Euphytica111: 105110, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.105Flow cytometric analysis of DNA content in Hosta reveals ploidy chimerasBen J.M. Zonneveld & Frank Van IrenInstitute of Molecular Plant Sciences, Leiden University, PO Box 9505, 2300 RA Leiden, The NetherlandsReceived 11 June 1998; accepted 17 June 1999Key words: ow cytometry, Hosta, ploidy chimeras, propidium iodideSummaryNuclear DNA content in 84 Hosta cultivars was measured by ow cytometry. As an internal standard we used theclosely related Agave americana, the latter was calibrated against other reference species. Propidium iodide valueswere 20% higher than DAPI values, indicating a lower AT/GC ratio for Hosta. Ten new tetraploid plants werefound in addition to the only tetraploid known so far; H. ventricosa. Small forms of the tetraploid H. ventricosaturned out to be triploid hybrids. Three other triploids were discovered. Three supposed tetraploids turned out to bediploid. In ve plants the ploidy levels of the germ layers L1 and L3 appeared to be different. In these cases thickleaves coincides with a tetraploid DNA content. The DNA contents as measured in these ploidy chimeras coincidewith differences in leaf colour, thus giving an opportunity to analyze the build-up of Hosta plants. In general,analysis of the nuclear DNA content can be used as a simple means to increase the possibilities for creating newcultivars in this economically important perennial.IntroductionHosta Tratt. the number one selling perennial in theUSA has some interesting characteristics for the studyofcell lineage. It isgenerallyacceptedthat dicoty-ledons are basically built from 3 germ layers namedL1 (forming mainly the epidermis), L2 (gametes andsome subepidermal tissues) and L3 (ground tissue andvascular tissues) (Hanstein, 1868; Satina et al., 1940;Poehtig, 1989). Stewart &Dermen(1979)demon-strated that also many monocotyledons are composedof threegermlayersinplantswhereL2differsinchloroplast or in DNA content fromL1 and L3. There-fore, we assume the monocotyledon Hosta to have asimilar build-up.Hostaisrather uniqueinthat bothchloroplast-andploidy-chimerismoccursometimesinthesameplant. In those cases the different germ layers can befollowed not only by determining DNA content, butalso by observing the differently coloured parts of theleaves. Moreover Hosta can be multiplied vegetativelyenabling the chimeras to be propagated permanently.Chimeras, viz plants with genetically differentgerm layers have since long interested researchers andbreeders alike (Winkler, 1907; Tillney-Bassett, 1978;Steward & Dermen, 1979). Chloroplast chimeras areimportant in ornamental plants as they result in plantswith variegated leaves. Ploidy is one of the parametersthat inuences cell size;higher ploidy levels usuallyresulting inproportionally larger cells. Sopolyploidplantsandploidychimerasmayhavethickerleavesandlargerfruitsandarethereforeofinteresttothebreedersofornamental plantsandthefruitgrowingindustry(Neilson-Jones, 1969). Hostaisgrownona large scale for their showy leaves in a tremendousarray of colours.Uptonow, ploidyhasnot playedanimportantroleinselectioninHosta, becausenosubstantiatedchanges in ploidy have been reported. Tetraploid chro-mosome numbers have been reported for H. ventricosaandtriploid numbers forH.clausa(Kaneko, 1968).Here we report the analysis by ow cytometry of thenuclear DNA content of 84 Hosta cultivars.Generally, microscopical Feulgen densitometry isconsidered the most reliable estimate of the DNA con-tent of a single nucleus (Bennett & Leitch, 1995). Sev-eral authors indeed found that ow cytometric DAPImeasurements correlated badly withFeulgen data in106wide cross sections of the angiosperms (Doleel et al.,1998). However, itseems likelythat withinalowertaxon like a genus the differences in AT content areusually much smaller (Buitendijk et al., 