Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspec...

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Title:
Somatic embryogenesis, tetraploidy, and variant leaf morphology in transgenic diploid strawberry (Fragaria vesca subspecies vesca ‘Hawaii 4’)
Series Title:
BMC Plant Biology
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Mixed Material
Creator:
Qian Zhang
Kevin M Folta
Thomas M Davis
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BMC Plant Biology
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Subjects / Keywords:
Fragaria
Strawberry
Transgenes
Leaf morphology
Tetraploidy
Somatic embryogenesis

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Abstract:
Background: The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca ‘Hawaii 4’ was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. ‘Hawaii 4’ is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the ‘Hawaii 4’ transformation and regeneration system. Results: Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established ‘Hawaii 4’ protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy. Conclusions: These results define opportunities to optimize the existing ‘Hawaii 4’ protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the ‘Hawaii 4’ model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.

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University of Florida
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University of Florida
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RESEARCHARTICLEOpenAccessSomaticembryogenesis,tetraploidy,andvariant leafmorphologyintransgenicdiploidstrawberry ( Fragariavesca subspecies vesca ‘ Hawaii4 ’ )QianZhang1,KevinMFolta2andThomasMDavis1*AbstractBackground: Thediploid(2n=2x=14)strawberrymodelplant Fragariavesca ssp. vesca ‘ Hawaii4 ’ was employedforfunctionalanalysisofexpressedDNAse quencesinitiallyidenti fiedasbeinguniqueto Fragaria andofunknownorpoorlyunderstoodfunction. ‘ Hawaii4 ’ isprominentinstrawberryresearchduetoitsease of Agrobacterium -mediatedtransformationandregenerability,anditsstatusasthesourceofthefirstcomplete strawberrygenomicsequence.Ourstudiesofasetoftransformantshavedocumentedintriguing,construct-associated effectsonleafmorphology,andprovideimportantandunexpectedinsightsintotheperformanceofthe ‘ Hawaii4 ’ transformationandregenerationsystem. Results: Following Agrobacterium -mediatedtransformationofleafexplantswithgeneconstructscarriedby Gatewayvectors,plantswereregeneratedusingamodifiedversionofanestablished ‘ Hawaii4 ’ protocol. Expandinguponthefindingsofprio rstudies,wedocumentedthatplantletregenerationwasoccurringviaa somaticembryogenicratherthananorganogenicdevelopmentalpathway.Amongtransformants,severalvariations inleafmorphologywereobserved.Unexpectedly,aparticularleafvarianttype,occurringin~17%ofallregenerants independentofconstructtype,wasfoundtobeattributabletotetraploidy.Thetetraploidy-associatedalteration inleafmorphologycouldbedifferentiatedfromtheleafmorphologyofdiploidregenerantsonthebasisofa quantitativeratioofleafdimensions:B/A,whereBisthewidthofthecentralleafletandAistheoverallwidthof thetrifoliateleaf.Varianteffectsonleafmorphologyoffourdifferenttransgenicconstructswerealsodocumented, andwereinallcasesdistinguishablefromtheeffectsoftetraploidy. Conclusions: Theseresultsdefineopportunitiestooptimizetheexisting ‘ Hawaii4 ’ protocolbyfocusingon treatmentsthatspecificallypromotesomaticembryogenesis.Thereportedmorphologicalmetricanddescriptions willguidefuturetransgenicstudiesusingthe ‘ Hawaii4 ’ modelsystembyalertingresearcherstothepotential occurrenceofpolyploidregenerants,andtodifferentiatingtheeffectsonleafmorphologyduetopolyploidyversus transgenicmanipulations.Finally,anintriguingspectrumofleafmorphologyalterationsresultingfrommanipulation ofexpressedsequencesofuncertainfunctionisdocumented,providingafoundationfordetailedstudiesofthe respectivegenesandtheirfunctionalroles. Keywords: Fragaria ,Strawberry,Transgenes,Leafmorphology,Tetraploidy,Somaticembryogenesis *Correspondence: tom.davis@unh.edu1DepartmentofBiologicalSciences,UniversityofNewHampshire,Durham, NH03824,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Zhangetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublicDomainDedication waiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwise stated.Zhang etal.BMCPlantBiology 2014, 14 :23 http://www.biomedcentral.com/1471-2229/14/23

