Regulation of embryo development in culture

由于胚胎的不可接近性，与植物胚胎发育相关的生理和分子事件难以在体内进行研究。体细胞胚胎发生，即在培养中由无性细胞产生胚胎，为理解控制胚胎发育的机制，特别是在早期阶段，开辟了新的途径。本研究计划在油菜（Brassica napus）和白云杉（Picea glauca）中利用这一策略来研究胚胎发生的两个重要方面：谷胱甘肽代谢在油菜小孢子衍生胚胎发育中的作用以及云杉体细胞胚胎分生组织基因的鉴定。谷胱甘肽是一种普遍存在的分子，它以两种形式存在：还原形式和氧化形式。这种代谢物参与许多细胞和发育过程。最近，我们已经证明了谷胱甘肽池向其氧化状态的转换改善了胚胎发育和根尖分生组织的形成。作为这项工作的延伸，我们感兴趣的是识别谷胱甘肽在胚胎发生过程中调节的基因，并通过转化研究表征它们的功能。这些工作将通过生物化学研究加以补充，这些研究将确定谷胱甘肽在嘌呤和嘧啶核苷酸生物合成中的作用，这些嘌呤和嘧啶核苷酸参与植物生长和发育的调节。由于分生组织的形成是胚胎发生过程中的一个关键事件，我们也提出了鉴定新的ARGONAUTE基因参与胚胎顶极的形成和维持。转化研究将用于揭示这些基因的表达改变如何影响分生组织的结构。这些基因在其他分生组织标记基因的调控中的作用将通过激光捕获显微解剖技术进一步研究。这项研究将阐明调节胚胎发育的重要机制，并将促进加拿大生物技术行业用于植物物种体外繁殖的新型组织培养程序的设计。

The physiological and molecular events associated to plant embryo development are difficult to study in vivo, due to the inaccessibility of the embryo. The use of somatic embryogenesis, i.e. the generation of embryos in culture from asexual cells, opens new avenues for understanding the mechanisms controlling embryo development, especially during the early stages. The proposed research program utilizes this strategy in canola (Brassica napus) and white spruce (Picea glauca) to investigate two important aspects of embryogenesis: the role played by glutathione metabolism during embryo development in canola microspore-derived embryos and the identification of meristem genes in spruce somatic embryos. Glutathione is a ubiquitous molecule which exists in two forms: a reduced form and an oxidized form. This metabolite is involved in many cellular and developmental processes. Recently we have demonstrated that a switch of the glutathione pool towards its oxidized state improves embryo development and apical meristem formation. As an extension of this work we are interested in identifying genes regulated by glutathione during embryogenesis and in characterizing their function through transformation studies. Such work will be complemented by biochemical studies which will define the role of glutathione in relation to the biosynthesis of purine and pyrimidine nucleotides, which participate in the regulation of plant growth and development. As meristem formation is a key event occurring during embryogenesis, we are also proposing to identify novel ARGONAUTE genes involved in the formation and maintenance of the embryonic apical poles. Transformation studies will be employed to reveal how altered expression of these genes affect the architecture of the meristems. The role of these genes in the regulation of other meristem marker genes will be further investigated using laser capture microdissection techniques. This research will elucidate important mechanisms regulating embryo development and will facilitate the design of novel tissue culture procedures to be used by the Canadian biotechnology industry dedicated to the in vitro propagation of plant species.