1997).DAPIisawidelyusedDNAstaininowcyto-metryof cell nuclei. Because uorescence of theunbound stain is relatively low, background problemsare less. However, because of its AT preference, es-timates depend strongly on the base composition andthis varies widely throughout the plant kingdom. Soit can be used only when comparing DNA contents ofsamples with the same AT/GC ratio.Propidium iodide isthe preferred stainforabso-lutenuclear DNAcontent measurements, asit hasnobasepreference andithasbeendemonstrated tocorrelatewell withDNAcontentsdeterminedwithFeulgen staining (Doleel et al., 1998;Buitendijk etal., 1997). However, becauseofsubstantialuores-cence of the unbound dye and lower specicity of thestaining, therearemorebackgroundproblemsthanwith DAPI. We applied both methods, in order to ob-tain accurate values for DNA amounts per nucleus (2Ccontent), andtoseewhetherAT/GCdifferences arepresent throughout the measured Hosta cultivars.Materials and methodsPlant materialPlants were from our collection or obtained from theDutch nurseries of P. Warmerdam at Noordwijk, M.Fransen at Ter Aar, J. van der Top at Barneveld, theEnglish nursery of A. and R. Bowden at Okehamptonand the American nurseries ofWaltersGardens Inc.Holland Michigan and Plant Delight Nursery, Raleigh,N. Carolina. Inmost cases leaves wereused, butoccasionally also roots were measured.Flow cytometry of nuclear DNA contentPropidium iodideFor the isolation of nuclei, about 0.5 cm2of an adultleaf was chopped with a new razor blade in a Petri dishin 0.25 ml nuclei-isolation buffer A of the Partec highresolution DNA kit (Partec, Mnster) to which per ml0.25mgRNAsewasadded. Agaveamericanawasused as an internal standard (see below). After adding0.75 ml PI solution (50 mg/l in water) the suspensionwith nuclei was ltered through a 30m mesh nylonlter. The uorescence of the nuclei was measured us-ing a Partec CA-II ow cytometer. The optical pathcontained the lters KG1, BG12, the dichroic mirrorTK500, and OG570 with a Leitz 50 1 water immer-sion objective. Relative uorescence intensity of thenuclei was analyzed using Partec DPAC software. Atleast three different samples were measured for eachplant. Most histograms revealed a CV of less than 5%.DAPIFor the isolation of nuclei about 0.5 cm2of an adultleafwaschopped withanewrazorbladeinaPetridish in a few drops of Partec buffer containing DAPI(4,6-diamidino-2-phenylindole) (De Laat et al., 1987).After adding 2 ml more DAPI buffer the suspensionwith nuclei was ltered through a 30mmesh sizenylonlter. Fluorescenceof thenuclei was meas-ured usinga Partec CA-IIow cytometer within 30minutes. The optical path contained the lters KG1,BG38 andUG1, the dichroic miffor TK420, andGG435witha20 0.66Partecobjective. Relativeuorescence intensity of the nuclei was analyzed us-ing Partec DPAC software. At least three, but in mostcases more than ve different samples were measuredfor each plant. Most histograms revealed a CV of lessthan 5%.Internal standardIn order to eliminate differences in signal intensitiesdue to light absorption, quenching and other variables,apieceofAgaveamericanaasaninternalstandardwas always chopped together with the material to beanalyzed. Agave americana was chosen as it is closelyrelated (Bogler & Simpson, 1995) and has about thesameDNAcontent. It hasthesamebasicnumberand size of chromosomes as Hosta and is year-roundavailable.Pisum sativum and Hordeum vulgare were used tocalibrate our Agave americana. Baranyi & Greilhuber(1996) foundnosignicant differencesinthenuc-lear DNA content of 38 accessions of Pisum sativum.We measured the commercially available cultivarsRondo (also included inthe Baranyi &Greilhuberstudy)andFrisson. Whenchoppedtogetherwith,and measured against Hordeum vulgare Sultan (ob-tainedfromI. Leitch, KewGardensEng.), wealsofoundnodifference; inbothcasestheratioof thepropidium signals from P. sativum and H. vulgare