Structural, physiological and biochemical aspects of embryo development in vivo and in vitro

顶端分生组织位于每个生长点，专门为植物身体产生新的细胞。分生组织在植物生长和发育中的重要性可以用伊恩·苏塞克斯教授的评论《分生组织造就植物》(Cell 56：225,1989)中的一句简单的话来概括。在过去的资助时期，我们证明了茎分生组织的启动对生长素的扰动很敏感。此外，在体外胚胎发生过程中，虽然形成了茎分生组织，但由于其组织结构随着培养时间的延长而恶化，因此并不稳定。另一方面，根分生组织的发育需要生长素。这些观察表明，分生组织基因的表达和细胞命运可能会受到生长素水平和胚胎环境因素的影响。我们还证实了细胞及其周围环境的氧化还原状态可以调节体外胚胎的发育。氧化还原环境可以影响产生的胚胎的质量，特别是茎的顶端分生组织。关于分生组织的启动及其决定过程，仍然有许多重要的悬而未决的问题。目前，我们已经收集了一批以GUS(β-葡萄糖醛酸酶)为标记基因的拟南芥分生组织相关增强子陷阱系。在下一个资助期，我们将利用这些增强子-陷阱系来研究分生组织的形成以及生长素和其他介质添加剂在体外发育过程中的作用。使用GUS标记基因的好处是，一旦该基因随后被翻译，我们就可以对产生蓝色的酶活性进行染色。*通过研究颜色模式的变化，我们可以研究分生组织发育过程中各种分生组织基因的活动。*我们将进一步研究氧化还原环境对胚胎发育的重要性。我们将使用白云杉系统。氧化还原化合物，即还原和氧化的谷胱甘肽在胚胎组织增殖和程序性细胞死亡中的作用将通过组织学、组织化学和生化方法进行研究。

Apical meristems are located at each growing point and are specialized for the production of new cells for the plant body.  The importance of meristems in plant growth and development can be summarized by a simple statement from Professor Ian Sussex's review "The meristems make the plant" (Cell 56: 225, 1989). In the past funding period, we demonstrated that shoot meristem initiation is sensitive to auxin perturbation.  Furthermore, during in vitro embryogenesis, although a shoot meristem is formed, it is not stably determined as its organization deteriorates upon prolong culture. The root meristem, on the other hand, requires auxin for development. These observations suggest that expression of the meristem genes and hence the cell fate could be altered by auxin levels and factors from the embryonic environment. We also established that the redox state of both the cell and its surrounding environment can modulate in vitro embryo development.  The redox environment can influence the quality of the embryos produced, especially the shoot apical meristem.There are still many important, unanswered questions regarding meristem initiation and its determination process.  At present, we have in our collection a number of Arabidopsis meristem-related enhancer-trap lines with GUS (beta-glucuronidase) as the marker gene.  In the next funding period, we will make use of these enhancer-trap lines to study meristem formation and the role of auxin and other medium additives during in vitro development.  The advantage of using the GUS marker gene is that once this gene is subsequently translated, we can stain for the enzyme activity which gives a blue color.  By studying changes in the color pattern, we can study the activities of various meristem genes during the course of meristem development.  We will further investigate the importance of the redox environment on embryo development.  The white spruce system will be used. The role of the redox compounds, i.e. reduced and oxidized glutathione on embryogenic tissue proliferation and programmed cell death will be studied using histological, histochemical, and biochemical methods.