A simple and versatile plasma membrane staining method for visualizing living cell morphology in reproductive tissues across diverse plant species

Abstract

Plant reproduction involves dynamic spatiotemporal changes that occur deep within maternal tissues. In ovules of Arabidopsis thaliana (A. thaliana), one of the two synergid cells degenerates at fertilization, while the fertilized egg cell (zygote) undergoes directional elongation followed by asymmetric division to initiate embryonic patterning. However, morphological analysis of these events has been hampered by the limitations of conventional cell wall staining, which fails to label cells lacking complete walls, and by the requirement for transgenic fluorescent reporters to visualize cell outlines. Here, we report that the membrane-specific fluorescent dye FM4-64 readily permeates ovules, allowing clear visualization of reproductive cell morphology both before and after fertilization. This staining method supports high-resolution time-lapse imaging and quantitative analysis of early embryogenesis in living tissues. Importantly, it is applicable not only to the angiosperm A. thaliana but also to the liverwort Marchantia polymorpha (M. polymorpha) and the fern Ceratopteris richardii (C. richardii), enabling the visualization of live reproductive cell structures within maternal tissues and revealing fertilization-associated morphological changes. This simple and robust method thus provides a valuable tool for spatiotemporal and quantitative analyses of reproductive processes across a broad range of plant species, without the need to generate transgenic lines.

Plant phenotyping relevance

生きた植物生殖組織の細胞形態を可視化・定量化する蛍光染色法を開発し、複数種で適用・検証しているため、植物フェノタイピング手法が中心である。

Code and data availability

The paper describes FM4-64 staining and microscopy of reproductive tissues, with image quantification done in Fiji, but no public phenotype/trait datasets, image deposits, author analysis code, or trained models are mentioned. Fiji (https://fiji.sc/) is a generic third-party tool, not a paper-specific asset.

No evidence-backed public reproduction asset is currently recorded.