Journal Article

Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

J. Doležel, J. Greilhuber, S. Lucretti, A. Meister, M. A. Lysák, L. Nardi and R. Obermayer

in Annals of Botany

Published on behalf of Annals of Botany Company

Volume 82, issue suppl_1, pages 17-26
Published in print December 1998 | ISSN: 0305-7364
Published online December 1998 | e-ISSN: 1095-8290 | DOI: http://dx.doi.org/10.1093/oxfordjournals.aob.a010312
Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

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  • Plant Sciences and Forestry
  • Ecology and Conservation
  • Evolutionary Biology

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Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C = approx. 0·3–30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument.

Keywords: DAPI; Feulgen densitometry; flow cytometry; plant genome size; nuclear DNA content; propidium iodide; standardization

Journal Article.  0 words. 

Subjects: Plant Sciences and Forestry ; Ecology and Conservation ; Evolutionary Biology

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