Aislamiento de núcleos para citometría de flujo en plantas con alto contenido de compuestos mucilaginosos: un ejemplo en el género Viola L. (Violaceae)un ejemplo en el género Viola L. (Violaceae)

  1. Cires Rodríguez, Eduardo
  2. Cuesta Moliner, Candela
  3. Fernández Casado, María de los Angeles
  4. Nava Fernández, Herminio S.
  5. Vázquez, Víctor M.
  6. Fernández Prieto, José Antonio
Aldizkaria:
Anales del Jardín Botánico de Madrid

ISSN: 0211-1322

Argitalpen urtea: 2011

Alea: 68

Zenbakia: 2

Orrialdeak: 139-154

Mota: Artikulua

DOI: 10.3989/AJBM.2273 DIALNET GOOGLE SCHOLAR lock_openDialnet editor

Beste argitalpen batzuk: Anales del Jardín Botánico de Madrid

Garapen Iraunkorreko Helburuak

Laburpena

Cires, E., Cuesta, C., Fernández, M.A., Nava, H.S., Vázquez, V.M. & Fernández, J.A. 2011. Isolation of plant nuclei suitable for flow cytometry from species with extremely mucilaginous compounds: an example in the genus Viola L. (Violaceae). Anales.Iard. Bot. Madrid 68(2): 139-154. Flow cytometry analysis has been widely appljed in the determination of nuclear DNA content and ploidy level in many organisms. Despite being the most appropriate method for DNA content measurement, flow cytometry also presents some limitations. A fairly common, but little-studied problem is the effect on measurements of the presente of secondary metabolites. A good example is the genus Viola, which is composed of 525-600 species distributed worldwide. These species have proved to be problematic for flow cytometric analyses due to the release of extremely mucilaginous compounds into the nuclear suspension. In this work, the genome size of 13 species of Viola using flow cytometry are presented for the first time. Despite obtaining histograms with high coefficients of variation, we here pre-sent an optimized protocol to remove cytoplasmic compounds, particularly mucilaginous ones, from plant nudei that paye the way for its application to estimate the genome size of other species exhibiting similar problems. Statistical analyses revealed significant differences between sections Viola and Melanium, and within each section (P < 0.001). Furthermore, statistically significant differences were not detected among samples of the same species.

