Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species

Abstract

Background and aims: After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry.

Methods: GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species.

Key results: In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed.

Conclusions: WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high-quality suspensions of intact nuclei suitable for DNA flow cytometry.

Fig. 1.

Histograms of relative fluorescence intensities (PI fluorescence, channel numbers) with overlays of distributions obtained with the general purpose buffer (GPB, red) and the woody plant buffer (WPB, blue). Mean channel numbers (FL) and coefficients of variation (CV,%) of G₀/G₁ (peaks 1 and 2) and G₂ peaks (peaks 3 and 4) are given.

Fig. 2.

Cytograms of forward scatter (logarithmic scale, FS log) vs. side scatter (logarithmic scale, SS log) (A, D), histograms of PI fluorescence intensity (PI fluorescence, channel numbers) (B, E), and cytograms of SS log vs. PI fluorescence (C, F) of nuclear suspensions of Rosa sp. obtained with WPB (A–C) and GPB (D–F). An effect similar to the ‘tannic acid effect’ (Loureiro et al., 2006b) was observed in nuclear suspensions obtained with GPB. Arrows indicate two additional populations of particles. The first population comprises nuclei to which weakly fluorescent particles were attached (higher SS and FL values). The second population consists of clumps of weakly fluorescent particles (higher SS and lower FL values). Mean channel numbers (Mean channel) and coefficients of variation (CV,%) of G₀/G₁ peaks are given.

Fig. 3.

Histograms of relative fluorescence intensities (PI fluorescence, channel numbers) obtained after simultaneous analysis of nuclei isolated from sample (peak 1) and internal reference standard (peak 2) using the buffer that performed better (see Table 3). The following reference standards were used: Solanum lycopersicum ‘Stupické’ (2C = 1·96 pg DNA) (A, C, D, F, I, K); Glycine max ‘Polanka’ (2C = 2·50 pg DNA) (G, L); Zea mays ‘CE-777’ (2C = 5·43 pg DNA) (H); Pisum sativum ‘Ctirad’ (2C = 9·09 pg DNA) (B, E); Vicia faba'Inovec' (2C = 26·90 pg DNA) (J). Mean channel number (Mean), DNA index (DI = mean channel number of sample/mean channel number of internal reference standard), and coefficients of variation (CV,%) of G₀/G₁ peaks are given.