AOBPreview originally published online on August 7, 2007
Annals of Botany 2007 100(4):875-888; doi:10.1093/aob/mcm152
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TECHNICAL ARTICLE |
Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species
el2
1 Laboratory of Biotechnology and Cytomics, CESAM and Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
2 Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská, Olomouc, CZ-77200, Czech Republic
* For Correspondence. E-mail jloureiro{at}ua.pt
Received: 20 March 2007 Returned for revision: 14 May 2007 Accepted: 8 June 2007 Published electronically: 7 August 2007
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.
Key words: Cytosolic compounds, flow cytometry, general purpose buffer, genome size, lysis buffers, nuclear DNA content, nuclear DNA staining, propidium iodide, woody plant buffer
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