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BackgroundThediploid(2n=2x=14)strawberryspecies Fragaria vesca hasbeenembracedasaconvenientmodelsystem forgenomicresearch.Itsfavorableattributesinclude a~240Mbreferencegenome,shortgenerationtime, smallplantsize,closekinshiptocommercialoctoploid strawberry Fragaria ananassa ,andmembershipinthe economicallyimportantRosaceaefamily[1,2].Molecular,genomic,andbiotechnologicalresearchinstrawberry hasmadesubstantialprogresswithrespecttoinvitro transformationsystems[3-5],geneticlinkagemaps[6-8], transcriptanalysis[9-11],andgenomicsequencing[12-14]. Inparticular,the F.vesca varietyknownas ‘ Hawaii4 ’ (PI551572=CFRA197)hascometoprominencedueto itseaseof Agrobacterium -mediatedtransformationand regenerability[4,15],thegenerationofaninbredderivative ‘ Hawaii4x4 ’ (SlovinJ.,unpublished),andchoiceofthe ‘ Hawaii4x4 ’ lineasthesourceofthefirstcompletestrawberrygenomicsequence[14].Assuch,geneticprotocol refinementfor ‘ Hawaii4 ’ isanimportantpriority. TheoriginalPI551572germplasmaccessionwascollectedinHawaiiin1983byR.Bringhurst,andwas maintainedbytheNationalClonalGermplasmRepository(NCGR),Corvallis,Oregon,underthelocalidentificationnumberCFRA197. Agrobacterium -mediated transformationandregenerationwasfirstreportedby HaymesandDavis[15].Thoseauthorsdescribedasingle transformant,selectedonkanamycinandcarryingneomycinphosphotransferase( nptII )and -glucuronidase ( gus )markergenes,andthesegregationofthesemarker genesinafirst(R1)generationprogenypopulation. PI551572hadbeenutilizedinthisinitialeffortinpart becauseoftwofavorablephenotypictraits:itwasasocalled ‘ Alpine ’ or semperflorens form,meaningthatithad aperpetual(day-neutral)floweringhabitascontrastedto theseasonal(shortday)floweringhabitofmostwild strawberrygenotypes;anditwasarunneringform,incontrasttonon-runnering ‘ Alpine ’ varietiessuchas ‘ Yellow Wonder ’ (PI551827), ‘ BaronSolemacher ’ (PI551507), ‘ Reugen ’ (PI551834),and ‘ Alexandria ’ (PI602923). F.vesca istypifiedbyredfruit;however, ‘ Hawaii4 ’ ,and ‘ Yellow Wonder ’ haveyellowfruit. Subsequently,PI551572wasfoundtobethemostfavorablyresponsiveamongfourteenF.vesca germplasm accessionsevaluatedfortransformabilityandregenerabilitybyOosumietal.[4],whoestablishedanoptimizedsystemthatemployedhygromycinresistanceas theselectablemarker.ThestudyofOosumietal.[4] wasaimedatestablishinganinvitrosystemthatwould facilitatehighthroughputdevelopmentofT-DNAinsertionlines[16]indiploidstrawberry.Ouraim[17-19] andapproachdifferedinthatwesoughttointroduce RNAiconstructs,andthatourvectorsystem[20] employedkanamycinresistance.Asapreludetofuture research,wealsosoughttoknowwhetherregeneration wasoccurringviaasomaticembryogenesisoranorganogenicpathway,adistinctionthathadnotbeenspecifiedbyOosumietal.[4]. Herewereportsignificantnewfindingsregardingthe responseof ‘ Hawaii4 ’ toinvitrotransformationandregeneration,includingexplicitevidencethatregeneration undertheemployedconditionsoccursviaasomaticembryogenicpathway,andthefirstreportthatasubstantial proportionoftheprimarytransformantsofthisdiploid linearetetraploid.Wealsoreportseveralvariantleaf morphologiesthataretransgene-specific,anddescribea simplemeansofdifferentiatingthealteredleafmorphologyresultingfromtetraploidyfromthoseofthewild typeandtransgene-relatedvariantforms.ResultsSelectionandGFPscreeningofplanttransformantsPriortoinitiatingtransformationprocedures,preliminaryexperimentswereconductedtoexaminetheeffectof explanttypes,combinationsofplantgrowthregulators, antibioticconcentrations,andcultureconditions.The resultsshowedthattheexplantsfromleavesandpetioles respondedbestonthebasalmediumwitheither3mg/l BAand0.2mg/lIBA,or2mg/lTDZand0.2mg/lNAA, onwhichbasisBAandIBAwerechosenforsubsequent useasthecytokininandauxinmediacomponents.The explantsrespondedmoreslowlyindarknessthanunder lightinthefirsttwoweeksofculture.Nodularembryos formedatcutedgesofexplantsaftertwoweeksofculture (Figure1A).Shootsregeneratedafterfourmoreweeksin culture.Inclusionofcarbenicillin(CB)at500mg/lhadno observedeffectonembryoorcallusformation.Inclusion ofkanamycin(50mg/l)causeddeathofmostexplantsby 5 – 6weeksinculture.Onthebasisoftheseresults, 500mg/lCB(forsuppressionof Agrobacterium growth) and50mg/lkanamycinfortransformantselectionwere includedwith3mg/lBAand0.2mg/lIBAintheselection medium.GFPsignalswereseenonexplantsafter18days ofco-cultivation.Bythethirdsubcultureonselective medium,mostoftheGFP-(andpresumablyuntransformed)cellmassesandembryogenicstructuresonthe explantshaddied.Ofthesurvivingcellmassesandembryonicstructures,mostbutnotallwereGFP+.EmbryogenesisandplantregenerationGlobularsomaticembryosformedalongsomecutedges oftheleafexplantsduringthefirstweekofcultureon non-selectivemedium,andthenappearedalongallcut edges(Figure1A-arrows)aftertransferofexplantsto selectivemediumandcultureunderlight.Isolatedsingle embryosorembryomasseswerealsoproducedonexplantsurfaces.After4 – 6weekspost-inoculation,most oftheexplantshadformedsomaticembryoclustersonZhang etal.BMCPlantBiology 2014, 14 :23 Page2of11 http://www.biomedcentral.com/1471-2229/14/23

