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Distinguishing 2N gamete nuclei from doublets in pollen using flow cytometry and pulse analysis
Author(s) -
Kron Paul,
Husband Brian C.
Publication year - 2015
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.22696
Subject(s) - gamete , pollen , flow cytometry , cytometry , pulse (music) , biophysics , biology , physics , botany , microbiology and biotechnology , optics , detector , sperm
The value of flow cytometry for quantifying unreduced (2N) pollen production in plants is well recognized; however, the approach has been limited by technical obstacles to obtaining high quality nuclei fluorescence histograms and the difficulty in distinguishing 2N nuclei from 1N doublets. Here, we use mathematical arguments and observations of fluorescence properties of angiosperm pollen nuclei to generate guidelines for applying pulse analysis to correct for doublets in pollen nuclei data. We show that the theoretical requirements for applying pulse analysis for doublet correction are met when nucleus fluorescence height and/or width measures in the unreduced gamete (2C DNA content) region exhibit bimodality (reflecting singlets and doublets) in combination with unimodal distributions of the same parameters in the reduced gamete (1C) region. These conditions are regularly met in the family Brassicaceae but not in the Asteraceae and Poaceae. We further show that when these requirements are met, pulse analysis estimates of doublet proportions are well correlated with estimates obtained with microscopy. We propose guidelines for doublet correction when estimating frequencies of unreduced male gametes. © 2015 International Society for Advancement of Cytometry