tomato transformation gupta van eck - bioRxiv.org · 2016-04-02 · Gupta and Van Eck 2 11 Abstract 12 Background: Tomato (Solanum lycopersicum) has rapidly become a valuable model

GuptaandVanEck 1

ModificationofplantregenerationmediumdecreasesthetimeforrecoveryofSolanum1

lycopersicumcultivarM82stabletransgeniclines2

3

SarikaGuptaandJoyceVanEck*4

TheBoyceThompsonInstitute,533TowerRoad,Ithaca,NY14853USA5

6

*Correspondingauthor,e-mail:[email protected];phone:607-254-1686;fax:607-254-12427

8

SarikaGupta,e-mail:[email protected]

10

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted April 2, 2016. . https://doi.org/10.1101/046839doi: bioRxiv preprint

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GuptaandVanEck 2

Abstract11

Background:Tomato(Solanumlycopersicum)hasrapidlybecomeavaluablemodelasaresult12

oftheavailabilityofahighqualityreferencegenome,extensivegeneticresources,andefficient13

genetransfermethodology.Ahigh-throughputmethodtoobtaintransgeniclinessoonerthan14

standardmethodshasthepotentialtogreatlyadvancegenefunctionstudies.15

16

Results:Thegoalofthisstudywastooptimizeourcurrentgenetransfer(transformation)17

methodfortomatobyinvestigatingmediumcomponentsthatwouldresultinadecreasedtime18

forrecoveryoftransgeniclines.Themethodologyreportedhereisbasedoninfectionof6-day-19

oldcotyledonexplantsfromSolanumlycopersicumcultivarM82invitro-grownseedlingswith20

AgrobacteriumtumefaciensstrainLBA4404containingthebinaryvectorpBI121.Thisvector21

containstheβ-glucuronidasereportergeneandtheneomycinphosphotransferaseIIselectable22

markergenethatconfersresistancetokanamycin.Modificationofourstandardplant23

regenerationmediumbytheadditionofindole-3-aceticacid(IAA)atconcentrationsofeither24

0.05mg/lor0.1mg/lgreatlydecreasedthetimeforrecoveryoftransgeniclinesby6weeksas25

comparedtoourstandardmediumthatcontainszeatinalone.Moreover,additionof1mg/l26

IAAtotherootinductionmediumresultedinfasterrootdevelopment.Weobserved50%and27

54%transformationefficiencywhenplantregenerationmediumcontaining0.05mg/land0.128

mg/lIAA,respectively,wasusedfortransformationexperiments.Transgeniclinesrecovered29

withtheoptimizedmethodweresimilarindevelopmenttoourstandardmethod.Nonegative30

effectswereobservedasaresultoftheadditionofIAAtothemedium.31

32


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Conclusions:TheresultsshowedthatadditionofIAAatconcentrationsofeither0.05or0.133

mg/ltotheplantregenerationmediumreducedthetotaltimeforrecoveryofstabletransgenic34

linesby6weeks.Theabilitytorecovertransgeniclinesinashortertimeresultsinhigher35

throughputfortheintroductionofgeneconstructsandhasthepotentialtodecreasethetime36

neededtocompleteinvestigationsofgenefunction.Theabilitytorecovertransgeniclinesfor37

eachgeneconstructinashortertimesaveslaborandresourceexpensesbecauseculture38

maintenanceisalsoreduced.39

40

Keywords:Agrobacteriumtumefaciens,indole-3-aceticacid,Solanaceae,Solanum41

pimpinellifolium,tomato42

43

Background44

Tomato,Solanumlycopersicum,isamemberoftheSolanaceaefamily,whichcontains45

approximately3,000plantspeciesandincludessomeofthemosteconomicallyimportantfood46

crops.ItisnativetoSouthAmericaandwasbroughttoEuropeinthe1500sandthentoNorth47

Americainthe1800s[1].Tomatoisaperennialplantthathastwodifferentgrowthhabits,48

determinateandindeterminate.Therearetwodifferentmarkettypesoftomatoes,fresh49

marketandprocessing.AccordingtotheAgriculturalMarketingResourceCenter,in2014the50

