Estimation of nuclear DNA content in plants using flow cytometry.
Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.
Dolezel
Jaroslav
J
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, Olomouc, Czech Republic. dolezel@ueb.cas.cz
Greilhuber
Johann
J
Suda
Jan
J
D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D059828:Plant Cells; D010944:Plants / Q000235:genetics; D013194:Staining and Labeling
D018744:DNA, Plant; D005456:Fluorescent Dyes