Analysis of Nuclear DNA content in plant cells by Flow cytometry
ENEA1
TL;DR: Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro and showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissue and variousin vitro cultures.
Abstract: Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.
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TL;DR: Current procedures for estimation of absolute DNA amounts in plants using flow cytometry are reviewed, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards.
844 citations
TL;DR: The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants, and confirmed an urgent need for an agreement on standards.
583 citations
ENEA1
TL;DR: It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal.
Abstract: Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.
569 citations
TL;DR: A new model for grass functional genomics is described based on Brachypodium distachyon, which in the evolution of the Pooideae diverged just prior to the clade of "core pooid" genera that contain the majority of important temperate cereals and forage grasses.
Abstract: A new model for grass functional genomics is described based on Brachypodium distachyon , which in the evolution of the Pooideae diverged just prior to the clade of “core pooid” genera that contain the majority of important temperate cereals and forage grasses. Diploid ecotypes of B . distachyon (2 n = 10) have five easily distinguishable chromosomes that display high levels of chiasma formation at meiosis. The B . distachyon nuclear genome was indistinguishable in size from that of Arabidopsis, making it the simplest genome described in grasses to date. B . distachyon is a self-fertile, inbreeding annual with a life cycle of less than 4 months. These features, coupled with its small size (approximately 20 cm at maturity), lack of seed-head shatter, and undemanding growth requirements should make it amenable to high-throughput genetics and mutant screens. Immature embryos exhibited a high capacity for plant regeneration via somatic embryogenesis. Regenerated plants display very low levels of albinism and have normal fertility. A simple transformation system has been developed based on microprojectile bombardment of embryogenic callus and hygromycin selection. Selected B . distachyon ecotypes were resistant to all tested cereal-adapted Blumeria graminis species and cereal brown rusts ( Puccinia reconditia ). In contrast, different ecotypes displayed resistance or disease symptoms following challenge with the rice blast pathogen ( Magnaporthe grisea ) and wheat/barley yellow stripe rusts ( Puccinia striformis ). Despite its small stature, B . distachyon has large seeds that should prove useful for studies on grain filling. Such biological characteristics represent important traits for study in temperate cereals.
508 citations
TL;DR: 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.
400 citations
Cites background from "Analysis of Nuclear DNA content in ..."
..., 1983), LB01 (Doležel et al., 1989), Otto (Ulrich and Ulrich, 1991; Doležel and Göhde, 1995) and Tris....
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...and Earle, 1991a) or spermine (Doležel et al., 1989)....
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References
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Book•
01 Jan 1985
TL;DR: Using Flow Cytometers: Applications, Extensions, and Alternatives as mentioned in this paper is an excellent overview of the history of the field of Flow Sorting, its applications, extensions, and alternatives.
Abstract: List of Tables and Figures. Preface to the Fourth Edition: Why You Should Read the Book - Or Not. Foreword to the Third Edition. Preface to the Third Edition. Preface to the Second Edition. Foreword to the First Edition. Preface to the First Edition. 1. Overture. 2. Learning Flow Cytometry. 3. History. 4. How Flow Cytometers Work. 5. Data Analysis. 6. Flow Sorting. 7. Parameters and Probes. 8. Buying Flow Cytometers. 9. Building Flow Cytometers. 10. Using Flow Cytometers: Applications, Extensions, and Alternatives. 11. Sources of Supply. 12. Afterword.
2,214 citations
TL;DR: The amount of nuclear DNA in the homogenates of monocotyledonous and dicotylingonous plants was accurately and rapidly determined by flow microfluorometry, and the distribution of nuclei involved in the cell cycle was charted for tissues selected from different physical locations or developmental stages.
Abstract: Mechanical chopping of plant tissues in the presence of mithramycin released intact nuclei representative of the cells within the tissues. The amount of nuclear DNA in the homogenates of monocotyledonous and dicotyledonous plants was accurately and rapidly determined by flow microfluorometry, and the distribution of nuclei involved in the cell cycle was charted for tissues selected from different physical locations or developmental stages.
1,836 citations
Book•
01 Jan 1979
TL;DR: Cytophysical Methods Cell Preparation Cytochemical Methods Analysis of Measurements Cell Biology Hematology and Immunology Applications in Oncology Index.
Abstract: Cytophysical Methods Cell Preparation Cytochemical Methods Analysis of Measurements Cell Biology Hematology and Immunology Applications in Oncology Index.
765 citations
TL;DR: An efficient method based on a measurement of the DNA content of interphase nuclei by flow cytometry turned out to be highly competitive in terms of simplicity, accuracy, and costs.
Abstract: An efficient method for the determination of the ploidy level is described, based on a measurement of the DNA content of interphase nuclei by flow cytometry. Both individual plants as well as plant populations can be used to obtain the desired DNA-histograms Compared to conventional chromosome counting flow cytometry turned out to be highly competitive in terms of simplicity, accuracy, and costs.
192 citations
"Analysis of Nuclear DNA content in ..." refers methods in this paper
...- value might be probably still further decreased by additional improvements of the lysis buffer as was shown by DE LAAT et al. (1987) who used a commercial Partec buffer....
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TL;DR: The principles, advantages and limitations and the major applications of flow cytometry in research and in clinical diagnosis are summarized, and some of the opportunities and challenges for the further development of this versatile technology are outlined.
Abstract: Flow cytometry combines many of the advantages of microscopy and biochemical analysis in a single high precision technique for rapid phenotypic analysis and sorting of individual cells, microorganisms and cell organelles. Flow cytometry has evolved over the past twenty five years from a few custom-made instruments designed to meet individual research needs to commercially available instruments, used routinely in many areas of contemporary biomedical research, and for the diagnosis and staging of clinical disease. This review summarizes the principles, advantages and limitations and the major applications of flow cytometry in research and in clinical diagnosis, and outlines some of the opportunities and challenges for the further development of this versatile technology.
149 citations