was0.872.In a recent and thorough study by four laboratories(Doleel et al., 1998) the nuclear DNA contents of P.sativum and H. vulgare were calculated to be 8.75 and10.04 pg respectively. The ratio between these values107Table 1. DNA content in Hosta cultivars measuredwith Propidium Iodide and ratio in DNA content asmeasured with Propidium Iodide and DAPIpg DNA per PI/DAPI2C nucleus ratioDiploid cultivarsBeatrice 25.7 1.17Bella 24.8 1.16Bunter Babette 25.7 1.23Butter Rim 25.2 1.22Blue Cadet 23.4 1.28Diamond Tiara 22.3 1.13Elata 26.7 1.20Fire Works 26.0 1.20Fall Bouquet 28.3 1.22Fortunei Albopicta 26.3 1.18Francee 25.0 1.20Fragrant Bouquet 25.9 1.25Francis Williams 24.2 1.18Ginko Craig 25.9 1.20Golden Tiara 22.4 1.12Gold Edger 20.9 1.12Gold Standard 26.7 1.26Great Expectations 24.2 1.13Hadspen Blue 27.1 1.24Hakyo 23.9 1.21Halcyon 27.2 1.27Hirao Majesty 24.4 1.22Hirao Supreme 24.6 1.24Hirao Tetra 23.8 1.17Hydon Sunset 24.1 1.18Inaho 25.2 1.27Invincible 26.0 1.26Kabitan 23.4 1.12Kirishima 23.9 1.21Little Jim 23.0 1.13Little Wonder 20.2 1.10Love Pat 24.5 1.17Maruba 27.8 1.18Masquerade 23.2 1.18Neat Splash 23.6 1.25On Stage 25.9 1.19Out House Delight 24.2 1.18Purple Profusion 26.4 1.28Regal Rhubarb 25.8 1.22Royal Standard 26.4 1.27Sagae 25.7 1.25Shining Tot 24.1 1.26Sitting Pretty 24.9 1.20Snowcap 27.5 1.19Snow Flakes 24.9 1.14Sparky 25.7 1.21Table 1. Continued.pg DNA per PI/DAPI2C nucleus ratioSweet Susan 25.9 1.22Tardiora Shining Tot 26.7 1.25Theos Blue 24.9 1.22Urajiro Hachyo 24.1 1.14Ultravoilet Light 22.3 1.23William Lachman 24.9 1.18White Tachi 24.3 1.25Triploid cultivarsHollys Honey 30.7 1.20Peedee Eln Bells 33.1 1.22Venucosa 32.7 1.24Tuckers Valentine 32.3 1.14clausa ventricosa 32.3 1.21venusta ventricosa 30.7 1.22clausa v clausa 31.3 1.18Sum and Substance 39.0 1.16Tetraploid cultivarsF. Minuteman 53.0 1.17F. Twilight 52.8 1.13F. Patriot 3 51.2 1.26F. Patriot 4 50.3 1.20F. Patriot Green 50.3 1.22F. Whirlwind 49.7 1.13F. Second Wind 49.8 1.14Grand Tiara 43.4 1.20ventricosa Aureomarginata 41.6 1.17Jolly Green Dwarf 40.8 1.16Little Blue 38.7 1.21is the same as we found, viz., 0.871. We adopted theirgures for the nuclear DNA content. Whether we co-chopped with P. sativum, or with H. vulgare, the resultfor our internal standard Agave americana was 15.9 pgper nucleus (2C value) as measured with PI.Results and discussionMany Hosta cultivars have yellow or white colouredleaf edges or leaf centres. These leaf parts were ana-lyzed separately to reveal possible differences in nuc-learDNAcontent. Ifthere isnodifference inDNAcontent between the edge or center of the leaf only asingle value is given. Table 1 shows the DNA contentof a large number of hosta cultivars ranging fromabout2028 pg in the diploids, 3133 pg in the triploids and108Figure 1. Histograms of uorescence intensity of nuclei from leaftissue of Agave americana (peak 1) and root tissue of Hosta Patriot1 (peak 2 and 3). Nuclei were isolated and stained simultaneously.A: stained with propidium iodide and B: stained with DAPI.4153 pg in the tetraploids. If different ploidy levelswithin a single sample were present, as in roots andinploidychimeras(Table2), morethanoneDNAcontent is presented.Due to endoreduplication many dicotyledons showmultipleDNAcontentsinleaves, especiallyinthevascular tissue. Therefore, also the veins of the leafwere analyzed separately. Because no difference withthe rest of the leaf was found, it was concluded thatendoreduplicationisabsent inHostaleavesasit isin the internal standard Agave. This is in accordancewith the results of Bharathan et al., (1994) for othermonocotyledons.Diploid cultivarsThe PI/DAPI ratio demonstrates that with PI the DNAcontents areabout 20%higher comparedwiththeDAPI measurements (also shown in Figure 1). So itappears likely that Agave americana (published valuesfor four other Agave species about 55% AT, CavalliniTable 