Improved Method of Enzyme Digestion for Root Tip Cytology

in HortScience

Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.

Contributor Notes

We gratefully acknowledge our sources of plant material: the US National Arboretum, Rogów Arboretum, NCGR-Corvallis, Monrovia Nursery, and Blue Heron Farm. Additional technical support provided by Tyler Hoskins and Sawyer Hurd in production and maintenance of the plant materials.

Graduate Research Assistant.

Associate Professor.

Corresponding author. E-mail: ryan.contreras@oregonstate.edu.

  • Bationo-KandoP.ZongoJ.-D.Siljak-YakovlevS.2016First genome size assessment, heterochromatin and rDNA chromosome mapping in the genus Sclerocarya (Anacardiaceae): Insight into the new basic chromosome numberBot. Lett.16311117

    • Search Google Scholar
    • Export Citation
  • BennettM.D.1998Plant genome values: How much do we know?Proc. Natl. Acad. Sci. USA9520112016

  • BennettM.D.LeitchI.J.2011Nuclear DNA amounts in angiosperms: Targets, trends and tomorrowAnn. Bot.107467590

  • CaiZ.Q.ZhangT.JianH.Y.2013Chromosome number variation in a promising oilseed woody crop, Plukenetia volubilis L. (Euphorbiaceae)Caryologia6615458

    • Search Google Scholar
    • Export Citation
  • CastiglioneM.R.CremoniniR.2012A fascinating island: 2n = 4Plant Biosyst.1463711726

  • ChenS.-C.CannonC.H.KuaC.-S.LiuJ.-J.GalbraithD.W.2014Genome size variation in the Fagaceae and its implications for treesTree Genet. Genomes10977988

    • Search Google Scholar
    • Export Citation
  • ContrerasR.N.FriddleM.LattierJ.D.2013Relative fertility and ploidy levels of selected rose-of-sharon cultivars. Proc. 58th Ann. SNA Res. Conf. 58:232–236

  • ContrerasR.N.RanneyT.G.TalluryS.P.2007Reproductive behavior of diploid and allotetraploid Rhododendron L. ‘Fragrant Affinity’HortScience423134

    • Search Google Scholar
    • Export Citation
  • ContrerasR.N.RuterJ.M.HannaW.W.2009An oryzalin-induced autoallooctoploid of Hibiscus acetosella ‘Panama Red’J. Amer. Soc. Hort. Sci.134553559

    • Search Google Scholar
    • Export Citation
  • ContrerasR.N.RuterJ.M.SchwartzB.M.2010Oryzalin-induced tetraploidy in Cryptomeria japonica (Cupressaceae)HortScience45316319

  • DahmerN.WittmannM.T.S.KaminskiP.E.2009Chromosome number and karyotype of the endangered Amazonian woody Centrolobium paraense Tul. speciesCrop Breed. Appl. Biotechnol.9382385

    • Search Google Scholar
    • Export Citation
  • DarlingtonC.D.WylieA.P.1956Chromosome atlas of flowering plants. Macmillan New York NY

  • FavreJ.M.BrownS.1996A flow cytometric evaluation of the nuclear DNA content and GC percent in genomes of European oak speciesAnn. Sci. For.53915917

    • Search Google Scholar
    • Export Citation
  • FleischmannA.MichaelT.P.RivadaviaF.SousaA.WangW.TemschE.M.GreilhuberJ.MüllerK.F.HeublG.2014Evolution of genome size and chromosome number in the carnivorous plant genus Genlisea (Lentibulariaceae), with a new estimate of the minimum genome size in angiospermsAnn. Bot.11416511663

    • Search Google Scholar
    • Export Citation
  • GamageH.K.SchmidtS.2009Short communication. A robust method for chromosome quantification and ploidy determination in woody speciesAustral. J. Bot.578793

    • Search Google Scholar
    • Export Citation
  • GarbariF.BediniG.PeruzziL.2012Chromosome numbers of the Italian flora. From the Caryologia foundation to presentCaryologia656271

  • GarciaS.GarnatjeT.HildalgoO.Mas de XaxarsG.PellicerJ.Sánchez-JiménezI.VitalesD.VallèsJ.2010First genome size estimations for some eudicot families and generaCollect. Bot.29716

    • Search Google Scholar
    • Export Citation
  • GarciaS.LeitchI.J.Anadon-RosellA.CanelaM.Á.GálvezF.GarnatjeT.GrasA.HidalgoO.JohnstonE.de XaxarsG.M.PellicerJ.Siljak-YakovlevS.VallèsJ.VitalesD.BennettM.D.2014Recent updates and developments to plant genome size databasesNucleic Acids Res.42D1D1159D1166