USdollarvalueforfreshmarkettomatoeswas1.14billionand1.325billionforprocessing51

types,whichareusedtomakeproductssuchasjuice,sauces,andketchup[2].Inadditionto52

beinganeconomicallyimportantfoodcrop,tomatoisanexcellentsourceofhealthbeneficial53

nutrientsincludingbeta-caroteneandlycopene.54


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GuptaandVanEck 4

Overtheyears,utilizationoftomatoasamodelplantspecieshasincreasedbecauseof55

readilyavailableresourcessuchasmutantpopulations[3],bioinformaticstools[4],andahigh56

qualityreferencegenome[5].Inaddition,sincetheveryfirstreportofAgrobacterium-57

mediatedtransformationoftomatobyMcCormicketal.[6],therehavebeenotherreportsof58

successfultransformationsofdifferentgenotypes[7-12].Akeyaspectfortheadoptionofa59

modelplantspeciesistheavailabilityofefficienttransformationmethodology.Thiswas60

certainlythecaseforArabidopsis,whichisbyfarthemostwidelyusedmodelforplantresearch61

programs[13].62

Whilethereareseveralmethodsavailableforplanttransformation,Agrobacterium63

tumefaciens-mediatedtransformationhasbecomethemostextensivelyusedmethod[14,15].64

Despiteitseffectivenessforgenetransferintomato,thereisstillneedforimprovement.65

Improvingmethodologytodecreasethetimefromintroductionofageneconstructofinterest66

torecoveryofstabletransgenicswouldimprovethethroughputandshortenthetimeframefor67

studiesthatutilizetomatotransgeniclines.68

Wewereinterestedinfindinganapproachtodecreasethetimetoobtaintransgeniclines69

oftheprocessingtypetomatoM82becausethisgenotypeisusedforgenefunctionstudiesin70

ourlabaswellasothers[16,17].Wechosetostartbyinvestigatingsupplementationofour71

standardplantregenerationandrootingmediawithagrowthregulatorthathadthepotential72

tospeedupplantdevelopment[12].73

Cytokininsandauxinsareimportanthormonesthatinfluencegrowthanddevelopmental74

processesinplants.Interactionsbetweencytokininsandauxinshavebeenshowntobe75

necessaryfortheshootapexgrowth[18,19].Auxinhasalsobeenshowntoplayaroleinthe76


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specificationoftherootapicalmeristem[18,20,21].Thehormonalinteractionscanbeutilized77

intheareaoftissueculturetoleveragethepresenceofthehormonesinthemedium.Inthis78

study,wereporttheeffectsoftheadditionoftheauxin,indole-3-aceticacid(IAA)onthe79

recoverytimeofM82transgeniclines.80

81

Methods82

Plantmaterial83

SeedsofSolanumlycopersicumcvM82weresurfacesterilizedin20%(v/v)bleachsolution84

containingTween-20for20minfollowedby3rinsesinsterilewater.Seedsweregerminatedin85

MagentaGA7boxes(CaissonLabs,Logan,UT)thatcontained50mlofMurashigeandSkoog86

(MS)[22](CaissonLabs)basedmediumcontaining2.15g/lMSsalts,100mg/lmyo-inositol,287

mg/lthiamine,0.5mg/lpyridoxine,0.5mg/lnicotinicacid,10g/lsucroseand8g/lSigmaagar88

(Sigma-Aldrich,St.Louis,MO).Culturesweremaintainedat24°Cundera16hlight/8hdark89

photoperiodat57–65uEm-2s-1.90

OnedaypriortoinfectionwithAgrobacterium,cotyledonexplantsandfeederlayerplates91

wereprepared.Feederlayerswerepreparedbeforecuttingtheexplantsbydispensing2mlof92

a1-week-oldNT1suspensioncultureontoKCMSmedium(4.3g/lMSsalts,100mg/lmyo-93

inositol,1.3mg/lthiamine,0.2mg/l2,4-dichlorophenoxyaceticacid,200mg/lKH2PO4,0.1mg/l94

kinetin,30g/lsucrose,5.2g/lAgargel(SigmaAldrich),pH6.0.Thesuspensionwascovered95

withasterile7cmWhatmanfilterpaper.Explantswereexcisedfrom6-day-oldseedlings96

beforethefirsttrueleavesemerged.Topreparetheexplants,seedlingswereplacedona97

sterilepapertowelmoistenedwithsterilewater.Cotyledonswereexcisedatthepetioles,cut98


