Journal Article

Using flow cytometry to estimate pollen DNA content: improved methodology and applications

Paul Kron and Brian C. Husband

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 110, issue 5, pages 1067-1078
Published in print October 2012 | ISSN: 0305-7364
Published online August 2012 | e-ISSN: 1095-8290 | DOI: http://dx.doi.org/10.1093/aob/mcs167
Using flow cytometry to estimate pollen DNA content: improved methodology and applications

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

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Background and Aims

Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.

Methods

The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.

Key Results

Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.

Conclusions

The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.

Keywords: Pollen; flow cytometry; unreduced gametes; genome size; nuclei extraction; 2n pollen; cell cycle

Journal Article.  9251 words.  Illustrated.

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

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