The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G₁/G₀ nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

中文翻译：

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流式细胞术估算植物核 DNA 绝对含量的参考标准


迄今为止，核 DNA 含量的估算是流式细胞术在植物中最流行的应用。由于流式细胞术测量 DNA 荧光染料染色的细胞核的相对荧光强度，倍性测定和绝对单位核 DNA 含量的估计都需要与已知 DNA 含量的参考标准进行比较。这意味着所获得结果的质量取决于标准的选择和使用。当同时分离、染色和测量未知样品的细胞核和参考标准时，内部标准化对于精确测量是必需的。由于代表样品和标准品的G ₁ /G ₀核的DNA峰出现在相同的荧光强度直方图上，因此它们在荧光强度轴上的位置的商提供了DNA量的商。为了以绝对单位估算 DNA 量，现在有许多完善的标准涵盖了已知植物基因组大小的范围。由于使用的标准不同，因此始终需要报告标准和指定的基因组大小。当现有标准都不适合时，就需要引入新的标准物种。为此，应优先考虑采用回归线方法或对候选标准与多个既定标准进行同步分析。 此外，新选择的标准生物体必须满足许多要求：它应该易于识别和维护，分类学上明确，全球可用，具有已知的基因组大小稳定性，缺乏有问题的代谢物，适合分离足够量的细胞核，并且能够以较低的 DNA 峰变异系数进行测量，因此适合制备高质量样品。