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intoapproximately1cmsections,placedadaxialsidedownontheKCMSfeederlayerplates,99

andmaintainedat24°Cundera16hlight/8hdarkphotoperiodat57–65uEm-2s-1.100

101

Bacterialstrainandbinaryvector102

ElectroporationwasusedtointroducethepBI121vectorintotheAgrobacteriumtumefaciens103

strainLBA4404.Asingle,well-formedcolonyfromtheselectionplatewastransferredto50ml104

ofYEPselectivemediumthatcontained50mg/lkanamycinandmaintainedinashaking105

incubatorat28°Cfor18–24hrsorthelengthoftimeneededtoreachanOD600of0.6-0.7.106

TheAgrobacteriumsuspensionwascentrifugedat8000rpmfor10minat20°C.Thepelletwas107

resuspendedin50mlof2%MSOmedium(4.3g/lMSsalts,100mg/lmyo-inositol,0.4mg/l108

thiamine,and20g/lsucrose)byvortexing.109

110

Agrobacterium-mediatedtransformation111

CotyledonexplantswereincubatedintheAgrobacterium/2%MSOsuspensionfor5min,112

transferredtoasterilepapertoweltoallowexcesssuspensiontobrieflydrain,placedback113

ontothefeederplateswiththeadaxialsidesdown,andco-cultivatedinthedarkat19°Cfor48114

hrs.Explants,adaxialsideup,weretransferredtoourstandardplantregenerationselective115

mediumdesignated2ZKthatcontained4.3g/lMSsalts,100mg/lmyo-inositol,1ml/lNitsch116

vitamins(1000x),20g/lsucrose,2mg/ltrans-zeatin,75mg/lkanamycin,300mg/ltimentin,117

and5.2g/lAgargel.Oneweeklater,theexplantsweretransferredonto2ZKmedium118

containingIAAateither0mg/l,0.01mg/l,0.05mg/l,0.1mg/l,or0.5mg/lIAA.119


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Aftertwoweeks,explantsweretransferredonto1ZKmediumthatcontained4.3g/lMS120

salts,100mg/lmyo-inositol,1ml/lNitschvitamins(1000x),20g/lsucrose,1mg/ltrans-zeatin,121

75mg/lkanamycin,300mg/ltimentin,5.2g/lAgargel,andIAAateither0mg/l,0.01mg/l,0.05122

mg/l,or0.1mg/lIAA,or0.5mg/linplatesorMagentaGA7boxesdependinguponthesizeof123

theshootsregeneratingfromthecotyledonexplants.124

ShootswereexcisedandtransferredtoselectiverootingmediumdesignatedRMK(4.3g/l125

MSsalts,1ml/lNitschvitamins(1000x),30g/lsucrose,pH6.0,8g/lDifcoBactoagar(Becton,126

DickinsonandCompany,FranklinLakes,NJ),75mg/lkanamycin,300mg/ltimentin,andIAAat127

either0mg/lor1mg/linMagentaGA7boxes.128

Unlessotherwisenoted,thepHofallmediawasadjustedto5.8beforeautoclaving.Forall129

media,thetrans-zeatin,IAA,kanamycin,andtimentinweredispensedfromfiltersterilized130

stocksolutionsintoautoclavedmediumthatwasallowedtocoolto55oC.Cotyledonexplant131

culturesweretransferredtofreshlypreparedmediumeverytwoweeks.132

133

GUShistochemicalassay134

Histochemicalassayofβ-glucuronidase(GUS)activitywasperformedonleavesfromputative135

transgenicandcontrol(non-transformed)plants.Leaveswerevacuuminfiltratedfor20–30136

mininbuffer(0.8g/l5-bromo-4-chloro-3-indolyl-𝛽 −D-glucuronide(X-Gluc),0.1MNa2HPO4,137

0.1MNaH2PO4phosphate,10mMethylenediaminetetraaceticacid(EDTA),1.6mM138

potassium-ferricyanideand1.6mMpotassium–ferrocyanide,5%v/vTritonX-100,and20%v/v139

methanol)beforeincubationat37°Covernight.Thechlorophyllwasremovedfromtheleaves140


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by3-4washeswith70%ethanolatroomtemperature.TheleaveswereexaminedwithaLeica141

S8APOstereomicroscopeoutfittedwithadigitalcamera.142

143

Polymerasechainreactionanalysis144

ToconfirmthepresenceoftheneomycinphosphotransferaseIIselectablemarkergene(nptII),145

