A Simple Automated Method for Microstructural Fluorescence Image Analysis to Determine the Degree of Polyploidy in Mono- and Dicotyledonous Plant Cells

Abstract

An evaluation of plant ploidy is an important task in breeding and biotechnology. Current methods of ploidy assessment (flow cytofluorometry and microscopy) are time-consuming and costly, and not applicable to real-world agricultural conditions. We developed an automated method for ploidy assessment based on fluorescence microscopy, which aims to accelerate and reduce the cost of plant ploidy analysis. The method is based on the automated selection of plant nuclei in fluorescence micrographs, followed by analysis of nuclear area, fluorescence intensity of the Hoechst DNA-binding probe, and nuclear geometry (circularity, roundness, solidity). The study was conducted on monocotyledonous and dicotyledonous plants with known genome sizes. Triticum aestivum Wt (6n, hexaploid) and Temp (4n, tetraploid) are monocotyledonous, and Capsella bursa-pastoris (4n, tetraploid) and Capsella rubella (2n, dT/iploid) are dicotyledonous. A simple fluorescent staining protocol combined with automated analysis using our ImageJ macro enables reliable separation of both monocotyledonous and dicotyledonous plants by genome size with an accuracy close (for dicots) or comparable (for monocots) to flow cytofluorometry. For ploidy separation in monocots, the most sensitive parameters are fluorescence intensity, nucleus area, and circularity. For ploidy separation in dicots, the most sensitive parameters are nucleus area, fluorescence intensity, and circularity.

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