Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants

Abstract

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1/G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well‐established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

A study of the chromosome number and genome size of the rare species Rhododendron keiskei var. hypoglaucum in Korea

“…Classical karyotyping methods using Feulgen staining are challenging in highly polyploid species of Cyperaceae, Juncaceae and Ericaceae due to small size of the chromosomes, which hinders chromosomal determination in polyploids and aneuploids (Chung and Im, 2020;Chung and Chung, 2021;Choi et al, 2022;Redpath et al, 2022). Flow cytometry is an alternative method for estimating genome size and ploidy level (Choi et al, 2021;Sliwinska et al, 2021;Temsch et al, 2021).…”

A dormant resource for genome size estimation in ferns: C‐value inference of the Ophioglossaceae using herbarium specimen spores

“…We prepared the nuclei extractions of the spore samples and internal standards from the leaf tissues separately because they required different mechanical pre‐treatments (i.e., bead‐vortexing vs. chopping) to acquire nuclei. These separate preparations have been defined as pseudo‐internal standardization (sensu Temsch et al, 2021 ). Nonetheless, the staining properties of the nuclei per se were unlikely to be changed by the different mechanical pre‐treatments.…”

Chromosome Number, Ploidy Level, and Nuclear DNA Content in 23 Species of Echeveria (Crassulaceae)

“…Due to the difficulty of obtaining isolated nuclei of Echeveria and the great differences in the content of nuclei between the Echeveria leaves and the internal standards, it was almost impossible to obtain a good relative proportion of nuclei. Therefore, it was decided to use the pseudo-standardization technique, where the nuclei solutions are prepared separately, check Temsch et al [ 59 ]. Between 120 and 250 mg of Echeveria leaf tissue was chopped with a razor blade in a Petri dish containing 1.5 to 2.00 mL of Otto 1 solution (1.5 mL of 0.1 M citric acid and 0.5% Tween 20), and then filtered through a 50 µm nylon mesh.…”

“…Between 120 and 250 mg of Echeveria leaf tissue was chopped with a razor blade in a Petri dish containing 1.5 to 2.00 mL of Otto 1 solution (1.5 mL of 0.1 M citric acid and 0.5% Tween 20), and then filtered through a 50 µm nylon mesh. Separately, 20–42 mg of internal standard plant was chopped into another Petri dish containing 1 mL of Otto1 solution and filtered in the same way [ 59 ]. Both solutions were incubated for 15 min at room temperature.…”