共培养促进青花菜顽固基因型小孢子胚胎发育的作用机理

The Broccoli breeding in China have been emerged in recent years, the germplasm resources for which is so scarce that most of the seeds for production are imported from abroad. Conventional breeding need more time and energy, and is a low efficiency of work. In contrast, the microspore culture for breeding can get lots of homozygous material in 1-2 years, increasing the chances to choose the beneficial genetic traits and accelerating the breeding process, so it has very important value. The broccoli DH lines was obtained successfully in previous work, but the microscopic changes that occur over the course of microspore embryo development and the molecular mechanism of embryos derived from different genotype materials is still not clear. Therefore, in this project, based on preliminary studies, different genotype material will be cultivated alone or co-cultivated with that the embryo easily derived, the real-time dynamic process of microspore embryo development will be observed by inverted microscope, fluorescence microscope and electron microscope. Differential displayed proteins involved in microspore development will be obtained by two-dimensional electrophoresis and the differential expression genes will be clear, the expression characteristics and function of which need to be further analysis and validation. The findings of this research project will help to find the key factors that constrain microspore embryogenesis and provide theoretical and practical basis for large-scale applications of microspore culture technology in the production.

青花菜在我国的育种工作起步晚，资源有限，生产上所用种子多从国外引进。常规育种方法费工费时，效率低。而小孢子培养能在1-2年时间内获得大量纯合材料，并能增加有益遗传性状选择几率，加快育种进程，具有十分重要的应用价值。申请人前期工作中成功获得了青花菜DH株系，但对小孢子胚胎发育过程中的显微结构变化及影响不同基因型材料出胚率的分子作用机制尚不清楚。故本项目拟在前期研究基础上，采用不同基因型材料单独培养及与易出胚材料共培养的方法，利用倒置显微镜、荧光显微镜及电镜观察小孢子胚胎发育的实时动态变化过程；采用双向电泳法获得与小孢子发育有关的差异蛋白，并根据获得的蛋白进行逆转录，以获得差异基因，并对此差异基因的表达特性进行分析和功能验证。本项目的研究成果将有助于找到制约小孢子胚胎发育的关键因素，为小孢子培养技术在生产上大规模应用提供理论和实践依据。

我国青花菜育种工作起步晚，种质资源有限，生产上所用种子多从国外引进，价格昂贵。常规育种方法费工费时，效率低。而小孢子培养能在1-2年获得DH株系，并能增加有益遗传性状选择几率，可加快育种进程，提高效率。. 本项目主要开展了青花菜小孢子胚胎发生的技术优化、易出胚甘蓝型油菜与难出胚青花菜品种小孢子混合培养、胚胎实时动态发育过程观察、再生植株群体倍性鉴定、胚胎发生早期小孢子转录组分析和基因功能注释及差异基因表达等系列研究。确定了小孢子处于单核晚期至双核早期时花蕾大小和形态、胚诱导培养基成分、热击温度和时间处理组合、胚状体在液体培养基中的滞留时间、胚转化培养基成分和琼脂浓度，以及生根培养基成分、组培苗驯化移栽等技术，建立了最佳的青花菜小孢子培养、胚成苗和驯化移栽系列技术体系；通过比较流式细胞仪DNA含量检测、FDA荧光染色和1%I-KI染色三种方法鉴定小孢子再生植株倍性的优劣，确定了1%I-KI染色法作为最适宜的检测手段；初步建立了油菜和青花菜共培养的方法，确定了花蕾数目比值为1:1、32.5℃热激1d时，每蕾平均出胚数明显高于对照，获得的青花菜再生株数最多；光学显微镜观察到小孢子热激后体积明显膨大，多次对称分裂形成原胚，15d后形成球形、心形或鱼雷形胚，25 d左右时，发育形成子叶型、球形和畸形胚状体，透射电镜发现了胚性小孢子自然加倍的过程，明确了小孢子胚胎发生的实时动态变化过程；对胚胎发育早期的小孢子开展了转录组分析，并把基因功能注释到GO、COG、KEGG数据库，初步明确了小孢子胚胎发育的代谢途径及差异基因表达情况，为进一步研究胚胎发生机理打下基础。. 以相关研究内容发表论文2篇，另1篇正在投稿中。获得国家授权发明专利3个：采用本技术体系成功获得了紫花菜小孢子再生植株（ZL201410371326.5）；共培养方法显著提高了羽衣甘蓝出胚率，并获得了大量再生植株（ZL201310338479.5）；建立的青花菜组培方法有助于小孢子再生植株中发现的雄性不育系材料保存和扩繁（ZL201310166265.4）。参加国际、国内相关会议各1次。