DNAwasextractedfromleavesofputativetransgeniclinesandcontrols(non-transformed)146

withtheQiagenDNeasyplantminikit(Hilden,Germany)asperthemanufacturer’s147

instructions.PrimersusedtodetectnptIIwereforward5’-GGCTGGAGAGGCTATTC-3’and148

reverse5’-GGAGGCGATAGAAGGCG-3’.Thediagnosticampliconsizeexpectedwiththese149

primersisapproximately700bp.ThePCRprogramstartedwithaone-stepcycleof2minat150

95oC,followedby29cyclesof30sat94oC,45sat57oC,50sat72oC,anda10minfinal151

extensionat72oC.DNAwasseparatedandvisualizedbyelectrophoresisthrougha1%152

agaraose,ethidiumbromide-stainedgel.153

154

ExperimentalDesign155

Atotalof5differentexperimentswereperformed.Threebiologicalreplicateswereusedfor156

eachIAAconcentrationineachexperiment.Atotalof750cotyledonexplantswereusedper157

IAAconcentrationinvestigated.Thestandarderrorwascalculated.158

159

160

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Results163

OptimizationofIAAconcentrationforrecoveryofstabletransgeniclines164

Followingtheco-cultivationperiod,cotyledonexplantsweretransferredtoourstandard165

selectiveplantregenerationmediumdesignated2ZKthatcontains2mg/ltrans-zeatinasthe166

onlyplantgrowthregulator.Oneweeklater,theexplantsweretransferredto2ZK167

supplementedwithdifferentIAAconcentrations(0mg/l,0.01mg/l,0.05mg/l,0.1mg/l,0.5168

mg/l)todetermineiftheadditionofIAAwoulddecreasethetimefrominfectionwith169

Agrobacteriumtorecoveryofstabletransgeniclines.Wecontinuedtousethissameseriesof170

IAAconcentrationsinthesubsequentselectiveplantregenerationmediumdesignated1ZK.171

MediumsupplementedwithIAAresultedinshootsthatweremorefullydevelopedearlierin172

thecultureprocessascomparedtomediumwithoutIAA(Fig.1A).InFigure1A,cotyledon173

culturesshownina–erepresentcontrolsthatwerenotinfectedwithAgrobacterium.We174

observedthatastheIAAconcentrationincreased,thelevelofplantregenerationfromthe175

controlsdecreased(Fig.1A,a–d).ForourstandardmethodwithoutIAA(Fig.1A,e),thelevel176

ofplantregenerationissignificantlylessincomparisonwithmediumthatcontainedIAA.177

CotyledonexplantsinfectedwithAgrobacteriumandculturedonmediumcontainingIAA178

exhibitedthesamepatternofshootdevelopmentasthecotyledonsnotinfected,inthatwe179

observedmorewell-developedshootsatanearlystageofculturepostinfection(Fig.11f–i).180

Ingeneral,earlieremergenceofwell-developedshootsfromAgrobacterium-infected181

cotyledonexplantsonmediumcontainingIAAtranslatedtotherecoveryofwholerootedplants182

inlesstimeascomparedwithmediumthatdidnotcontainIAA(Table1).Mediumcontaining183

either0.05mg/lor0.1mg/lIAAresultedintheshortesttime,11wks,forrecoveryofstable184


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transgeniclines.ThereappearedtobeathresholdofIAAconcentrationandeffectonrecovery185

timebecauseat0.5mg/lIAAthetimewassimilartoourstandardmethod.Weobserveda186

similardecreaseintimewhentransformationsofothertomatogenotypeswereperformedby187

differentlabmemberswhotestedplantregenerationmediumthatcontained0.1mg/lIAA188

(dataunpublished).189

190

EffectofIAAontransformationefficiencyandrooting191

Theformulabelowwasusedtocalculatetransformationefficiency(TE).192

Transformationefficiency(%):193Totalnumberofrootedshoots x100194

TotalnumberofcotyledonexplantsinfectedwithAgrobacterium195196

Overall,theTEwaslowerwhenmediumcontainingIAAwasusedascomparedtotheTEof88%197

whenmediumcontainingtrans-zeatinastheonlygrowthregulatorwasused(Table1).198

Whenputativetransgeniclineswereapproximately3-4cmtall,theywereremovedfrom199

thecotyledonexplantsandtransferredtoeitherourstandardselectiverootingmedium(RMK)200

