Department of Life Sciences, University of Bath;
Department of Life Sciences, University of Bath.
J Vis Exp. 2023 Jun 30(196). doi: 10.3791/65280.
Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.
有性生殖在开花植物中需要花粉粒和柱头表面之间的初始相互作用，在相互作用的伙伴之间建立分子对话。对一系列物种的研究表明，一系列分子检查点调节花粉-柱头相互作用，以确保只有相容的、通常是种内的花粉成功实现受精。在具有“干燥柱头”的物种中，如模式植物拟南芥，授粉后的第一个预合子相容性检查点是花粉水合作用的建立。授粉的这个阶段受到严格调控，花粉粒发出的信号促使柱头释放水分，从而允许花粉水合。准确测量和跟踪花粉随时间水合的能力是设计旨在理解繁殖过程中这一关键步骤调控的实验的关键。已发表的方案经常利用从母体植物上切下的花朵，在液体或固体培养基上保持，并进行大量授粉。本文描述了一种非侵入性、体内授粉生物测定法，可高分辨率地实时跟踪单个拟南芥花粉粒的分钟级水合作用。该测定法具有高度可重复性，能够检测花粉水合轮廓的非常细微变化，因此适合分析影响授粉途径的突变体。尽管该方案比批量授粉描述的方案更长，但它提供的精度和可重复性，以及其体内性质，使其成为详细剖析授粉表型的理想选择。
