title: Glycerol-treated nuclear suspensions--an efficient preservation method for flow cytometric analysis of plant samples.
abstract: Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.
authors.affiliation: Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01 Prague, Czech Republic. filip.kolar@gmail.com
authors.forename: Filip
authors.initials: F
authors.lastname: Kolář
authors.forename: Magdalena
authors.initials: M
authors.lastname: Lučanová
authors.forename: Jakub
authors.initials: J
authors.lastname: Těšitel
authors.forename: João
authors.initials: J
authors.lastname: Loureiro
authors.forename: Jan
authors.initials: J
authors.lastname: Suda
mesh_terms: D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D018744:DNA, Plant / Q000032:analysis*; D005434:Flow Cytometry; D005990:Glycerol / Q000494:pharmacology*; D059828:Plant Cells / Q000737:chemistry / Q000187:drug effects; D011309:Preservation, Biological / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D018744:DNA, Plant; D005990:Glycerol