3D Imaging of Whole-Mount Ovules at Cellular Resolution to Study Female Germline Development in Rice.

Abstract

Recent advances in fluorescence-based staining of cellular compartments coupled with confocal microscopy imaging have allowed the visualization of three-dimensional (3D) structures with cellular resolution in various intact plant tissues and species. Such approaches are of particular interest for the analysis of the reproductive lineage in plants including the meiotic precursor cells deeply embedded within the ovary of the gynoecium enclosed in the flower. Yet, their relative inaccessibility and the lack of optical clarity of plant tissues prevent robust staining and imaging across several cell layers. Several whole-mount tissue staining and clearing techniques are available. One of them specifically allows staining of cellular boundaries in thick tissue samples while providing extreme optical clarity, using an acidic treatment followed by a modified Pseudo-Schiff propidium iodide (mPS-PI) method. While commonly used for Arabidopsis tissues, its application to other species like the model crop rice required protocol adaptations for obtaining robust staining that we present here. The procedure comprises six steps: (a) Material sampling; (b) Material fixation; (c) Tissue preparation; (d) Staining; (e) Sample mounting; and (d) Microscopy imaging. Particularly, we use ethanol and acetic anhydride as fixative reagents. A modified enzymatic treatment proved essential for starch degradation influencing optical clarity hence allowing acquisition of images at high resolution. This improved protocol is efficient for analyzing the megaspore mother cells in rice (Oryza sativa) ovary but is broadly applicable to other crop tissues of complex composition, without the need for tissue sectioning.

Plant phenotyping relevance

イネの胚珠を細胞解像度で3Dイメージングするための染色・透明化・固定・顕微鏡プロトコルを他種向けに改良しており、植物組織の状態取得法が研究の中心である。

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