Journal Article

Anthocyanin Inhibits Propidium Iodide DNA Fluorescence in <i>Euphorbia pulcherrima</i>: Implications for Genome Size Variation and Flow Cytometry

Michael D. Bennett, H. James Price and J. Spencer Johnston

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 101, issue 6, pages 777-790
Published in print April 2008 | ISSN: 0305-7364
Published online December 2007 | e-ISSN: 1095-8290 | DOI:
Anthocyanin Inhibits Propidium Iodide DNA Fluorescence in Euphorbia pulcherrima: Implications for Genome Size Variation and Flow Cytometry

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


Show Summary Details



Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics.


DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia).

Key Results

There were large differences in PI staining (35–70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2·8–6·9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1·69–1·76 pg) than green leaves (1·81 pg). Chopping pea or poinsettia tissue in buffer with 0–200 µm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin.


Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation.

Keywords: Anthocyanin; cyanidin-3-rutinoside; DNA staining inhibitors; Euphorbia pulcherrima; flow cytometry; genome size artefacts; nuclear DNA amount; genome plasticity

Journal Article.  9398 words.  Illustrated.

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

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