withoutIAAorRMKsupplementedwith1mg/lIAA.Wechosethisconcentrationbasedon201

previousworkwithtomatotransgeniclinesrecoveredfromafewgenotypesthatdidnotroot202

aswellasM82onourstandardrootingmedium(datanotpublished).Weobservedthatshoots203

culturedonRMKsupplementedwithIAAresultedintheemergenceofrootsafter6-7daysas204

comparedto11-14daysonmediumwithoutIAA.TheadditionofIAAtothemediumdidnot205

resultinanyphenotypicdifferencesoftheplantsascomparedtomediumthatdidnotcontain206

IAA.207

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Characterizationofputativetransgeniclines209

ThefirstlevelofanalysistoconfirmtherecoveredplantsfromAgrobacterium-infected210

cotyledonsweretransgenicwasahistochemicalassayfortheGUSreporterprotein.Whole211

leavesfromplantsrootedonRMKwereusedfortheanalysis.AllleavesexhibitedGUSactivity,212

althoughweobservedvariationinthelevelofintensitywithsomeleavesexhibitingadarker213

colorationthanothers(Fig1B).GUSactivitywasnotobservedinleavesfromnon-transformed214

controlplants.215

Tofurtherconfirmtherecoveredplantswereindeedstabletransgeniclines,wedidPCR216

analysisforthepresenceofthenptIIselectablemarkergeneinplantsfoundtobepositivefor217

GUSactivity.TotalgenomicDNAwasisolatedfromtheleavesoftheGUS-positivelinesand218

non-transformedcontrolplants.PCRamplificationofthenptIIgenewasdetectedinplantsthat219

werealsoGUSpositive.NoamplifiedproductwasdetectedinDNAfromthecontrol,(non-220

transgenic)plants(Fig.1C).221

222

Modifiedprotocol223

Basedonourfindings,wenowfollowamodifiedprotocolasoutlinedinFigure2forall224

Agrobacterium-mediatedtomatotransformations.IAAconcentrationsof0.5and0.1mg/lIAA225

bothresultedina6-weekdecreaseforrecoveryofstabletransformants,however,wechoseto226

use0.1mg/lIAAinourmodifiedprotocolbecauseofthe54%transformationefficiency(Table227

1).InadditiontoM82,wehaveappliedthisprotocoltoothertomatogenotypesandalso228

observedadecreaseintimeforrecoveryoftransgeniclinesascomparedtoourprevious229

tomatotransformationmethodology(datanotpublished).230


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Discussion231

Fordevelopmentofstabletransformationmethodology,theforemostfactorstobe232

consideredaretransformationefficiencyandthetimefrominfectionwithAgrobacterium233

tumefaciensuntiltherecoveryoftransgeniclines.Methodsthatprovidebothhighefficiency234

andtheshortesttimetorecoveryoftransgeniclinesleadtoahigh-throughputpipelinethat235

allowsearlierevaluationofgenefunction.Inturn,ahigh-throughputpipelinedecreasesthe236

amountoflaborandresourcesneeded,whichcantranslateintosignificantfinancialsavings.237

Thefocusofourstudywastoinvestigatemediumcomponentsthathadthepotentialto238

decreasethetimeforrecoveryofstabletomatotransgeniclines.Ourstandardmethod,which239

hasahightransformationefficiencyatapproximately90%,takes17weeksforrecoveryof240

transformants.Theinterestinoptimizationofourmethodsstemmedfromanincreasedneed241

fortransgeniclinesbecausetomatohasbecomethemodelspeciesofchoiceformanystudies242

thatincluderipening,abioticandbiotictolerance,andnutritionalcontent[23-26].Inaddition,243

withtherecentdemonstrationofsuccessfulgenomeeditingbyCRISPR/Cas9intomato,the244

interestinapplyingthistechnologyforthestudyofgenefunctionwillincrease[16,17,27].245

Therefore,atransformationmethodologythatcandelivermodifiedlinesinashortertime246

framewillhelptoadvancethesestudies.247

OurstandardprotocolisamodifiedversionofmethodsreportedbyFillattietal.[8]in248

whichzeatinistheonlygrowthregulatorincorporatedintotheplantregenerationmedium249

[12].Wechosetostartourinvestigationbyexaminingadditionalgrowthregulatorsthat,in250

combinationwithzeatin,wouldgreatlyreducethetimeforrecoveryofstabletransgeniclines251

butnothaveasignificantnegativeeffectontransformationefficiency.Inaliteraturesearch,252


