表达的基因参与了微管的运动、细胞或亚细胞成分的运动，具有蛋白质复合物结合、微管结合、四吡咯结合、微管蛋白结合等微管活性。主要参与内质网蛋白加工和类苯丙烷生物合成。芽的形成受编码b3结构域、ap2/ERF家族和WUS同源结构域蛋白的转录因子调控。这一结果为理解诱导黄瓜雌核发生再生植株的分子机制提供了有价值的资源。