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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
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
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
GuptaandVanEck 3
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
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
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
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
GuptaandVanEck 5
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
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
GuptaandVanEck 6
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
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
GuptaandVanEck 7
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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GuptaandVanEck 8
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
161
162
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GuptaandVanEck 9
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
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
GuptaandVanEck 10
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
208
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
GuptaandVanEck 11
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
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
GuptaandVanEck 12
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
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
GuptaandVanEck 13
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
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
GuptaandVanEck 14
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
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
GuptaandVanEck 15
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
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
GuptaandVanEck 16
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398
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GuptaandVanEck 19
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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GuptaandVanEck 20
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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