Spokevicius Antanas, Taylor Lynette, Melder Emma, Van Beveren Kim, Tibbits Josquin, Creux Nicky, Bossinger Gerd
School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne;
School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne.
J Vis Exp. 2016 Oct 5(116):54553. doi: 10.3791/54553.
Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree's lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant's secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.
树木的次生茎生长及相关木材形成从生物学和商业角度来看都非常重要。然而，对于其发育的分子调控机制，我们了解得还相对较少。部分原因在于次生生长过程的研究常常受到物理、资源和时间的限制。许多体外技术已被用于木本和非木本植物物种，涉及植物部分或整个植物系统。然而，这些技术在次生茎生长过程研究中的适用性、某些物种的难处理性以及劳动强度等问题，对于中高通量应用来说往往是阻碍。此外，在研究次生茎发育和木材形成时，所研究的特定性状可能要在树木生长数年的生命周期后期才能够测量。为应对这些挑战，已开发出替代的体内实验方案，即诱导体细胞扇形分析，该方案直接在植物的次生茎中创建转基因体细胞组织扇形区。本方案的目的是提供一种高效、简便且相对快速的方法，用于创建转基因次生植物组织，以进行基因和启动子功能表征，可应用于一系列树种。此处呈现的结果表明，转基因次生茎扇形区可在多种树种次生茎的所有活组织和细胞类型中创建，并且次生茎中的木材形态特征以及启动子表达模式能够轻易评估，有助于中高通量功能表征。