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theircutedgesorsurfaces(Figure1B).Theexplants withGFP+embryoclustersweretransferredtononselectivemediumforfurtherdevelopment.Budsand youngshootsgrewwellonmediumwith1 – 3mg/lBA and0.1-0.2mg/lIBA.GFPscreeningandmicroscope observationconfirmedthatsomaticembryoswent throughheart,torpedo,anddicotyledonstageanddevelopedintowholeplantlets(Figure1C-G).Secondary somaticembryoswereformedonsomeprimarydicotyledonstageembryos(Figure1D-arrow).Inorderto preventmoresecondaryregeneration,youngshootsand smallplantletsweresubculturedontohormone-free medium.Over1500well-developedGFP+plantswere obtained,andabout1000plantsweretransferredtosoil andgrowntotheflowering/fruitingstage.Intheexceptionalcase,theconstructFLNH-C08yieldedGFPpositivecallusthateventuallydiedincultureevenif transferredtokanamycin-freemedium,anddidnotyield anysomaticembryosorregenerantsinfourexperiments with468inoculatedleafexplants.Morphologicalvariationamongregenerated transformantsBythetimeplantshadreachedthethree-true-leafstage ofplantletdevelopment,considerablemorphological variationwasevidentamongtransformants,bothwithin andbetweenconstructs.Unexpectedly,aparticular broad-leafletvariantform(Figure2-right)appeared amongthetransformantsrecoveredfromeveryconstructinwhichregenerationwasobtained.Uponsexual maturity,thesebroad-leafletplantsdisplayedenlarged flowersandnegligiblefruitset.VariationduetopolyploidyBaseduponourpriorfamiliaritywithcolchicine-induced autotetraploidyanditsconsequencesin F.vesca [21],it Figure1 Stagesofsomaticembryogenicdevelopment. Photographsin1A,1Fand1Gweretakenonadissectingmicroscope,whilethose in1Bthrough1EdepictGFPfluorescenceasseenunderUVilluminationviaconfocalmicroscopy. A .Globularembryonicnodules(redarrows) formingalongcutedgesofleafexplants. B .Aclusterofglobularembryosonaleafexplantsurface. C .Anearlytorpedostageembryo. D .Secondary embryo(bluearrow)emergingfromaprimaryembryo. E .Anadvancedstage,bipolarembryowithcotyledonarydevelopment. F .Bipolarembryo attachedtoexplantatbase(rootend). G .Advanced,bipolarembryoswithleafdevelopmentandexpandinghypocotyl. Figure2 Distinctive,broadleafletform(right)ofatetraploid regenerantascomparedwiththenormalleafletform(left)ofa diploidregenerant. Zhang etal.BMCPlantBiology 2014, 14 :23 Page3of11 http://www.biomedcentral.com/1471-2229/14/23

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washypothesizedthatthebroad-leafletregenerantplants wereautotetraploid.Atestofthishypothesis,employing nuclearDNAcontentmeasurementsexpressedasproportionsoftheknownstandard,indicatedthattheputativetetraploidplantshadDNAcontentvaluesthatwere twicethoseofknowndiploidcomparators.Amongarepresentativesamplingof47putativelydiploidandtetraploidtransformantplants,DNAcontentvaluesranged from0.45to0.48forputativetetraploids,andfrom0.23 to0.25forputativediploids(Table1).AroottipchromosomecountofoneplantwithanelevatednuclearDNA contentshowedthatitwasindeedtetraploid(2n=4x=28) (Figure3). Incomparisonsamongthe47plantsthatwerefirst establishedbynuclearDNAtestingtobediploidor tetraploid,B/Aratios(Figure4)weredeterminedand foundtovaryfrom2.5to3.8forthediploids,andfrom 1.8to2.4forthetetraploids(Table1).Thediploidand tetraploidmeansof2.95and2.11,respectively,differed highlysignificantly(p<0.01).ThelowerB/Aratiocharacteristicoftetraploidsresultedfromtheincreasedwidthof thecentralleaflet(dimensionA)relativetotheoverall widthofthetrifoliateleaf(dimensionB)(Figure4).Of992 transformantsrepresentingallsixteenconstructs,173 plants,orabout17%ofthetotal,wereclassifiedastetraploidonthebasisoftheirB/Aratios.Construct-specificleafvariantsWithfourconstructs(FLNHnumbersE08,D09,F10, andE10),variantleafmorphologieswereobservedthat wereconstruct-specific,clearlydifferentfromwildtype (Figure5A),andclearlydistinctfromtheformassociated withtetraploidy.AmongtheGFP-positiveregenerants obtainedwiththesefourconstructs,onlyafew(<10%) Table1B/Aratios,nuclearDNAcontentvalues,oftransgenic F.vesca ‘ Hawaii4 ’ plantsNameofplantlineB/AratioInferredploidyDNAratiowith internalstandard NameofplantlineB/AratioInferredploidyDNAratiowith internalstandard D09-66B3.82x0.24E08-642.44x0.48 E08-523.52x0.24B07-12.34x0.46 D02-303.42x0.23F10-402.34x0.46 E08-303.32x0.25E10-562.34x0.46 E10-443.12x0.23E11-162.34x0.48 B10-63.12x0.23E08-42.34x0.48 E08-513.12x0.24E08-162.34x0.46 G10-63.02x0.23F06-72.24x0.45 F06-132.92x0.24F06-602.24x0.48 D02-72.92x0.23D09-282.24x0.48 C12-52.92x0.23D09-532.24x0.48 F06-1742.82x0.23D09-872.24x0.48 C10-672.82x0.23E10-532.14x0.47 B10-32.82x0.25G10-22.14x0.46 E08-12.72x0.23D02-352.14x0.48 E08-372.72x0.23C12-92.14x0.47 F10-1132.62x0.23F10-102.04x0.46 D09-142.62x0.24E10-12.04x0.45 B07-562.52x0.23G10-1312.04x0.46 F06-1212.52x0.23C10-2412.04x0.46 Mean2.950.23 D02-252.04x0.47 B10-202.04x0.47 C12-22.04x0.47 B07-121.94x0.48 C10-561.94x0.46 A05-71.84x0.47 F10-421.84x0.45 Mean2.110.47TheinternalstandardusedinDNAcontentmeasurementswas Ilexcrenata ‘ Fastigiata ’ .Zhang etal.BMCPlantBiology 2014, 14 :23 Page4of11 http://www.biomedcentral.com/1471-2229/14/23

