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.
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Acknowledgments
This work was supported and funded by the Commission for Technology and Innovation (CTI grant 16997), the Baugarten Stiftung Zürich, the University of Zürich, and the Swiss National Science Foundation (SNF grant 31003A_149974). We acknowledge Professor Ueli Grossniklaus (UG) for scientific support and insightful discussions and technical assistants of the department for organizational support and assistance with microscopy imaging (Christoph Eichenberger, Valeria Gagliardini, Arturo Bolaños, Peter Kopf).
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Mendocilla-Sato, E., She, W., Baroux, C. (2017). 3D Imaging of Whole-Mount Ovules at Cellular Resolution to Study Female Germline Development in Rice. In: Schmidt, A. (eds) Plant Germline Development. Methods in Molecular Biology, vol 1669. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7286-9_3
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DOI: https://doi.org/10.1007/978-1-4939-7286-9_3
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