Jaroslav Dolezel, Johann Greilhuber, and Jan Suda Overview
The rise of plant flow cytometry since 1973 is testimony to the impact of a single elementary methodological innovation - the use of a razor blade instead of enzymes for isolation of nuclei. From 1983 onwards, this innovation led to an avalanche of applications of sophisticated instrumentation originally designed for biomedical research, and rarely before employed in the plant sciences. Owing to the physical size of the cell nucleus, its stainability with nucleic acid dyes and its genetic hegemony, nuclear DNA flow cytometry still dominates the field. Modern, small and affordable multiparameter instruments that measure fluorescence and light scatter allow convenient and rapid ploidy screening of living and dried plant samples, high precision genome size and endopolyploidy measurements, as well as cell cycle studies. Simultaneous measurement of side scatter allows improved analysis by discriminating clean nuclear fractions from samples corrupted with fluorescent debris particles which stick to the nuclei. The unambiguous detection of aneuploidy requires a high resolution technology, and is possible only under favorable cytogenetic circumstances. The estimation of the AT/GC ratio in nuclear DNA with a pair of fluorochromes remains a special field of research yielding sometimes controversial results. Since the nature of (multicellular) plant architecture is such that it is difficult to isolate individual cells, cell studies are often carried out with protoplasts (e.g. analysis of gene expression using fluorescent proteins). The study of subcellular processes (photosynthesis with plastids, respiration with mitochondria, membrane studies, and apoptosis-associated events) is mostly carried out using isolated organelles. When particles become too small to be analyzed as individuals, or when multiple antibodies are to be analyzed at one time, the innovative bead-based technologies can be applied. Thus, in phytosanitary screening with the microsphere immunoassay, dual-fluorochrome-tagged beads loaded with various covalently bound antibodies for microbial pests are employed. As up to 100 different bead classes can be made discernible by tagging them with various proportions of fluorochromes, a large number of different microbes can theoretically be quantified. The method can also be used for the analysis of other molecules and viruses. The autofluorescence of various photopigments together with light scatter of the cells makes phyto-plankton (together with abiotic particles and zooplankton) amenable to flow cy-tometry in situ. In this area of research, the main approach is the discrimination of particles by their bi- or multivariate fluorescence/scatter pattern or Coulter-sizing, and their association with classified organisms. Time-resolved pulse analysis even makes cell counting of linear algal colonies (e.g. diatoms) possible. Buoyant or submersed vehicles with purpose-designed flow cytometers are put to service for the control of environmental changes in the sea. A hitherto generally deplored disadvantage of flow cytometry is a lack of visualization of the objects being measured, but this has been overcome by integrated video imaging in recent instrumental developments. It is only a matter of time before this innovation becomes standard in bench-top flow cytometers which stand in botany laboratories.
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