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The rising world of flow cytometric analysis of pollen grains

Key terms flow cytometry; genome size; plant sciences; pollen grains; pulse analysis FLOW cytometry is nowadays recognized as a vital tool in plant sciences, with more and more applications in both basic and applied research (1). Due to the unique characteristics of plant material, i.e., complex three-dimensional tissues with the presence of rigid cell walls, specific methods had to be developed to produce suspensions of single particles of interest (2). These particles of interest include, nuclei, mitochondria, chloroplasts, and chromosomes, with most of the applications being focused around the estimation of nuclear DNA content (either in absolute or relative amounts), leading to increasing impacts in the fields of plant breeding and population biology. One of the few single particles produced by plants is the pollen grain. Still, most works focused on studying pollen grains have involved the extraction of nuclei or sperm cells, rather than using the intact pollen. This is mostly due to the autofluorescence and/or specific staining of the pollen exine, and to the particle size limitations of some flow cytometers (3). To add to this, isolation of high-quality nuclei from pollen grains is often difficult, at least in comparison with somatic tissues. Together, these difficulties have led to a very minute number of publications involving flow cytometric analysis of pollen grains in the last 25 years.