In Vitro Organogenesis

植物细胞和组织在适当的条件下去分化并长出新芽和根的能力构成了现代植物生物技术的基础。顶端分生组织的形成是产生新的功能茎和根的关键事件。顶端分生组织负责初级植物机体的组织和器官形成--初级植物机体是植物所有细胞的母体。虽然在理解顶端分生组织在正常植物发育过程中的形成和功能方面已经取得了进展，但我们仍然不能完全理解在体外环境中导致根和茎发育的分生组织形成。这项研究计划的总体目标是提供对体外分生组织和器官形成的机制的理解。我们利用拟南芥手杖阶段的合子胚胎和根尖作为外植体，设计了新的芽再生方案。我们的方法与传统方法的不同之处在于不定芽诱导培养基中含有脱落酸(ABA)。这会导致形成绿色的器官来源愈伤组织，从中可以持续地生长出新芽。这种器官来源愈伤组织的精确鉴定使我们能够详细地研究不定芽的形成过程。此外，我们对拟南芥的使用使我们能够利用许多可用的转化系和已定义的抗体来自信地研究发育过程的细节。本研究的目的是：(1)研究器官发生愈伤组织的个体发育，确定负责不定芽形成的器官发生细胞；(2)研究与不定芽形成有关的结构和分子事件；(3)研究不定芽器官发生过程中激素的分布和水平的变化；(4)研究ABA在再生过程中的作用方式。正如伊恩·苏塞克斯教授在他的评论(Cell 56：225,1989)中所说的那样，“分生组织构成了植物”。没有功能分生组织的形成，就不能声称在任何系统中取得了任何生物技术的成功。所获得的结果将对顶端分生组织的形成提供良好的理论理解，并改进生物技术方案的设计。

The ability of plant cells and tissues to de-differentiate and give rise to shoots and roots under appropriate conditions forms the basis of modern day plant biotechnology. The key event in the generation of new functional shoots and roots is the formation of apical meristems. Apical meristems are responsible for tissue and organ formation of the primary plant body - the mother of all cells in plants. Although recent advances have been made in understanding apical meristem formation and function during normal plant development, we still do not fully understand meristem formation leading to root and shoot development from an in vitro environment. The overall goal of this research program is to provide a mechanistic understanding of in vitro meristem and organ formation. We have devised new protocols for shoot regeneration by using Arabidopsis zygotic embryos at the walking-stick stage and root tips as explants. Our protocol differs from the conventional procedure in that the shoot induction medium contains abscisic acid (ABA). This results in the formation of a green organogenic callus wherefrom shoots will consistently develop. This precise identification of the organogenic callus allows us to study the process of shoot formation in detail. Moreover, our use of Arabidopsis allows us to take advantage of the many available transformed lines and defined antibodies to study details of the developmental processes with confidence. The objectives of the proposed research are (1) to study the ontogeny of the organogenic callus and to characterize the organogenic cells that are responsible for shoot formation, (2) to study the structural and molecular events associated with shoot formation, (3) to study changes in the distribution and levels of hormones during the course of shoot organogenesis, and (4) to study the mode of action of ABA in the regeneration process. As Professor Ian Sussex stated in his review (Cell 56: 225, 1989) "The meristems make the plant". Without the formation of functional meristems, one cannot claim to have any biotechnological success in any system. The results obtained will provide a sound theoretical understanding of apical meristem formation and improved designs of protocols for biotechnology.