2. DNAcontent in ploidy chimeras of Hosta, measuredwith Propidium IodideHosta cultivars pg DNA per nucleus2C 4C 8CRoyal Super leaf edge 26.1 aleaf center 26.5 52.6 root 53.6 105.6Cheatin Hart leaf edge 23.0 leaf center 22.5 44.4 root 44.8 91.3U. Middle Ridge leaf edge 52.3 leaf center 25.4 51.2 root 24.8 51.6 Fortunei Patriot 1 leaf edge 52.8 leaf center 26.7 54.8 root 27.7 55.9 Fortunei Patriot 2 leaf edge 52.3 leaf center 27.6 52.8 root 27.7 52.8 plantaginea leaf edge 53.2 Heaven Scent leaf center 30.0 56.7 root 28.5 54.1 a = ploidy level absent.et al., 1996) has a higher AT content than Hosta. Noclearpatterns inthe ratios are found between Hostawithalowerand thosewithahigher DNAcontent.ThissuggeststhatthehigherDNAcontent insomecultivars was due to an increase with DNA of a similarAT content as found in the plants with the lower DNAcontent.Triploid cultivarsH. Hollys Honey, Peedee Eln Bells, Tuck-ers Valentine, andVenucosa are reportedto besmall forms or hybrids of the apomictic H. ventricosa(Schmid, 1991). TheDNAvaluesclearlyshowallof themtobetriploid. Therelativelylowamountof DNAof theabovementionedtriploids andthesemi-bellshaped owers, suggests that they are indeedhybrids with the tetraploid H. ventricosa, just as thetwo triploid species hybrids (made by the rst author)inTable2. Basedonitslowpollenstainabilityandseemingly aneuploid offspring Sumand Substancemust be considered a triploid. However, the high DNAcontent indicates that H. ventricosa is not one of theparents.109Tetraploid cultivarsSomeHostaplantshave beendescribed withleavesthicker than usual (The genus Hosta. List of registeredcultivars, 1993 and additions).Several plantswithreportedlythickleavesweremeasured and eleven turned out to be new tetraploids(Table1). H. LittleBlue, small, withbell-shapedowers like H. ventricosa, and H. Jolly Green Dwarfwith thick owerstalks, have a DNA amount slightlylower than H. ventricosa. This lower DNA content andlow pollen fertility (Zonneveld, unpublished) suggestthat they are aneuploids.Hirao (1981) reported that he treated Hosta withcolchicine inferring that the resultant plants were tet-raploid. The amount of DNAfoundinH. HiraoTetra, HiraoSupreme, andHiraoMajesty are,however, clearly at the diploid level. Moreover, theyall have a low pollen stainability (data not shown). Sodespite the colchicine treatment they have not becometetraploid.RootsTherootsofHostaalwaysshow25%to50%cellswithdoubletheDNAcontent irrespectivewhetherthe plants are diploid, triploid or tetraploid (Fig-ure 1). Usually this doubling is ascribed to G2 arrest(dAmato, 1964; Torrey, 1961). The proportion of G2cells also varies within a single root. After strippingthe cortex from the stele, the G2 arrested cells withdouble DNA content turned out tobe present in thecortex only. This phenomenon has been found in rootsin many other plants also mainly if not exclusively inthe cortex.Ploidy- or chromosomal chimeras (Table 2)H. RoyalSuperisthickleavedwithayellowleafedge and a sport from the all green H. Royal Stand-ard. The yellow leaf edge, containing only epidermalL1cells, hasadiploidDNAcontent. Thecenterofthe leafblade istetraploid, apart froma smallfrac-tion of diploid cells derived from the epidermis. Thetetraploidy of the L3 is corroborated by the roots inwhich only tetraploid (4C) and 8C cells are present.Diploid cells (2C) are absent. This also indicates thatL1 does not participate in root formation. Obviously,a tetraploid L3 in an otherwise diploid leaf was suf-cient to lead to a thickened leaf. The fact that the plantis sterile indicates that the L2 is diploid. H. CheatinHart turned out to have the same composition, with adiploid L1 and a tetraploid L3.H. Undulata Middle Ridge a plant with a whiteleaf center was just the opposite to H. Royal Superwith respect to its ploidy distribution. In the green leafedge nuclei were found to have double