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displayedconstruct-specificvariantforms,;however,for threeofthesefourconstructs(E08,D09,F10),multipleindependenttransformantsthatdisplayedtherespective variantphenotypeswereobtained,indicatingthatthese featureswereduetotheeffectsofintroducingtheconstructitselfratherthanarisingfrominsertionalmutation. Forthefourthconstruct,E10,onlyasinglevarianttransformantwasobtained.ConstructE08Adistinctivesuiteofmorphologicaleffectswasgenerally consistentamongsixindependenttransformantsobtainedwithconstructE08(Figure5B),oneofwhichwas tetraploid.Perhapsmostobviousamongtheseaberrant featuresispetiolulelength,(petiolule=stalkconnecting leaflettopetioleinacompoundleaf),whichisdistinctly elongatedinbothdiploidandtetraploidE08transformants whileinconspicuousinwildtype ‘ Hawaii4 ’ (Figure5A). TheE08variantplantsrarelyproducerunners,whilewild type ‘ Hawaii4 ’ producesmanyrunners.Thevariantleaf formassociatedwithconstructE08wasstablethrough Figure3 Mitoticchromosomespreaddisplaying28chromosomes inroottipcellofatetraploidregenerant. A B Figure4 DepictionofthemeasuredleafdimensionsAandB usedinthemorphometricassay. Imagesoffullyexpanded trifoliateleavesweremeasuredalongtheAandBaxesasindicated, andtheratioB/Awascomputedforeachleaf. Figure5 Variantleafmorphologiesindiploid(2x – left)and tetraploid(4x – right)wildtype(A),andtransformants(B-E) carryingvariousconstructs.A .Diploid(left)andtetraploid(right) formsof ‘ Hawaii4 ’ B .ConstructE082xand4xtransformants displayingelongatedpetiolules(arrow)andsubtlealterationsin leafletshape.LeafletsoftetraploidE08regenerantswerebroadened asassociatedwithtetraploidy. C .TheleavesofconstructF10 transformantshavearuffledappearance,whichismagnifiedinthe 4xform.Elongatedpetiolulesarealsosometimespresentinthe4x form. D .InconstructD092xtransformants,leavesaresmallerthan wildtype,primarilyduetonarrowingoftheleaflets. E .Inconstruct E10transformants,thereisavariabletendencytodevelopleaves withfourorfiveleaflets(seealsoFigure6). Zhang etal.BMCPlantBiology 2014, 14 :23 Page5of11 http://www.biomedcentral.com/1471-2229/14/23

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vegetativepropagation,andwastransmittedtoaproportionofprogenyderivedfromself-pollination.ConstructF10InthecaseofconstructF10,fiveindependenttransformants – fourdiploidsandonetetraploid – displayeda subtle,variantleafform(Figure5C).Thediploidshad thinnerleaflaminas,lightercolor,andmarkedlyshorter petiolulesascomparedwithwildtype ‘ Hawaii4 ’ .Inthe tetraploid,leafletswerewiderthanintherespectivediploidsandhadanoticeably “ ruffled ” orcorrugatedlook (Figure5C).ConstructD09Threeindependentdiploidtransformantswereobtained, andthesesharedmultiplemorphologicaldistinctions (Figure5D)thatwerestablethroughvegetativepropagation.Ascomparedwith ‘ Hawaii4 ’ ,theselineshadreducedvigor;smaller,narrowerleaves;smallfruitsof ovoidshaped,veryfewrunners,andgreatlyreducedseed set.Notably,mostoftheseedsthatweresetbythese linesdidnotdisplayGFP-fluorescence,andthesuiteof mutantcharacterswasnottransmittedtoprogeny plants.Therecoveryofthreeindependenttransformants sharingthesamesuiteoffeaturessuggeststhatthese featuresmaybeduetotheeffectsoftheconstructitself, ratherthanarisingfrominsertionalmutation.ConstructE10Anoteworthyphenotypeconsistingofvariablyincreasedleafletnumberwasassociatedwithconstruct E10(Figures5E,6).Thisphenotypeappearedtovarying degreesinthreemature(flowering)transformantplants thoughttohavearisenfromasingletransformationevent, andthevariantphenotypewasalsotransmittedtovegetativelypropagatedrunnerplants.Althoughallsuchplants hadmostlytrifoliateleaves,someleavesalsohadfour,five, orevensevenleaflets,andwhenaleafhadmorethan threeleafletssometimestwoadjacentleafletswerepartiallyfused(Figure6).DiscussionOurdetailedobservationsoftheearlystagesoftransgenicplantregenerationundertheemployedconditions clearlyrevealapathwayofsomaticembryogenesis, encompassingheart,torpedo,anddicotyledonstages. ThisfindingcontrastswiththatofOosumietal.,[4], whoachievedtransformationandregenerationof F. vesca ‘ Hawaii4 ’ ,butwhodescribedaregenerationsequencebeginningwithorganogenicshootformationand