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wefoundseveralreportsthatdemonstratedapositiveeffectontomatoplantregenerationand253

transformationefficiencywhenindole-3-aceticacid(IAA)wasincorporatedintozeatin-254

containingplantregenerationmedium[10,28,29].However,theydidnotreportanyeffects255

observedonthetimerequiredtorecovertransgenicplants.256

Wefoundthatadditionofeither0.05or0.1mg/lIAAtoourstandardplantregeneration257

mediumthatcontainstrans-zeatinastheonlygrowthregulatordecreasedthetimefor258

recoveryofstabletransgeniclinesfrom17to11weeks.Previousreportshavedemonstrated259

thatshootapicalmeristemdevelopmentinvolvesinteractionsamongcytokininsignaling260

pathwaycomponents,auxin,andseveralfamiliesoftranscriptionfactors[18].Itispossiblethat261

theadditionofIAAtoourstandardplantregenerationmediumfacilitatesinteractionsamong262

thecytokininsignalingandauxinregulatedgenes,whichresultsinfastershootdevelopment263

fromthecotyledonexplants.264

AlthoughtherewasareductionintransformationefficiencywiththeadditionofIAAfrom265

approximately90%toabout50%,thislevelisacceptableconsideringtransgeniclinescanbe266

evaluatedsignificantlyearlierthanwhenourstandardmethodwasused.Thisdecreaseintime267

allowsresearcherstotesttheirmaterialearlierandmakechangestotheirapproachessoonerif268

resultsareunsatisfactoryfortheirgenesofinterest.269

InadditiontosupplementationofthestandardplantregenerationmediumwithIAA,we270

alsoinvestigatedeffectsofaddingIAAtotherootingmedium,whichwasnotacomponentin271

ourstandardrootingmedium.InclusionofIAAininvitrorootingmediumhasbeenreported272

fortomato,however,itisnotroutinelyaddedbecausetomatoreadilydevelopsrootsinculture273

mediumwithoutgrowthregulators[9].Ourinterestwastodetermineifsupplementation274


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decreasedthetimetorooting,whichwedidobserve.Auxinisproducedinbothshootsand275

rootsandtheauxinproducedintherootshelpsinrootdevelopment[30-32].Itispossiblethat276

IAA,whenexogenouslyadded,increasesthelevelsofauxinintheplants,henceresultinginthe277

cellsdifferentiatingearliertoformroots.However,researchneedstobeconductedtoconfirm278

thishypothesis.279

280

Conclusions281

Interestintomatoasamodelhasincreasedovertheyearsandwehaveseenariseinthe282

numberofresearchgroupsthatrequirestabletransgeniclinesforvariousstudies.283

Modificationofourstandardplantregenerationmediumthroughtheadditionofeither0.05or284

0.1mg/lAAshortenedtherecoveryoftransgeniclinesby6weeksfortheM82tomatocultivar.285

Applicationofthismodificationfortransformationofothertomatogenotypesinourlabalso286

resultedinadecreasedtimeforrecoveryofstabletransgeniclines.287

Ashorterrecoverytimeforstabletransgeniclinesishighlydesirableforfunctionalstudiesto288

allowearlierdeterminationofthegenesandnetworksinvolvedinphenotypesofinterest.A289

decreaseinrecoverytimewouldalsoprovideahigherthroughputprocess,whichhasthe290

potentialforcostsavingsrelatedtolaborandresources.Optimizationstudiesofstandard291

transformationmethodologiesfordifferentplantspeciesshouldalwaysbeconsideredinorder292

toalleviatebottlenecksforgenerationofstabletransgeniclines[33].Availabilityofefficient293

transformationmethodsisespeciallycriticalwiththerapiddevelopmentofgenomeediting294

technologies,whichwillresultinanincreaseddemandforgenerationoftransgeniclinesfor295