Erreferentzia bibliografikoak

  • Ajalin, I., Kobza, F. & Doležel, J. 2002. Ploidy identification of doubled chromosome number plants in Viola × wittrockiana Gams. M 1-generation. Hortscience 29: 35-40.
  • Aldasoro, J.J. 1992. Números cromosomáticos de plantas occidentales, 668-680. Anales del Jardín Botánico de Madrid 50(2): 247-249.
  • Ballard, H.E., Sytsma, K.J. & Kowal, R.R. 1999. Shrinking the violets: phylogenetic relationships of infrageneric groups in Viola (Violaceae) based on internal transcribed spacer DNA sequences. Systematic Botany 23: 439-458. http://dx.doi.org/10.2307/2419376
  • Becker, W. 1910a. Violenstudien I. Beihefte zum Botanischen Centralblatt 26: 1-44.
  • Becker, W. 1910b. Violenstudien II. Beihefte zum Botanischen Centralblatt 26: 289-390.
  • Becker, W. 1925. Viola L. In: Engler, A. & Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, Vol. 21. Wilhelm Engelmann, Leipzig, 363-376.
  • Bennett, M.D. & Leitch, I.J. 2005. Angiosperm DNA C-values database (release 4.0, Dec. 2005), http://www.kew.org/cvalues/homepage.html (retrieved 07 Sept. 2009).
  • Castroviejo, S. & Valdés-Bermejo, E. (eds.) 1991. Números cromosomáticos de plantas vasculares ibéricas. Archivos de Flora Ibérica 1: 1-202.
  • Clausen, J. 1927. Chromosome number and the relationship of species in the genus Viola. Annals of Botany 41: 677-714.
  • Dizerbo, A.H. 1972. Les Viola de la section Nomimium du Massif Armoricain. Bulletin de la Société Scientifique de Bretagne 47(3-4): 137-139.
  • Doležel, J. & Bartoš, J. 2005. Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany 95: 99-110. http://dx.doi.org/10.1093/aob/mci005 PMid:15596459
  • Doležel, J., Binarová, P. & Lucretti, S. 1989. Analysis of nuclear DNA content in plant cells by flow cytometry. Biologia Plantarum 31(2): 113-120. http://dx.doi.org/10.1007/BF02907241
  • Doležel, J. & Göhde, W. 1995. Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry. Cytometry 19: 103-106. http://dx.doi.org/10.1002/cyto.990190203 PMid:7743889
  • Doležel, J., Greilhuber, J., Lucretti, S., Meister, A., Lysák, M.A., Nardi, L. & Obermayer, R. 1998. Plant genome size estimation by flow cytometry: inter-laboratory comparison. Annals of Botany 82 (suppl. A): 17-26. http://dx.doi.org/10.1006/anbo.1998.0730
  • Doležel, J., Greilhuber, J. & Suda, J. 2007a. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols 2(9): 2233-2244. http://dx.doi.org/10.1038/nprot.2007.310 PMid:17853881
  • Doležel, J., Greilhuber, J. & Suda, J. 2007b. Flow cytometry with plants: an overview. In: Doležel, J., Greilhuber, J. & Suda, J. (eds.), Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley-VCH Verlag, Weinheim, 41-65.
  • Doležel, J., Sgorbati, S. & Lucretti, S. 1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625-631. http://dx.doi.org/10.1111/j.1399-3054.1992.tb04764.x
  • Erben, M. 1996. The significance of hybridization on the forming of species in the genus Viola. Bocconea 5: 113-118.
  • Fernández Casado, M.A. 1982. Revisión de las especies del género Viola L. en la Península Ibérica. PhD Thesis, Universidad de Oviedo, Oviedo.
  • Fernández Casado, M.A. 1984. Estudios sobre el género Viola L. en la Península Ibérica. I. Cariología. Fontqueria 5: 23-32.
  • Gadella, F. & Kliphuis, E. 1963. Chromosome numbers of flowering plants in the Netherlands. Acta Botanica Neerlandica 12(2): 195-230.
  • Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P. & Firoozabady, E. 1983. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1049-1051. http://dx.doi.org/10.1126/science.220.4601.1049 PMid:17754551
  • García Fernández, R., Fernández Casado, M.A. & Nava, H.S. 1990. Viola gr. silvestris en Asturias. Anales del Jardín Botánico de Madrid 47(2): 339-348.
  • González Zapatero, M.A., Elena Roselló, J.A. & Navarro Andrés, F. 1986. Estudios cariológicos en táxones del CW español pertenecientes a Quercetea ilicis. Lazaroa 9: 61-68.
