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，专门为植物体生产新细胞。分生组织在植物生长发育中的重要性可以用Ian Sussex教授的评论“The meristems make The plant”（Cell 56: 225, 1989）中的一句话来概括。在过去的资助期内，我们证明了茎分生组织的形成对生长素的扰动很敏感。此外，在离体胚发生过程中，虽然形成了茎分生组织，但随着培养时间的延长，茎分生组织的组织结构逐渐退化，并不能稳定地确定。另一方面，根分生组织的发育需要生长素。这些观察结果表明，生长素水平和胚胎环境因素可能改变分生组织基因的表达，从而改变细胞命运。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。氧化还原环境会影响胚的质量，尤其是茎尖分生组织的质量。关于分生组织的发生及其决定过程，仍有许多重要的、未解的问题。目前，我们收集了许多以β -葡萄糖醛酸酶（GUS）为标记基因的拟南芥分生组织相关增强诱捕株系。在下一个资助期内，我们将利用这些增强诱捕系来研究分生组织的形成以及生长素和其他培养基添加剂在体外发育中的作用。使用GUS标记基因的优点是，一旦该基因随后被翻译，我们就可以染色酶活性，从而获得蓝色。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性。我们将进一步探讨氧化还原环境对胚胎发育的重要性。将使用白云杉系统。氧化还原化合物，即还原性和氧化性谷胱甘肽对胚胎性组织增殖和程序性细胞死亡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.