followedbyrootingupontransfertohormone-free medium.Inoneoftheirfigures,thelatterauthorsindicatedthepresenceof “ embryoniccallus ” [4:legendof Figurethree],butdidnotemploytheterm “ somatic embryogenesis ” orexplicitlyspecifythenatureoftheir regenerationpathwayintheirreport. Somaticembryogenesishasbeenreportedintheoctoploid,cultivatedstrawberry[22-26].Importantly,Husaini andAbdin[24]foundthattheregenerationpathwayfrom leafletexplantsincultivar ‘ Chandler ’ wassteeredtoward eithersomaticembryogenesisordirectshootformation dependingupontheconcentrationofjustonekey mediumcomponent:thidiazuron(TDZ).Thisresearch groupthenreportedtheoptimizationofTDZconcentrationforthepromotionofsomaticembryogenesisin ‘ Chandler ’ [25],statingthatthiswasthepreferred Figure6 VariableleafphenotypesassociatedwithconstructE10. Notepresenceoffourorfiveleaflets,withinstancesofpartialfusion betweenadjacentleaflets(arrow). Zhang etal.BMCPlantBiology 2014, 14 :23 Page6of11 http://www.biomedcentral.com/1471-2229/14/23

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regenerationpathwayfortheirappliedresearchpurposes. Otherfactorsfoundtopromoteembryogenesisinstrawberryincludeaperiodofcultureinthedark[22,27],and coldtreatment[27]. Futureeffortstowardoptimizationofpost-transformation plantletregenerationinthewidelyused ‘ Hawaii4 ’ variety ofthediploidmodelspecies F.vesca willlikelybenefit fromatargetedapproachthatseekstooptimizeeitheran organogenicorasomaticembryogenicpathway,dependinguponwhichpathwaybestservestheprojectneeds. Undertheconditionsemployedinthepresentstudy,we documentedregenerationviathelatterpathway,thereby establishingadefinedbaselineforfuturemethodological enhancementofthesomaticembryogenicapproach.Obviousdirectionsforsucheffortswouldbetoevaluatethe dose-responsivenessof ‘ Hawaii4 ’ explantstoTDZin comparisontoBA,andtheeffectsofcultureindarkness orundervaryinglightregimes. Thefrequent(~17%)occurrenceoftetraploidsamong post-transformationregenerantswasanunexpectedoutcome.Althoughnumerousreportsof Fragaria regenerationhaveappeared,nonehasyetreportedploidy changes.Inourstudy,theoccurrenceoftetraploidtransformantswasnotspecifictoanyparticularconstructsequence,astetraploidsoccurredamongthetransformants thatyieldedregenerantsregardlessofconstructtype.The detectionoftetraploidywasanunanticipated,adhocobservation,forwhichreasonwedidnotexaminethepotentiallycausalaffectsofexperimentalfactorssuchasvector systemorculturalconditions.Therefore,wecannotseparatethevariousaspectsofthetransformationandregenerationprocedures,consideredaloneorinconcert,as possiblecausalfactorsintheinductionoftetraploidy baseduponavailabledata.However,severalintriguing questionsaresuggested,asdiscussedbelow. First,is ‘ Hawaii4 ’ particularlysusceptibletotheinductionoftetraploidy,orissuchsusceptibilityageneralizedphenomenonin F.vesca ,indiploid Fragaria ,orin Fragaria ingeneral?Thefactthatelevatedploidiesfollowinginvitromanipulationshavenotbeenreported previouslyin Fragaria suggeststhepossibilitythatthe phenomenonwedocumentedin ‘ Hawaii4 ’ mayhaveat leastanelementofgenotype-and/ortaxon-specificity. Alternatelyoradditionally,theemployedvectoranddeliverysystemmayhavebeenafactorinthetetraploid outcomes.WeemployedaspecificGatewayvector[20], whereasOosumietal.,[4]employedthepCAMBIA1304binaryvector,whileourstudyandtheirsboth employedan Agrobacterium -baseddeliverysystem. Oosumietal.[4]didnotreporttheoccurrenceoftetraploidyamongtheirtransformants;however,acareful examinationofthefourtransformantsthatwerephotographicallydocumentedbytheseauthorssuggeststous thatthetransformantdepictedintheirFigureeight-b[4] maybeatetraploid,assuggestedbythedistinctiveleaf morphologythatwehaveshowntobeindicativeof tetraploidy. Thetetraploidtransformantsof ‘ Hawaii4 ’ thatweexaminedallsharedadistinctiveleafmorphology,which manifestedasaquantifiablyalteredratio(B/A)ofcentral leaflettooverallleafwidth.Increasedcellsizeisawidely documentedandgeneralconsequenceofploidyelevation, atleastfromthediploidtothetetraploidlevel.Itispossiblethatincreasedcellsizealonemayaccountforthedisproportionatebroadening(versuslengthening)ofallthree leafletsofthestrawberrytrifoliateleaf,thenetresultof whichisthatthebroadeningofthecentralleafletisproportionatelygreaterthantheoverallbroadeningofthe trifoliateleaf.Asyetwehavenotdefinedthetetraploidyassociatedchangeinleafmorphologyatthecellularlevel. Wehave,however,thoroughlydocumentedthefactof tetraploidy,andhaveprovidedasimplemorphological metricthatallowsitsdetectionanddistinctionfromdiploidplantsamongregeneranttransformantsof ‘ Hawaii4 ’ Therecognitionthattetraploidyoccursfrequentlyand thatithasadistinctivephenotypewhenitdoesoccurin ‘ Hawaii4 ’ transformantswillenhancetheabilityofresearcherstoidentifymutant,non-tetraploidformsinmutagen-treatedand/ortransformed ‘ Hawaii4 ’ plants. Inthepresentstudy,weobtainedtransformantswith variouslyalteredleafmorphologies,andonthebasisof ourcharacterizationoftheeffectsoftetraploidywereable topartitionploidy-relatedfromconstruct-associatedalterationsinleafmorphology,evenwhenbothoccurredin thesameplant.Thus,thepetioluleelongationassociated withintroductionofconstructE08waspresentinboth diploidandtetraploidregenerants(Figure5B),andthe tetraploidformwasclearlydistinguishablebyitswidened leaflets(Figure5B-right)andalteredB/Aratio.Contrastingly,theruffledleafletphenotypeassociatedwithconstructF10wasconsiderablymagnifiedinthetetraploid,as comparedwiththediploid,form(Figure5C). AusefulcatalogueofmorphologicalfeatureswasprovidedbySlovinetal.[28],pertainingto F.vesca inbred line5AF7,ayellow-fruitedandrunnerless ‘ Alpine ’ genotypecloselyrelatedto ‘ Hawaii4 ’ ,whichitselfisarunneringvariety.Thiscomprehensivephenotypicdescription wasintendedtoprovideabaselinetowhichotherforms includingderivativemutantformscouldbecompared. Petiolelengthwasamongthefeaturesdescribed;however, nomentionwasmadeofpetiolulelength.Thepetiolule elongationdisplayedbyE08transformantsrevealsand providesdefinitiontoanadditionalvariabletraittobe foundin Fragaria ,thusaddingtotheusefultraitcatalogue contributedbySlovinetal.[28]. AnotherinterestingleafphenotypeoccurredinassociationwithconstructD09(Figure5D),andthisphenotypewasrepresentedonlyindiploidregenerants.Here,Zhang etal.BMCPlantBiology 2014, 14 :23 Page7of11 http://www.biomedcentral.com/1471-2229/14/23

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theleaveswereofslightlyreducedsize,mostlydueto narrowingoftheleaflets,givingtheplantanoverallgangly look.Finally,severalregenerantscarryingconstructE10 exhibitedavariableincreaseinleafletnumber,fromthe usualthreetofour,five,orevenseven,whereinadjacent leafletsweresometimespartiallyfused(Figures5E,6). Pentafoliateleavesareadefiningfeatureofdiploid Fragaria species F.pentaphylla ,aformindigenousto theTibetanregion.However,in F.pentaphylla ,the additionalleafletsarequitesmallorvestigial,andare attachedmuchloweronthepetiole[29],whileinE10 theadditionalleafletsareattachedatmore-or-lessthe samepointasthenormalthreeleaflets(Figure6).The instabilityoftheE10variantformanditsoccurrence inonlyoneconfirmedindependenttransformantinvokes thepossibilityofaninsertionalorothermutageniccause. Asomaclonalvariantofcultivatedstrawberry( Fragaria ananassa )variety ‘ Redcoat ’ withasimilarphenotypewas describedbyNehraetal.[30].ConclusionsTheresultsreportedheredefineopportunitiestooptimize theexisting ‘ Hawaii4 ’ protocolbyfocusingontreatments thatspecificallypromotesomaticembryogenesis.Thereportedmorphologicalmetricsanddescriptionswillguide futuretransgenicstudiesusingthe ‘ Hawaii4 ’ modelsystembyalertingresearcherstothepotentialoccurrenceof polyploidregenerants,andtodifferentiatingtheeffectson leafmorphologyduetopolyploidyversustransgenicmanipulations.Theresultsreportedhereraisemanyintriguingquestionsaboutthetransformationsystemandhost genotypeemployedinthepresentstudy.Wastetraploidy inducedbysomeaspectoftheregenerationsystem,ordid thepolyploidsarisefrompolyploidcellspre-existingin theexplantmaterial?Towhatextentwasthehostgenotypeacontributingfactor?Forconstructsassociatedwith alteredphenotypes,whydidonlyasmallproportionofregenerated,GFP+plantsdisplaytheassociatedaberrant phenotype?Yetasidefromthegenerationofbiological andmethodologicalpuzzlesthatinvitefurtherstudy,the presentinvestigationalsoyieldedintriguingmutantlines displayingaremarkablespectrumofleafmorphologyalterationsandresultingfrommanipulationsofpreviously uncharacterizedgenes,therebyopeningnewavenuesto thecharacterizationofnovelgenefunctions.MethodsVectorconstructionandmanipulationExpressedsequencesofunknownfunctionwereidentifiedinpartialsequencesfrom5 endsequencingoffruit andflowercDNAlibraries.Sequenceswerecharacterizedas “ unknown ” ifBLASTanalysisrevealedanexpectation(E)value<10-3.Preferencewasgiventosequences lackingdomainstructure,asdeterminedbytestsagainst INTERPRO(http://www.ebi.ac.uk/interpro/)databases. AsapreludetofuturefunctionalanalysesusingRNAi suppression,constructscontainingthesequencesof interestwereintroducedinto F.vesca ‘ Hawaii4 ’ using Agrobacterium-mediatedtransformation,asdescribed below. TheGatewayvectorsystem(Invitrogen,Carlsbad, California)[20]wasemployed.Entryclones(indonorvector,pDONR ™ 222)withconstructsofsixteen “ unknown ” genefragmentsintherangeof700 – 1800bpweredevelopedusingstandardprocedures.ConstructsweremobilizedtotheRNAidestinationvector,[pK7GWIWG2D (II),0][20]byLRreactionaccordingtothemanufacture ’ s instruction(GatewayLRClonase ™ IIenzyme,Invitrogen). ToverifyLRproductsizes/identitiesforeachconstruct, theplasmidswereisolatedfrom4to8clonesanddigested withrestrictionenzyme Bsr GIandanalyzedonagarose gels. Theexpressionclonesweretransformedinto Agrobacterium strainGV3101byelectroporation. Agrobacterium colonieswereverifiedbycolonyPCRusingGFPmarker geneprimersGFP-F1(5 -CGGCGGCGGTCACGAAC TC)andGFP-R1(5 -CACCTACGGCAAGCTGACCCT GAA);andconstruct-specificprimers(notshown).PlantinvitroculturemediaMB5medium(MSsalts,[31]withB5vitamins[32]was usedasbasalmedium,with0.8%(w/v)agarinculture platesandvessels.Plantgrowthregulators,vitaminsand antibioticsolutionswerefilter-sterilizedandaddedinto thebasalmediumpost-autoclaving,whenthemedium temperaturehaddroppedtoabout50C.PlantmaterialfortransformationFragariavesca ‘ Hawaii4 ’ seedsweresterilizedin30% (v/v)commercialbleach(6%NaOCl)solutionwith2 dropsTween20for10minutesfollowedbyrinsing themfourtimeswithsteriledistilledwater,andthen inoculatedontoagarplatesofMB5atpH5.8with3% (w/v)sucrose.Germinatingseedswereculturedinitially at22Cunderdimlight(8 molm-2s-1)fortwoweeks, thenat25Cunder12hoursphotoperiod(lightintensity 124 molm-2s-1).Seedlingswithoneormoretrue leavesweretransferredintoMagentaGA7boxescontainingbasalmediumtoattainincreasedsize.Theseedlingswereroutinelytransferredintofreshmediumtwo weekspriortotheiruseasexplantsources.Explantco-cultivationTheco-cultivationmedium(coMB5)wasMB5atpH5.5, with2%(w/v)sucrose,towhichothercomponentswere addedonlyasspecifiedbelow.Young,fullyexpandedleafletswereplacedadaxialsideupontwolayersofsterilefilterpaperwettedwithsterilewaterinaPetridishandZhang etal.BMCPlantBiology 2014, 14 :23 Page8of11 http://www.biomedcentral.com/1471-2229/14/23

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slicedwithascalpeltoproducemultiplecutsacrossand/ oralongthesecondaryveins,therebyproducinganabundanceofwoundsiteswhilekeepingtheoverallstructure oftheleafletintactasaunittoenableeaseofhandling. Thethreeleafletsofeachleafwerealsokeptconnected untilandduringtheincubationwith Agrobacterium ,but wereseparatedbycuttingafterincubationor(best)after washingoffthe Agrobacterium .Freshlypreparedexplants wereuseddirectlyforco-cultivationwith Agrobacterium thenplatedontoselectivemediumasdescribedbelow, with20leafexplantsperplate.About50 – 100leafletswere usedasexplantsforeachconstruct. Agrobacterium withspecificconstructswerecultured in3mlLBmediumwith30mg/lrifampicin,50mg/l gentamycinand100mg/lspectinomycinat28Cina shakerforabout20 – 22.5hours(OD600,1.1-2.8).The bacteriawerewashedwithliquidcoMB5mediumand thenresuspendedin25mlcoMB5mediumwith10ul 0.25 Macetosyringone.ThefinalOD600ofthecells usedforco-cultivationwas0.15-0.34. Forco-cultivation,thefreshlyslicedexplantswere placedintothe Agrobacterium suspensionandincubated onashaker(50 – 90rpm)for25 – 40minutesatroom temperature.Theexplantswerethenquicklyblotteddry onsterilizedfilterpaperandthenplacedabaxialsideup in0.8%(w/v)agarcultureplatesofcoMB5plus3mg/l 6-benzylaminopurine(6-BA)and0.2mg/lindole-3butyricacid(IBA).Afterco-culturefor2 – 3daysatroom temperatureinthedark,theexplantswererinsed2 – 3 timeswith15 – 30mlliquidcoMB5mediumwith 500mg/lcarbenicillin(CB),thenblotteddryonsterilizedfilterpaper,thencutintosmallerpiecesandputon MB5mediumatpH5.8with2%(w/v)sucrosewith 500mg/lCB,3mg/l6-BAand0.2mg/lIBAtodevelop for7 – 8daysatroomtemperatureinthedark.SelectionoftransformantsTheexplants,whichhadexpandedinsizeduringculture,werethencutagainintosmallerpiecesbefore transfertoMB5selectivemediumcontaining50mg/l kanamycinand500mg/lCB,andsubsequentculture underthelight.Forallsubsequentproceduresofselectionandregeneration,theMB5mediumwasatpH5.8, with3%(w/v)sucroseandothercomponentsasspecifiedbelow. Theselectionplateswereculturedunderlightfor 12hoursphotoperiod(lightintensity124umolm-2s-1) at25C.Theexplantswerefirstculturedfor8 – 12days onMB5mediumwith3mg/l6-BA,0.2mg/lIBA, 50mg/lkanamycinand500mg/lCB,thensubcultured twiceattwotothree-weekintervalsonthismedium, followedbyrepeatedsubcultureatareducedlevelof 25mg/lkanamycinand250mg/lCB.GFPfluorescence wasperiodicallyassayedafterfiveweeksinculture. Subsequently,theGFPpositiveclustersorindividual shootsweretransferredtothesamemediumwithout kanamycin,butthatwassupplementedwithCBat 100mg/lifany Agrobacterium contaminationwasevident,andsubculturedevery4weeks.Ingeneral,shoots developedintoplantletsonagarmediumwithouthormones;however,upto0.2mg/lBAwasincludedto stimulategrowthwhenshootdevelopmentwasnotvigorous.Plantletswithrootsweretransferredintosoil afterwashingofftheagar.Allexplantswerescreenedfor