  • Greilhuber, J. 1986. Severely distorted Feulgen DNA amounts in Pinus (Coniferophytina) after nonadditive fixations as a result of meristematic self-tanning with vacuole contents. Canadian Journal of Genetics and Cytology 28: 409-415.
  • Greilhuber, J. 2008. Cytochemistry and C-values: the less-wellknown world of nuclear DNA amounts. Annals of Botany 101: 791-804. http://dx.doi.org/10.1093/aob/mcm250 PMid:17951594 PMCid:2710206
  • Greilhuber, J., Doležel, J., Lysák, M.A. & Bennett, M.D. 2005. The origin, evolution and proposed stabilization of the terms ‘genome size’ and ‘C-value’ to describe nuclear DNA contents. Annals of Botany 95: 255-260. http://dx.doi.org/10.1093/aob/mci019 PMid:15596473
  • Greilhuber, J., Temsch, E.M. & Loureiro, J.C.M. 2007. Nuclear DNA content measurement. In: Doležel, J., Greilhuber, J. & Suda, J. (eds.), Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley-VCH Verlag, Weinheim, 67-101.
  • Griesinger, R. 1937. Über hypo- und hyperdiploide Formen von Pe tunia, Hyoscyamus, Lamium und einige andere Chromoso - menzählungen. Berichte der Deutschen Botanischen Gesellschaft 55: 556-571.
  • Grime, J.P., Shacklock, J.M.L. & Band, S.R. 1985. Nuclear DNA contents, shoot phenology and species co-existence in a limestone grassland community. New Phytologist 100: 435-445. http://dx.doi.org/10.1111/j.1469-8137.1985.tb02792.x
  • Harmaja, H. 2003. A deviating cytotype of Viola riviniana from Finland. Annales Botanici Fennici 40: 395-400.
  • Hodálová, I., Mered’a, J.P., Mártonfi, P., Mártonfiová, L. & Danihelka, J. 2008. Morphological characters useful for the delimitation of taxa within Viola subsect. Viola (Violaceae): a morphometric study from the West Carpathians. Folia Geobotanica 43: 83-117. http://dx.doi.org/10.1007/s12224-008-9005-x
  • Karlsson, T., Marcussen, T., Wind, P. & Jonsell, B. 2008. Violaceae. In: Karlsson, T. (ed.), Flora Nordica Vol. 6. Stockholm: The Bergius Foundation (http://www.floranordica.org/Review/-Review_public/accounts/Viola.html).
  • Knaben, G. & Engelskjön, T. 1967. Chromosome numbers of Scandinavian arctic-alpine plant species. II. Acta Borealia, ser. A, 21: 1-57.
  • Kuta, E. 1990. Biosystematic studies on the genus Viola L., section Plagiostigma Godr. 1. Karyological analysis of V. epipsila Ledeb., V. palustris L. and their hybrids from Poland. Acta Biologica Cracoviensia. Series Botanica 31: 29-44.
  • Laínz, M. 1962. Aportaciones al conocimiento de la flora cántabro-astur, VI. Boletín de Ciencias de la Naturaleza. Real Instituto de Estudios Asturianos 5: 3-43.
  • Laínz, M. 1967. Aportaciones al conocimiento de la flora gallega, V. Anales del Instituto Nacional de Investigaciones Agrarias. Serie Forestal 12: 1-51.
  • Laínz, M. 1992. Sobre la distribución ibérica de Viola reichenbachiana Jord. ex Boreau. Anales del Jardín Botánico de Madrid 50(2): 267-268.
  • Larsen, K. 1954. Chromosome numbers of some European flowering plants. Botanisk Tidsskrift 50: 163-174.
  • Lee, H.C. & Lin, T.Y. 2005. Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue using a filtration column. Plant Molecular Biology Reporter 23: 53-58. http://dx.doi.org/10.1007/BF02772646
  • Leitch, I.J. & Bennett, M.D. 2007. Genome size and its uses: the impact of flow cytometry. In: Doležel, J., Greilhuber, J. & Suda, J. (eds.), Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley-VCH Verlag, Weinheim, 153-176.
  • Loureiro, J. 2007. Flow cytometric approaches to study plant genomes. PhD Thesis, University of Aveiro, Aveiro.
  • Loureiro, J., Rodriguez, E., Doležel, J. & Santos, C. 2007a. Two new nuclear isolation buffers for plant DNA flow cytometrya test with 37 species. Annals of Botany 100: 875-888. http://dx.doi.org/10.1093/aob/mcm152 PMid:17684025 PMCid:2749623