basicresearchstudiesthatcanleadtocropimprovement.296


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Competinginterests297

Theauthorsdeclarethattheyhavenocompetinginterests.298

299

Authors’contributions300

SGandJVEdesignedtheexperiments,SGperformedtheexperiments,SGandJVEwrotethe301

manuscript.Bothauthorsreadandapprovedthemanuscript.302

303

Acknowledgements304

WethankCynthiaDuforassistancewithpartoftheexperimentalprocess.WethankCynthia305

Du,PatriciaKeen,andMichelleTjahjadifortheircriticalreviewofthemanuscript.Supportfor306

thisworkwasthroughagrantfromtheNationalScienceFoundationPlantGenomeResearch307

Program(IOS-1237880).308

309


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33. AltpeterF,SpringerNM,BartleyLE,BlechlAE,BrutnellTP,CitovskyV,ConradLJ,Gelvin395SB,JacksonDP,KauschAPetal:Advancingcroptransformationintheeraofgenome396editing.PlantCell2016,inpress.397

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FigureLegends401

Figure1402

ResultsfortherecoveryofSolanumlycopersicumcvM82stabletransgenicsfrom403

Agrobacteriumtumefaciens-infectedcotyledonexplantsculturedonplantregeneration404

mediumsupplementedwithdifferentconcentrationsofindole-3-aceticacid(IAA).(A)405

Agrobacteriumtumefaciens-infectedcotyledonexplants(approximately5weekspostinfection)406

culturedonselectiveplantregenerationmediumcontainingthefollowingamountsofIAAin407

mg/l(f)0.01,(g)0.05,(h)0.1,(i)0.5,and(j)0.Imagesa–erepresentthecorrespondingnon-408

infectedcontrolsforeachIAAconcentration,respectively.409

(B)HistochemicalanalysisforGUSexpressioninleavestakenfromindependenttransgeniclines410

designated1-9recoveredfromselectiveplantregenerationmediumthatcontained0.1mg/l411

IAA.GUSexpressionwasnotobservedinthenon-transformedcontrols.412

(C)AgarosegelofPCRproductsshowingtheexpected~700bpproductamplifiedfromthenptII413

selectablemarkergenein10independenttransgeniclines(lanes1–10).Theselineswere414

recoveredfromselectiveplantregenerationmediumthatcontained0.1mg/lIAA.C=the415

control416

417

418

419

420

421

422


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Figure2423

SchematicrepresentationoftheoptimizedAgrobacteriumtumefaciens-mediated424

transformationmethodologyforSolanumlycopersicumcvM82.SeetheMaterialsand425

Methodsfordetailsonseedsterilizationandallmediacompositions.426

427

Tables428

Table1:ResultsforrecoveryofstabletransgeniclinesofSolanumlycopersicumcvM82from429

Agrobacteriumtumefaciens-infectedcotyledonexplantsculturedonselectiveplant430

regenerationmediumsupplementedwithdifferentindole-3-aceticacid(IAA)concentrations.431

IAA

(mg/l)

Totalnumber

explants

Totalnumberrootedplants

Averagetransformationefficiency(+)SE*

Totaltimeforrecoveryoftransgeniclines

(wks)0 750 660 88+2.2 17

0.01 750 390 52+1.0 150.05 750 375 50+1.5 110.1 750 405 54+1.2 110.5 750 360 48+2.0 16

432

*Averagetransformationefficiencywascalculatedaspercentofstabletransgeniclines433

recoveredfromthetotalnumberofcotyledonexplantsinfectedwithAgrobacterium434

tumefaciens.Transformationefficiencyvaluesshownaretheaveragefrom5experiments±the435

standarderror(SE)calculatedfrom3biologicalreplicates.436


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a b dc e

f h jig

A

12345678910CC

nptII

B

1 2 3 4 5

Control9876

GuptaandVanEck_Figure 1


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6days

24hrs

2days

1wk

2wks

~4wks

5min

~3wks

Germinationofsterilizedseedsinvitro

Preparationofexplantsfromcotyledons

IncubationinAgrobacteriumsuspension

Co-cultivationofexplants

Transferexplantsto2ZKselectiveplantregenerationmedium

Transferexplantsto2ZIKselectiveplantregenerationmedium

Transferexplantsto1ZIKselectivemedium

RemoveshootsfromexplantstransfertoRMIK

Well-rootedplants

GuptaandVanEck_Figure 2


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tomato transformation gupta van eck - bioRxiv.org · 2016-04-02 · Gupta and Van Eck 2 11 Abstract 12 Background: Tomato (Solanum lycopersicum) has rapidly become a valuable model