Department of Integrative Biology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.
Cytometry A. 2010 Aug;77(8):725-32. doi: 10.1002/cyto.a.20902.
Flow cytometry (FCM) is commonly used to determine plant genome size estimates. Methodology has improved and changed during the past three decades, and researchers are encouraged to optimize protocols for their specific application. However, this step is typically omitted or undescribed in the current plant genome size literature, and this omission could have serious consequences for the genome size estimates obtained. Using four bryophyte species (Brachythecium velutinum, Fissidens taxifolius, Hedwigia ciliata, and Thuidium minutulum), three methodological approaches to the use of FCM in plant genome size estimation were tested. These included nine different buffers (Baranyi's, de Laat's, Galbraith's, General Purpose, LB01, MgSO(4), Otto's, Tris.MgCl(2), and Woody Plant), seven propidium iodide (PI) staining periods (5, 10, 15, 20, 45, 60, and 120 min), and six PI concentrations (10, 25, 50, 100, 150, and 200 microg ml(-1)). Buffer, staining period and staining concentration all had a statistically significant effect (P = 0.05) on the genome size estimates obtained for all four species. Buffer choice and PI concentration had the greatest effect, altering the 1C-values by as much as 8% and 14%, respectively. As well, the quality of the data varied with the different methodology used. Using the methodology determined to be the most accurate in this study (LB01 buffer and PI staining for 20 min at 150 microg ml(-1)), three new genome size estimates were obtained: B. velutinum: 0.46 pg, H. ciliata: 0.30 pg, and T. minutulum: 0.46 pg. While the peak quality of flow cytometry histograms is important, researchers must consider that changes in methodology can also affect the relative peak positions and therefore the genome size estimates obtained for plants using FCM.
流式细胞术（FCM）常用于测定植物基因组大小估计值。在过去的三十年中，该方法已经得到改进和改变，研究人员被鼓励根据自己的具体应用来优化方案。然而，在当前的植物基因组大小文献中，这一步通常被省略或未描述，这可能对获得的基因组大小估计值产生严重后果。本研究使用了四种苔藓植物（Brachythecium velutinum、Fissidens taxifolius、Hedwigia ciliata 和 Thuidium minutulum），测试了三种使用 FCM 进行植物基因组大小估计的方法。这些方法包括九种不同的缓冲液（Baranyi's、de Laat's、Galbraith's、General purpose、LB01、MgSO4、Otto's、Tris.MgCl2 和 Woody Plant）、七种碘化丙啶（PI）染色时间（5、10、15、20、45、60 和 120 min）和六种 PI 浓度（10、25、50、100、150 和 200 microg ml-1）。对于所有四个物种，缓冲液、染色时间和染色浓度均对获得的基因组大小估计值具有统计学上的显著影响（P = 0.05）。缓冲液选择和 PI 浓度的影响最大，分别改变了 1C 值的 8%和 14%。此外，不同方法学的使用也会导致数据质量的变化。在本研究中，使用被确定为最准确的方法学（LB01 缓冲液和 PI 染色 20 min，浓度为 150 microg ml-1），获得了三个新的基因组大小估计值：B. velutinum：0.46 pg，H. ciliata：0.30 pg 和 T. minutulum：0.46 pg。虽然流式细胞术直方图的峰值质量很重要，但研究人员必须考虑到方法学的变化也会影响相对峰值位置，从而影响使用 FCM 的植物的基因组大小估计值。