  • Loureiro, J., Rodriguez, E., Gomes, A. & Santos, C. 2007b. Genome size estimations on Ulmus minor Mill., Ulmus glabra Huds. and Celtis australis L. using flow cytometry. Plant Biology 9: 541-544. http://dx.doi.org/10.1055/s-2007-965165 PMid:17642035
  • Löve, A. & Kjellqvist, E. 1974. Cytotaxonomy of Spanish plants. IV. Dicotyledons: Caesalpiniaceae-Asteraceae. Lagascalia 4(2): 153-211.
  • Lysák, M.A. & Doležel, J. 1998. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52: 123-132.
  • Marcussen, T. 2003. A new violet species (Violaceae) from the south-west Alps. Botanical Journal of the Linnean Society 142: 119-123. http://dx.doi.org/10.1046/j.1095-8339.2003.00160.x
  • Marcussen, T. & Nordal, I. 1998. Viola suavis, a new species in the Nordic flora, with analyses of its relation to other species in the subsection Viola (Violaceae). Nordic Journal of Botany 18: 221-237. http://dx.doi.org/10.1111/j.1756-1051.1998.tb01875.x
  • Mered’a, P., Hodálová, I., Mártonfi, P. & Kolarcik, V. 2006. Reports (17-22). In: Mráz, P. (ed.), Chromosome number and DNA ploidy level reports from Central Europe - 2. Biologia, Bratislava 61(1): 116-117.
  • Mered’a, P., Hodálová, I., Mártonfi, P., Kučera, J. & Lihová, J. 2008. Intraspecific variation in Viola suavis in Europe: parallel Isolation of suitable plant nuclei from species with mucilaginous compounds evolution of white-flowered morphotypes. Annals of Botany 102: 443-462.
  • Merxmüller, H. 1974. Veilchenstudien. I-IV. Phyton 16: 137-158.
  • Merxmüller, H. & Lippert, W. 1977. Veilchenstudien V-VII. Mitteilungen (aus) der Botanischen Staatssammlung München 13: 503-531.
  • Miyaji, Y. 1930. Betrachtungen über die Chromosomenzahlen von Viola, Violaceen und verwandten Familien. Planta 11: 631-649. http://dx.doi.org/10.1007/BF01912564
  • Montserrat, P. 1992. Las violas y los problemas que plantean. Anales del Jardín Botánico de Madrid 49(2): 298-301.
  • Moore, D.M. 1982. Flora Europaea check-list and chromosome index. Cambridge University Press, Cambridge. http://dx.doi.org/10.1017/CBO9780511735493
  • Morgan, H.D. & Westoby, M. 2005. The relationship between nuclear DNA content and leaf strategy in seed plants. Annals of Botany 96: 1321-1330. http://dx.doi.org/10.1093/aob/mci284 PMid:16230323
  • Muñoz Garmendia, F., Montserrat, P., Laínz, M. & Aldasoro, J.J. 1993. Viola. In: Castroviejo, S., Aedo, C., Cirujano, S., Laínz, M., Montserrat, P., Morales, R., Muñoz Garmendia, F., Navarro, C., Paiva, J., Soriano, C. (eds.), Flora Iberica. Plantas vasculares de la Península Ibérica e Islas Baleares Vol. III. Real Jardín Botánico, CSIC, Madrid, 276-317.
  • Noirot, M., Barre, P., Duperray, C., Hamon, S. & De Kochko A. 2005. Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation. Annals of Botany 95: 111-118. http://dx.doi.org/10.1093/aob/mci006 PMid:15596460
  • Noirot, M., Barre, P., Duperray, C., Louarn, J. & Hamon, S. 2003. Effects of caffeine and chlorogenic acid on propidium iodide accessibility to DNA: consequences on genome size evaluation in coffee tree. Annals of Botany 92: 259-264. http://dx.doi.org/10.1093/aob/mcg139 PMid:12876189
  • Noirot, M., Barre, P., Louarn, J., Duperray, C. & Hamon, S. 2000. Nucleuscytosol interactions - a source of stoichiometric error in flow cytometric estimation of nuclear DNA content in plants. Annals of Botany 86: 309-316. http://dx.doi.org/10.1006/anbo.2000.1187
  • Noirot, M., Barre, P., Louarn, J., Duperray, C. & Hamon, S. 2002. Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide. Annals of Botany 89: 385-389. http://dx.doi.org/10.1093/aob/mcf056 PMid:12096798
  • Nordal, I. & Jonsell, B. 1998. A phylogeographic analysis of Viola rupestris: three post-glacial immigration routes into the Nordic area? Botanical Journal of the Linnean Society 128: 105-122.
