Kron Paul, Husband Brian C
Department of Integrative Biology, University of Guelph, Ontario, N1G 2W1, Canada.
Cytometry A. 2015 Oct;87(10):943-57. doi: 10.1002/cyto.a.22696. Epub 2015 Jun 12.
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.
流式细胞术在量化植物中未减数（2N）花粉产量方面的价值已得到广泛认可；然而，该方法受到获取高质量细胞核荧光直方图的技术障碍以及区分2N细胞核与1N双峰的困难的限制。在此，我们运用数学论证以及对被子植物花粉细胞核荧光特性的观察，来生成应用脉冲分析以校正花粉细胞核数据中双峰的指导原则。我们表明，当未减数配子（2C DNA含量）区域的细胞核荧光高度和/或宽度测量值呈现双峰性（反映单峰和双峰），同时减数配子（1C）区域相同参数呈现单峰分布时，应用脉冲分析进行双峰校正的理论要求得以满足。十字花科经常满足这些条件，但菊科和禾本科则不然。我们进一步表明，当满足这些要求时，脉冲分析对双峰比例的估计与显微镜观察获得的估计高度相关。我们提出了在估计未减数雄配子频率时进行双峰校正的指导原则。
