基于外植体褐化效应的水曲柳体胚发生发育生物学机制解析

Taking Fraxinus mandshurica which with evident explant browning effects as model tree species, the biological mechanism of tree somatic embryogenesis was studied through treating the explants with browning induction and inhibition regents. Taking explant browning degree as indexes to divide the initiation and development stage, the cell morphology, physiological and chemical status, and gene expression during the process of the already differentiated explant somatic cell reversing their developmental program and transiting to embryogenic cell followed differentiating and forming somatic embryo (SE) were studied. Taking explant browning status and SE status as indexes to divide the SE induction type, the cell morphology, physiological and chemical status, and gene expression during the differentiation and transition process of the explant somatic cell were studied. Hence, the biological mechanism was revealed for determining what kind and status of explant somatic cell were easy to transit to embryogenic cell and forming SE, what kind of exogenous regents and its treatments can stimulate explant somatic cell transiting to embryogenic cell and forming SE quickly and synchronously. The deduced mechanism will be confirmed by the SE induction of tree species and Fraxinus mandshurica explant type with low SE induction rate. Thereby, the biological mechanism of tree somatic embryogenesis will be elucidated and it can provide scientific support for the establishment of high-efficiency system of somatic embryogenesis.

以具有明显外植体褐化效应的水曲柳为模式树种，以外源褐化诱导和阻遏剂处理外植体，研究树木体胚发生发育生物学机理。以培养前和培养中水曲柳外植体表观褐化程度为指标划分体胚发生发育过程阶段，研究外植体体细胞转化为胚性细胞并形成体细胞胚过程中细胞形态、生理生化状态和基因表达状况；以经过和不经过外源处理剂处理的外植体表观褐化状态与体胚发生状态划分典型类型，研究不同类型外植体的细胞形态、生理生化状态和基因表达状况差异及其与体胚发生发育相关关系；从而推测确定水曲柳什么来源和状态的外植体体细胞容易转化为胚性细胞并形成体细胞胚、什么种类和处理方式的外源处理能够高效刺激外植体体细胞快速同步转化为胚性细胞并正常发育形成体细胞胚的生物学机制；用不褐化、低体胚发生率树种和水曲柳外植体类型进行体胚发生试验，验证前述推测的代表性和普适性。从而明确解析树木体胚发生生物学机理，为确定合理高效体胚发生调控技术提供科学依据。

项目的背景：水曲柳是我国生态和经济重要珍贵乡土树种。水曲柳体胚发生始终伴随着外植体褐化，这一特殊现象可被用来联系表观表现和内部生理生化与分子生物学机理的纽带而增加树木体胚发生生物学机理解析的效率。.主要研究内容：主要针对培养基渗透压、植物生长调节剂（PGRs）、抗坏血酸等抗褐化剂与水曲柳外植体褐化和体胚发生伴随现象相关的表观表现、解剖表现、生理生化和转录组表达等进行解析，同时对不同母树来源外植体的不同表现进行探索，对间接体胚发生及植株再生和该过程中胚性愈伤组织超低温保存技术进行了研究，用暴马丁香对水曲柳中发现的现象进行了初步的验证。.重要结果和关键数据：（1）确认PGRs是水曲柳体胚发生必需，其中生长素是必备条件，细胞分裂素是促进条件；高渗透压条件下外植体褐化程度与体胚发生率同步。（2）验证了水曲柳体胚发生中PCD、线粒体形态和结构、H2O2及氧化还原酶之间的相关性。（3）证实抗坏血酸(ASA)与水曲柳外植体褐化与体胚发生之间具有显著相关性，外源ASA增加了体胚诱导初期细胞内多酚的含量、降低了PPO和PAL的活性；ASA在胞内代谢和胞外降解同时进行，胞外降解为主要代谢过程，在体胚发生时期主要以抗氧化循环为主，说明体胚发生中ASA非单一作为抗氧化剂起作用。（4）突破间接体胚发生瓶颈，实现植株再生；建立胚性愈伤组织超低温保存技术。（5）解析了不同基因型外植体在褐化和体胚发生方面的响应格局，确认了外植体表观褐化程度、解剖结构和体胚发生之间有紧密的关联性，初步解析了加激素与否的转录组富集途径差异。（6）暴马丁香结果表明外植体中适量的多酚积累和较高POD活性有利于愈伤组织诱导和体胚发生。.科学意义：该项研究成果，特别是找到与水曲柳体胚发生和外植体褐化有特殊关联的外源调控物质抗坏血酸，在强化直接体胚发生技术基础上，取得了间接体胚发生途径进行植株再生的突破，为进一步取得水曲柳体胚发生生物学机理与调控技术及规模化扩繁技术的突破奠定了强有力的基础。.