Glycerol-treated nuclear suspensions--an efficient preservation method for flow cytometric analysis of plant samples
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Cell Nucleus drug effects genetics MeSH
- Time Factors MeSH
- DNA, Plant analysis MeSH
- Glycerol pharmacology MeSH
- Cryoprotective Agents pharmacology MeSH
- Preservation, Biological methods MeSH
- Flow Cytometry * MeSH
- Plant Cells chemistry drug effects MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
- Glycerol MeSH
- Cryoprotective Agents MeSH
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.
See more in PubMed
Cytometry A. 2006 Apr;69(4):273-80 PubMed
Am J Bot. 2007 Aug;94(8):1391-401 PubMed
Mol Ecol. 2007 Sep;16(18):3902-25 PubMed
Cytometry A. 2005 Apr;64(2):72-9 PubMed
Methods Cell Biol. 1990;33:105-10 PubMed
J Plant Res. 2007 Nov;120(6):721-5 PubMed
Curr Protoc Cytom. 2006 Nov;Chapter 7:Unit7.30 PubMed
Ann Bot. 2006 Sep;98(3):515-27 PubMed
Cytometry A. 2010 Aug;77(8):717-20 PubMed
Am J Bot. 2008 Jan;95(1):50-8 PubMed
Chromosome Res. 2011 Aug;19(6):825-42 PubMed
Ann Bot. 2005 Jan;95(1):99-110 PubMed
Mol Phylogenet Evol. 2007 Jan;42(1):92-103 PubMed
New Phytol. 2011 Sep;191(4):1150-1167 PubMed
Preslia. 2009;81(3):309-319 PubMed
Nat Protoc. 2007;2(9):2233-44 PubMed