  • Otto, F. 1990. DAPI staining of fixed cells for high-resolution flow cytometry of nuclear DNA. In: Crissman, H.A. & Darzynkie - wicz, Z. (eds.), Methods in Cell Biology (Vol. 33). Academic Press, New York, 105-110.
  • Pettet, A. 1964. Studies on British pansies. I. Chromosome numbers and pollen assemblages. Watsonia 6: 39-50.
  • Pfosser, M., Amon, A., Lelley, T. & Heberle-Bors, E. 1995. Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21: 387-393. http://dx.doi.org/10.1002/cyto.990210412 PMid:8608738
  • Price, H.J., Hodnett, G. & Johnston, J.S. 2000. Sunflower (Helianthus annuus) leaves contain compounds that reduce nuclear propidium iodide fluorescence. Annals of Botany 86: 929-934. http://dx.doi.org/10.1006/anbo.2000.1255
  • Sanso, A. & Seo, M. 2005. Chromosomes of some Argentine angiosperms and their taxonomic significance. Caryologia 58: 171-177.
  • Schmidt, A. 1961. Zytotaxonomische Untersuchungen an europäischen Viola-Arten der Sektion Nominium. Öesterreichische Botanische Zeitschrift 108: 20-88.
  • Schmidt, A. 1964. Chromosomenzahlen su¨deuropai¨scher Viola- Arten der Sektion Melanium. Flora 154: 158-162.
  • Schöfer, G. 1954. Untersuchungen u.ber die Polymorphie einheimischer Veilchen. Planta 43: 537-565.
  • Skalinska, M., Pogan, E. & Pogan, A.L. 1966. Further studies in chromosome numbers of Polish angiosperms Acta Biologica Cracoviensia. Series Botanica 9: 31-58.
  • Stork, A.L. 1971. Viola reichenbachiana Jord. on Åland - chromosome counts. Svensk Botanisk Tidskrift Utgifven af Svenska Botaniska Foreningen 65(2): 226-228.
  • Suda, J. 2004. An employment of flow cytometry into plant biosystematics. PhD Thesis, Charles University in Prague.
  • Suda, J., Kron, P., Husband, B.C. & Trávníček, P. 2007. Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. In: Doležel, J., Greilhuber, J., Suda, J. (eds.), Flow cytometry with plant cells. Analysis of genes, chromosomes and genomes. Wiley-VCH Verlag, Weinheim, 103-130.
  • Suda, J., Kyncl, T. & Jarolímová, V. 2005. Genome size variation in Macaronesian angiosperms: forty percent of the Canarian endemic flora completed. Plant Systematics and Evolution 252: 215-238. http://dx.doi.org/10.1007/s00606-004-0280-6
  • Suda, J. & Trávníček, P. 2006. Estimation of relative nuclear DNA content in dehydrated plant tissues by flow cytometry. In: Robinson, J.P., Darzynkiewicz, Z., Dobrucki, J., Hyun, W., Nolan, J., Orfao, A. & Rabinovitch, P. (eds.), Current Protocols in Cytometry. John Wiley & Sons, New York, 7.30.1-7.30.14.
  • Talent, N. & Dickinson, T.A. 2005. Polyploidy in Crataegus and Mespilus (Rosaceae, Maloideae): evolutionary inferences from flow cytometry of nuclear DNA amounts. Canadian Journal of Botany 83: 1268-1304. http://dx.doi.org/10.1139/b05-088
  • Tjio, J.H. & Levan, A. 1950. The use of oxiquinoleine in chromosome analysis. Anales de la Estación Experimental de Aula Dei 2: 21-64.
  • Valentine, D.H. 1941. Variation in Viola riviniana Rchb. New Phytologist 40: 189-209. http://dx.doi.org/10.1111/j.1469-8137.1941.tb07043.x
  • Valentine, D.H., Merxmüller, H. & Schmidt, A. 1968. Viola L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D.M., Valentine, D.H., Walters, S.M. & Webb, D.A. (eds.), Flora Europaea 2. Cambridge University Press, Cambridge, 270-282.
  • Van den Hof, K., van den Berg, R.G. & Gravendeel, B. 2008. Chalcone synthase gene lineage diversification confirms allopolyploid evolutionary relationships of European rostrate violets. Molecular Biology and Evolution 25(10): 2099-2108. http://dx.doi.org/10.1093/molbev/msn157 PMid:18650224
  • Yockteng, R., Ballard, H.E., Mansion, Jr.G., Dajoz, I. & Nadot, S. 2003. Relationships among pansies (Viola section Melanium) investigated using ITS and ISSR markers. Plant Systematics and Evolution 241: 153-170. http://dx.doi.org/10.1007/s00606-003-0045-7