GFPexpressionsignalsaftereachsubcultureonselective mediumbyviewingplatesunderUVilluminationona confocalmicroscope.FluorescenceconfocalmicroscopyConfocalmicroscopywasperformedattheUniversityof NewHampshireConfocalImagingCenter.Theselection ofGFPpositivetransformantswasdonebyobservation oftissueswithaZeissLSM510MetaConfocallaser scanningmicroscopeequippedwithafluoresceinisothiocyanate(FITC)filter.NuclearDNAcontentmeasurementsRelativequantificationofnuclearDNAcontentswasprovidedbyPlantCytometryService s(Schijndel,Netherlands). Young,foldedleaveswereharvested,placedinlabeled microfugetubesonice,andshippedviaovernightcurrier totheserviceprovider.Intheprovidedassay,thefluorescentstain,4 ,6-diamidino-2-phenylindole(DAPI)was usedtobindthenucleiandDNAratiosbetweeneach sampleinternalstandardandaknownstandard, Ilexcrenata Thunb.f. ‘ Fastigata ’ ,weredetermined.Samplesfrom various Fragaria speciesofknownploidy,including2x and4xformsof F.vesca ‘ BaronSolemacher ’ [21]werealso assayedascomparators.LeafmorphologymeasurementsBaseduponinitialobservationthattheleafletsof4x plants(asconfirmedbynuclearDNAcontentmeasurement)lookedbroaderthanthoseof2xplants,aquantitativemorphometricassaywasdevised,asdepictedin Figure2.Measurementwasfacilitatedbygenerating convenientlypreservable,actual-sizescannedimagesof eachleaf.Thedimensions “ A ” (widthofcentralleafletat itswidestpoint)and “ B ” (transectbetweentipsofthe twolateralleaflets)weremeasured(Figure1),andthe ratioB/Awascomputedforonerepresentativeleafper plantfor992transformants.TheB/Aratiosofinitially identifiedsetsof2xand4xplantswerecomparedusing aStudent ’ sT-test(one-tailed).ChromosomecountRoottipsweretakenfromtetraploidtransformant F06-75,andaceto-orcein-stainedsquashpreparationsZhang etal.BMCPlantBiology 2014, 14 :23 Page9of11 http://www.biomedcentral.com/1471-2229/14/23

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werepreparedasdescribedbyNathewetetal.[33].Activelygrowingroottipswerecollectedandpre-treatedin 0.002M8-hydroxyquinolinesolutionatroomtemperaturefor1hrandsubsequentlystoredat4Cfor15hrs). Theroottipswerethenrinsedin0.075MKClbrieflyand fixedin3:1ethanol:aceticacidat4Cforatleast24hrs. Fixedroottipsweresoftenedin1NHClat60Cfor 20min,thenrinsedbrieflyindistilledwaterpriortoslide preparationandexamination.Abbreviations 6-BA: 6-benzylaminopurine;CB:Carbenicillin;GFP:Greenfluorescentprotein; IBA:Indolebutanoicacid;NAA:1-naphthaleneaceticacid;TDZ:Thidiazuron; MSmedium:MurashigeandSkoogmedium;MB5:MSsaltsandB5vitamins; LBmedium:Luria-bertanibroth;FLNH:FloridaNewHampshire. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions TMDandKMFdesignedandmanagedtheresearch,QZperformedmostof theexperimentalworkanddatagathering.Allthreeauthorscontributedto manuscriptwriting,andapprovedthefinalmanuscript. Acknowledgements ThisworkwassupportedinpartbyagrantfromtheNationalScience Foundation(#0701488;KMF&TMD)andbyNewHampshireAgricultural ExperimentStationProjectNH00433.ThisisScientificContribution2536from theNewHampshireAgriculturalExperimentStation(NHAES).Wethank JennyJingforherassistanceindatacollection,BoLiuforperformingthe chromosomecount,MarkTownleyforassistancewithuseoftheUNH ConfocalMicroscopyFacility,andMelanieShieldsforeditorialassistanceon themanuscript. Authordetails1DepartmentofBiologicalSciences,UniversityofNewHampshire,Durham, NH03824,USA.2HorticulturalSciencesDepartmentandtheGraduate PrograminPlantMolecularandCellularBiology,1301FifieldHallUniversity ofFlorida,Gainesville,FL32611,USA. Received:15October2013Accepted:18December2013 Published:13January2014 References1.FoltaKM,DavisTM: Strawberrygenesandgenomics. 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30.NehraNS,KarthaKK,StushnoffC,GilesKL: Theinfluenceofplant-growth regulatorconcentrationsandcallusageonsomaclonalvariationin callus-cultureregenerantsofstrawberry. PlantCellTissueandOrgan Culture 1992, 29 (3):257 – 268. 31.MurashigeT,SkoogF: Arevisedmediumforrapidgrowthandbioassays withtobaccotissuecultures. PhysiolPlant 1962, 15 (3):473 – 497. 32.GamborgOL,MillerRA,OjimaK: Nutrientrequirementsofsuspension culturesofsoybeanrootcells. ExpCellRes 1968, 50 (1):151 – 158. 33.NathewetP,YanagiT,SoneK,TaketaS,OkudaN: Chromosome observationmethodatmetaphaseandpro-metaphasestagesindiploid andoctoploidstrawberries. ScientiaHorticulturae 2007, 114 (2):133 – 137.doi:10.1186/1471-2229-14-23 Citethisarticleas: Zhang etal. : Somaticembryogenesis,tetraploidy, andvariantleafmorphologyintransgenicdiploidstrawberry( Fragaria vesca subspecies vesca ‘ Hawaii4 ’ ). BMCPlantBiology 2014 14 :23. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Zhang etal.BMCPlantBiology 2014, 14 :23 Page11of11 http://www.biomedcentral.com/1471-2229/14/23