the amount ofDNAwithrespecttothewhiteleafcenter. Thetet-raploid DNA content in 25% of the cells of the rootsis not dissimilar from wholly diploid plants indicatingagain that the L1 does not contribute to the roots.Also H. plantaginea Heaven Scent a sport fromH. plantagineahasatetraploidyellowedgeandadiploid green leaf center.The same combination of a tetraploid L1 and dip-loid L3 was found in two accessions of H. Patriot andboth have diploid roots (mainly 2C, Figure 1). H. Pat-riot is, just as the tetraploid H. Minuteman a sportfromthediploidH. Francee. Theirmuchbroaderwhite edges as compared to the small white edges ofH. Francee are clearly due to the L1 being tetraploidin both sports. On the other hand, two other accessionsof H. Patriot and also an unnamed green sport of itturned out tobe fully tetraploid, including the roots(Table 1).Summarizing, the absence of endoreduplication inthe above-ground parts enables the analysis of cell lin-eage in ploidy chimeras of Hosta, viz., the build-up ofthe various tissues and organs from the L1, L2, and L3germ layers.In addition, the analysis of nuclear DNA contentincreases thepossibilities for raisingnewtypes ofcultivars in this economically important perennial.ReferencesBennett, M.D. &I.J. Leitch, 1995. Nuclear DNAamounts inAngiosperms. Ann of Bot 76: 113176.Bennett, M.D. &I.J. Leitch, 1997. Nuclear DNAamounts inAngiosperms 583 new estimates. Ann of Bot 80: 169196.Bharathan, G., G. Lambert & D.W. Galbraith, 1994. Nuclear DNAcontent of monocotyledons and related taxa. Am J Bot 81: 381386.Bogler, D.J. & B.B. Simpson, 1996. Phylogeny of Agavaceae basedon its rDNA sequence variation. Am J Bot 83: 12251235.Buitendijk, J.H., E.J. Boon & M.S. Ramanna, 1997. Nuclear DNAcontent in twelve species of Alstroemeria L. and some of theirhybrids. Ann Bot 79: 343353.DAmato, F., 1964. Endopolyploidy as afactor inplant tissuedevelopment. Caryologia 17: 4151.Cavallini, A., L. Natali, G. Cionini &I. Castorena-Sanchez,1996. Cytophotometric andbiochemical analyses of DNAinpentaploid and diploid Agave species. Genome 39: 266271.Doleel, J.,S. Sgorbati & S. Lucretti, 1992. Comparison of threeDNAuorochromesforowcytometricestimationofnuclearDNA content in plants. Physiol Plant 85: 625631.Doleel, J., J.G. Greilhuber, S. Lucretti, A. Meister, M.A. Lysak&R. Obermayer, 1998. Plantgenomesizeestimationby ow110cytometry: inter-laboratory comparison. Ann Bot 82 (Suppl A):1726.Hanstein, J., 1868. Die Scheitelzellgruppe im Vegetationspunkt derPhanerogamen. Festschr Niederrhein Ges Natur und Heilk: 109134.Hirao, S., 1981. Inducing tetraploids. The American Hosta SocietyBulletin 12: 36.Kaneko, K., 1968. Cytological studiesonsome speciesofHostaIII. Karyotypes of H. clausa, H. clausav. normalis andH.ventricosa. Bot Mag Tokyo 81: 396403.De Laat, A.N.M., W. Ghde & M.J.D. Vogelzang, 1987. Determ-ination of ploidy of single plants and plant populations by owcytometry. Plant Breeding 99: 303307.Neilson-Jones, W., 1969.PlantChimeras1124. Methuen&Co.London.Poehtig, S., 1989. Geneticmosaics andcell lineageanalysis inplants. Trend Genet 5: 273277.Satina, S., A.F. Blakeslee& A.G. Avery, 1940. Demonstrationofthe three germ layers in the shoot apex of Datura by means ofinducedpolyploidyin periclinalchimeras.Am J Bot 27: 895905.Schmid, G.H., 1991. ThegenusHosta. TimberPress, Portland,Oregon.Stewart, R.N. & H. Dermen, 1979. Ontogeny in monocotyledons asrevealedby studies of the developmentalanatomy of periclinalchloroplast chimeras. Am J Bot 66: 4758.Thegenus Hosta. List of registeredCultivars, 1993andyearlyadditions. ed The American Hosta Society.Tilney-Bassett,R.A.E., 1963. The structure of periclinal chimeras.Heredity 18: 265294.Torrey, J.G., 1961. Kinetin as trigger for mitosis in mature endom-itotic plant cells. Exp Cell Res 23: 281299.Winkler, H., 1907. ber Prpfbastarde und Panzliche Chimren.Ber Deutsch Bot Ges 25: 568576.