Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Curr Protoc. 2022 Jan;2(1):e349. doi: 10.1002/cpz1.349.
Questions in developmental biology are most frequently addressed by using fluorescent markers of otherwise invisible cell states. In plants, such questions can be addressed most conveniently in leaves. Indeed, from the formation of stomata and trichomes within the leaf epidermis to that of vein networks deep into the leaf inner tissue, leaf cells and tissues differentiate anew during the development of each leaf. Moreover, leaves are produced in abundance and are easily accessible to visualization and perturbation. Yet a detailed procedure for the perturbation, dissection, mounting, and imaging of developing leaves has not been described. Here we address this limitation (1) by providing robust, step-by-step protocols for the local application of the plant hormone auxin to developing leaves and for the routine dissection and mounting of leaves and leaf primordia, and (2) by offering practical guidelines for the optimization of imaging parameters for confocal microscopy. We describe the procedure for the first leaves of Arabidopsis, but the same approach can be easily applied to other leaves of Arabidopsis or to leaves of other plants. © 2022 Wiley Periodicals LLC. Support Protocol 1: Preparation of plant growth medium Support Protocol 2: Preparation of growth medium plates Basic Protocol 1: Seed sterilization, sowing, and germination, and seedling growth Support Protocol 3: Preparation of IAA-lanolin paste Basic Protocol 2: Application of IAA-lanolin paste to 3.5-DAG first leaves Basic Protocol 3: Dissection of 3- to 6-DAG first leaves and leaf primordia Basic Protocol 4: Dissection of 1- and 2-DAG first-leaf primordia Basic Protocol 5: Mounting of dissected leaves and leaf primordia Support Protocol 4: Quality check of mounted leaves and leaf primordia by fluorescence microscopy Basic Protocol 6: Imaging of mounted leaves and leaf primordia by confocal microscopy.
发育生物学中的问题通常通过使用荧光标记物来解决，这些标记物可以标记原本不可见的细胞状态。在植物中，最方便的方法是在叶片中解决这些问题。事实上，从叶片表皮内的气孔和毛状体的形成到叶脉网络深入到叶片内部组织的形成，每个叶片的发育过程中，叶片细胞和组织都会重新分化。此外，叶片大量产生，并且易于可视化和干扰。然而，尚未描述用于发育叶片的干扰、解剖、安装和成像的详细程序。在这里，我们通过提供用于向发育叶片局部应用植物激素生长素的稳健、逐步的方案，以及用于常规解剖和安装叶片和叶原基的方案，解决了这一限制（1），并提供了用于共聚焦显微镜成像参数优化的实用指南。我们描述了拟南芥第一片叶子的程序，但同样的方法可以很容易地应用于拟南芥的其他叶子或其他植物的叶子。©2022 威立公司。支持方案 1：植物生长培养基的制备支持方案 2：生长培养基板的制备基本方案 1：种子消毒、播种和萌发以及幼苗生长支持方案 3：IAA-羊毛脂膏的制备基本方案 2：将 IAA-羊毛脂膏应用于 3.5-DAG 第一片叶子基本方案 3：3-6-DAG 第一片叶子和叶原基的解剖基本方案 4：1-和 2-DAG 第一片叶子原基的解剖基本方案 5：解剖叶片和叶原基的安装支持方案 4：通过荧光显微镜检查安装叶片和叶原基的质量检查基本方案 6：通过共聚焦显微镜对安装叶片和叶原基进行成像。
