Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
Acta Histochem. 2011 Feb;113(2):221-5. doi: 10.1016/j.acthis.2009.10.005. Epub 2010 Feb 10.
Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G(0)/G(1) peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.
利用碘化丙啶（PI）作为核 DNA 染色染料，通过流式细胞术测量植物基因组大小。然而，一些作者报告了基因组大小估计错误的发生，特别是在富含次生代谢物的植物中，如咖啡树。在这种情况下，我们测试了一种替代的细胞测量方案，使用 SYBR Green I 作为荧光染料，对咖啡属（2x）和阿拉伯咖啡（4x）的核双链 DNA 进行化学计量染色。结果表明，用 SYBR Green I 和碘化丙啶染色的核分别测量的平均基因组大小在统计学上是相同的。然而，用 SYBR Green I 染色的核的 G(0)/G(1)峰的变异系数（1.57-2.85%）低于用碘化丙啶染色的核（2.75-4.80%）。变异系数统计数据表明，SYBR Green I 适合使用该方法进行化学计量核染色。我们的结果提供了证据，表明 SYBR Green I 可用于植物的流式细胞术测量，具有最小化核 DNA 含量定量误差、相对更快染色、高亲和力和比碘化丙啶更少致突变性的优点。
