Plant material selection, collection, preservation, and storage for nuclear DNA content estimation

Abstract

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed.

Application‐based guidelines for best practices in plant flow cytometry

“…In principle, genome sizes can be estimated from any part of a plant that contains intact nuclei. 22 Most measurements of genome size are typically made using leaves, but other parts of the plant have been used, including flower stalks, petioles, petals, tree phloem, roots, or seeds. For some species, preserved or fixed samples (e.g., herbarium vouchers, silica-gel-dried or deep-frozen samples) can provide an approximate estimation of genome size.…”

“…Figure 2). Deviations from this recommended PI concentration and the optimal amount of material used (to be estimated empirically for a given tissue type and species) 22,38 may lead to nonstoichiometric binding and should be avoided. The PI-DNA binding can also be affected by numerous metabolites commonly present in the cytosol of plant cells and that are released during nuclei isolation.…”

“…• Where possible, selecting only the most healthy, fresh, and secondary metabolite-free parts of the sample and standard tissue for measurements. 22,38 • Using a plant internal standard whose genome size is close to that of the species being studied. This gives more reproducible results in repeated analyses of the same individual than when the genome size of the internal standard is more distant from the species being analyzed.…”

“…Nevertheless, since FCM is an indirect method for ploidy estimation, it has to be supported by chromosome counting (see below and reference 21 for example). Advantages of FCM over other methods of ploidy estimation include (i) no need for special preparation of plant material (most tissues of the growing plant can be used), 22 (ii) rapid sample preparation, allowing daily processing of dozens or even hundreds of samples, (iii) an ability to measure DNA content/ploidy in mitotically inactive cells (as compared to chromosome counting, which requires mitotic cells), (iv) nondestructive sampling (a very small sample, about 5–10 mg fresh weight, is sufficient to provide thousands of nuclei for ploidy determination), which enables investigation of rare and endangered species with minimal damage, and allows the same individual to be used for further analyses, (v) the possibility of analysis of bulked samples of pooled individuals at one time, thereby providing large‐scale screening, and (vi) very modest operating costs. It must be realized, however, that it is only possible to estimate ploidy levels from FCM measurements within a certain taxonomical rank (mostly within closely related species).…”

“…In principle, genome sizes can be estimated from any part of a plant that contains intact nuclei 22 . Most measurements of genome size are typically made using leaves, but other parts of the plant have been used, including flower stalks, petioles, petals, tree phloem, roots, or seeds.…”