Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment

Abstract

Flow cytometry has become the method of choice to measure the DNA content (genome size) in plants. Ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics and biotechnology. Although the cell wall is a problem when isolating plant cells, cytometry remains a powerful tool in plant sciences. Based on our 30-years’ experience in this field, this review will focus at first on genome size measurement using simply isolated nuclei: the good practice for acquisition, nuclei isolation, appropriate buffers, kind of tissues to use. The second part will briefly review what kind of measurements it is possible to make in plant cytometry, and for what purpose: base composition, ploidy level, cell cycle, endoreplication, seed screening, and nuclei/chromosomes sorting. We will address troubleshooting. The commonly-used mathematic tools will be discussed.

Gene flow between diploid and tetraploid junipers - two contrasting evolutionary pathways in two Juniperus populations

“…‘Sultan’ (2C value = 9.81 pg) [ 94 ]. Around 30 mg of the IS and juniper leaves were simultaneously chopped in 600 ul of cold Gif nuclear isolation buffer-GNB [ 95 ]. The nuclei suspension was filtered using a nylon mesh (50 μm) and stained with 100 μg/ml propidium iodide (PI).…”

A new spontaneous hybrid between the cultivated and wild iris species from Serbia

“…The total nuclear DNA amount was assessed by flow cytometry according to Bourge et al (2018) using fresh leaves of samples and internal standards, Triticum aestivum L. "Chinese Spring" (2C = 30.9 pg) (Marie & Brown 1993).…”

“…Small parts of leaves (approx. 0.5 cm²) of both internal standard and target species were simultaneously chopped using a razor blade in a plastic Petri dish with 600 µl of Gif nuclei-isolation buffer (Bourge et al 2018): [45 mM MgCl2, 30 mM sodium citrate, 60 mM MOPS (4-morpholine propane sulphonate, pH 7), and 1% (w/v) polyvinylpyrrolidone 10,000, pH 7.2] containing 0.1% (w/v) Triton X-100 and supplemented with 5 mM sodium metabisulphite and RNAse (2.5 U/ml). The nuclei suspension was filtered through a 30 μm nylon mesh.…”

Molecular Study of Selected Taxonomically Critical Taxa of the Genus Iris L. from the Broader Alpine-Dinaric Area

“…For most species involved in our study C-values are measured in several studies [ 27 , 28 , 29 ]. Different methods were used for the measurement of plant DNA content, but flow cytometry has become the method of choice due to its reliability, simplicity, and relatively low cost [ 30 , 31 ].…”