    • Search Google Scholar
    • Export Citation
  • GilloolyD.A.RanneyT.G.2015Genome size and ploidy Levels in the genus KalmiaHortScience5014261428

  • HizumeM.KondoT.ShibataF.IshizukaR.2001Flow cytometric determination of genome size in the Taxodiaceae, Cupressaceae sensu stricto and SciadopityaceaeCytologia66307311

    • Search Google Scholar
    • Export Citation
  • HuF.R.LiuH.H.WangF.BaoR.L.LiuG.X.2015Root tip chromosome karyotype analysis of hyacinth cultivarsGenet. Mol. Res.1431086310876

  • JauharP.P.2003Haploid and doubled haploid production in durum wheat by wide hybridization p. 161–166. In: M. Maluszynski K.J. Kasha B.P. Forester and I. Szarejko (eds.). Doubled haploid production in crop plants: A manual. Kluwer Academic Publishers Boston MA

  • JonesJ.R.RanneyT.G.LynchN.P.2007Ploidy levels and genome sizes of diverse species, hybrids, and cultivars of rhododendronJ. Amer. Rhod. Soc.614220227

    • Search Google Scholar
    • Export Citation
  • KaoK.N.1975A nuclear staining method for protoplasts p. 60–64. In: O.L. Gamborg and L.Z. Wetter (eds.). Plant tissue culture methods. L.R. National Research Council of Canada. Prairie Regional Laboratory Saskatoon SK Canada

  • LattierJ.D.RanneyT.G.LynchN.P.2013History and cytological reassessment of Rhododendron canadenseJ. Amer. Rhod. Soc.6729298

  • LattierK.S.2016Inducing and evaluating phenotypic and cytometric variation in landscape plants: Observations from Acer Ornithogalum and Penstemon. Oregon State University Corvallis M.S. Diss

  • OatesK.M.RanneyT.G.TouchellD.H.2014Campsis × tagliabuana ‘Chastity’: A highly infertile triploid trumpet vineHortScience49343345

    • Search Google Scholar
    • Export Citation
  • OchattS.J.2008Flow cytometry in plant breedingCytometry73A581598

  • ParrisJ.K.RanneyT.G.BairdW.V.2010Ploidy levels, relative genome sizes, and base pair composition in magnoliaJ. Amer. Soc. Hort. Sci.135533547

    • Search Google Scholar
    • Export Citation
  • PellicerJ.FayM.F.LeitchI.J.2010The largest eukaryotic genome of them all?Bot. J. Linn. Soc.1641015

  • PeruzziL.CaparelliK.F.BediniG.2014A new index for the quantification of chromosome number variation: An application to selected animal and plant groupsJ. Theor. Biol.3535560

    • Search Google Scholar
    • Export Citation
  • RanneyT.G.LynchN.P.FantzP.R.CapielloP.2007Clarifying taxonomy and nomenclature of Fothergilla (Hamamelidaceae) cultivars and hybridsHortScience42470473

    • Search Google Scholar
    • Export Citation
  • RibeiroT.BarãoA.ViegasW.Morais-CecílioL.2008Molecular cytogenetics of forest treesCytogenet. Genome Res.120220227

  • RockingerA.SousaA.CarvalhoF.A.RennerS.S.2016Chromosome number reduction in the sister clad of Carica papaya with concomitant genome size doublingAmer. J. Bot.103610821088

    • Search Google Scholar
    • Export Citation
  • RothleutnerJ.J.FriddleM.W.ContrerasR.N.2016Ploidy levels, relative genome sizes, and base pair composition in CotoneasterJ. Amer. Soc. Hort. Sci.141457466

    • Search Google Scholar
    • Export Citation
  • RounsavilleT.J.RanneyT.G.2010Ploidy levels and genome sizes of Berberis L. and Mahonia Nutt. species, hybrids, and cultivarsHortScience4510291033

    • Search Google Scholar
    • Export Citation
  • SchneiderJ.V.PauleJ.GitaíJ.DresslerS.GusmãoC.L.S.Benko-IsepponA.M.2015Divergent genome sizes reflect infrafamilial subdivision of the Neotropical woody MarcgraviaceaeBot. J. Linn. Soc.177114

    • Search Google Scholar
    • Export Citation
  • ShearerK.RanneyT.G.2013Ploidy levels and relative genome sizes of species, hybrids, and cultivars of dogwood (Cornus spp.)HortScience48825830

    • Search Google Scholar
    • Export Citation
  • Van LaereK.KhrustalevaL.HuylenbroeckJ.V.Van BockstaeleE.2010Application of GISH to characterize woody ornamental hybrids with small genomes and chromosomesPlant Breed.129442447

    • Search Google Scholar
    • Export Citation
  • WrightR.D.1986The pour-through nutrient extraction procedureHortScience21227229

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