Cellular and molecular aspects of brassica napus microspore embryogenesis

虽然胚胎发生是生物体发育的中心过程，但在植物中，早期事件很难研究，因为它们发生在胚珠内。获得足够数量的早期胚胎组织以在细胞和分子水平上研究该过程也是一个挑战。在过去的20年里，已经为几种植物开发了小孢子（未成熟花粉）培养系统，其产生大量在组成、形态和功能上与合子胚非常相似的胚。此外，在小孢子培养中，该过程的所有阶段都很容易获得，起始材料是单细胞的，培养基是液体的（并且容易改性）。对于大多数小孢子培养系统，发育触发因素是简单的胁迫处理。例如，甘蓝型油菜小孢子培养物中的胚发育通过温和的热处理诱导。由于这些原因，小孢子培养物是研究植物胚胎发生早期阶段的良好模型系统。此外，小孢子培养被广泛用于产生植物育种计划的双单倍体群体。 在过去的五年中，我们的小组已经开发了流式细胞术分析，以确定细胞，开始胚胎发生程序后3天，我们已经开发了细胞分选程序，以获得纯群体的胚胎发生细胞。我们的研究表明，与那些留在花粉发育途径中的细胞相比，胚性细胞变得基本，我们已经确定了100个在胚性细胞中上调的基因。拟议的工作的目标将是确定如何重定向的发展，从花粉成熟到胚胎发生，在细胞和分子水平上的小孢子培养物中启动和持续。从拟议的研究中获得的信息将有助于植物胚胎发生的最早阶段的基本理解，并可应用于增加目前对小孢子培养不敏感的基因型的反应，但对植物改良的目的是理想的。

Although embryogenesis is an organism's central development process, in plants the early events are difficult to study because they occur within the ovule. It is also a challenge to obtain sufficient quantities of early stage embryo tissue to study the process at cellular and molecular levels. In the last 20 years microspore (immature pollen) culture systems have been developed for several plant species which produce large numbers of embryos that are very similar to zygotic embryos in their composition, morphology and function. In addition, in microspore cultures, all stages of the process are readily accessible, the starting material is single-celled and the culture medium is liquid (and easily modified). For most microspore culture systems the developmental trigger is a simple stress treatment. For example, embryo development in Brassica napus microspore cultures is induced by a mild heat treatment. For these reasons microspore cultures are good model systems for studying the early stages of embryogenesis in plants. In addition, microspore cultures are widely used to produce double haploid populations for plant breeding programs. In the last five years our group has developed flow cytometric assays to identify cells that begin the embryogenic program as soon as 3 days after the heat treatment and we have developed cell sorting procedures to obtain pure populations of embryogenic cells. Our studies have shown that embryogenic cells become basic compared to those that remain in a pollen development pathway and we have identified 100 genes that are upregulated in embryogenic cells. The objective of the proposed work will be to determine how the redirection of development, from pollen maturation to embryogenesis, is initiated and sustained in microspore cultures at cellular and molecular levels. The information gained from the proposed studies will contribute to the basic understanding of the earliest stages of plant embryogenesis and could be applied to increase the responsiveness of genotypes that are currently recalcitrant to microspore culture but are desirable for plant improvement purposes.