Histological Study of Organogenesis in Cucumis Melo L. After Genetic Transformation: Why Is It Diﬀicult to Obtain Transgenic Plants? V Chovelon, V

Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it diﬀicult to obtain transgenic plants? V Chovelon, V. Restier, N. Giovinazzo, Catherine Dogimont, J. Aarouf

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V Chovelon, V. Restier, N. Giovinazzo, Catherine Dogimont, J. Aarouf. Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it diﬀicult to obtain transgenic plants?. Plant Cell Reports, Springer Verlag, 2011, 30 (11), pp.2001-2011. 10.1007/s00299-011-1108-9. hal- 01332269

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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study e-mail : 301 RueBaruchdeSpinoza,BP21239,84916Avignoncedex9,France UAPV Avignon,LaboratoiredePhys J. Aarrouf e-mail : 84143 MontfavetCedex,France INRA Avignon,UR1052,UnitédeGé V. Chovelon why isitdifficulttoobtaintransgenicplants? Histological StudyofOrganogenesisin in :Plant Cell Reports Version définitive dumanuscrit publiédans/Final version ofthemanuscript published of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to

[email protected] [email protected]

. V. Restier , 2011, vol 30 (11) :2001-2011 Comment citer cedocument: . N.Giovinazzo

nétique etd’Amélioration iologie desFruitsetLégumes, PôleAgrosciences,

. Cucumis melo C. DogimontJ. Aarrouf

DOI: 10.1007/s00299-011-1108-9 L.aftergeneti des FruitsetLégumes,BP94, c transformation:

: Cucumis melo Cucumis NptII xplants cultivatedintheseconditi Comment citer cedocument: L.)iswidelyconsidered asarecalcitrant speciesforgenetic and onducted onexplants2,7,15and28daysafterco-culturewith (1.13 ±0.02plantsperexplant) A genes A genes uid Agrobacterium tumefaciens L..Agrobacterium is period,mostofthemeri

Agrobacterium rashige andSkoog(MS)medium supplemented on GUS-positive cellstoregenerate transgenic e difficultiesencountered of regeneration, however, only few transgenic of regeneration,however,onlyfewtransgenic ation. This disruption of theGUS-positive d 0.2mg/l dimethylallylaminopurine (2-iP). cess atdifferentsteps different regenerationandtransformation ons: 70-90%ofexpl infectiononplantregeneration, was obtainedusingcotyledon stematic structures showed uid e GV2260pBI101strain). A by histologicalstudies.

to regeneratemelon reporter gene were ants expresseda .Genetic Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study such asthepresenceofmeristematic protuberan Palenius etal.2008)and theexplants source highly dependentofthegenotype(Galperinet and physicalfactorsaffectthe 2004; Akasaka-Kennedyetal.2004 pathways (Guisetal.1997,2000; described onawiderangeofmelon cultivars, using organogenesis orsomatic embryogenesis melon (FangandGumet 1990),more than40invitro melon regeneration and asuitablemethod fortransformation. Sinceth through involved inhorticultura and transfernovelcharacteristicsforbreeding Genomic toolsexist for thisspecies andplant cultivars withhighlevelsofdiseaseresist breeding hasbeendonebytraditionalhybridi chromosomes and arelativelysma genetic diversity in cultivate and researchsincetheseventeenthcentury.Mel horticultural speciesafterthe family include,i.e.,cucumber,watermelon, and Cucumis melo Introduction al. 2005;Ntui etal.2010). Transformation capacityvarieswidely according tothecultivar 2006) havebeenincorporated intodi 1997; Silvaetal.2004; Curuk salt tolerance(Bordaset al et al.1997;Huttner2001;Yalcin-Mendi virus resistance(FangandGrumet 1993;Yoshioka Grumet 1990;Grayetal.1995;Akasaka-Kennedy (Adelberg etal.1994;Nunez- tissue culture and high fre et al.2001).Explantsoriginaffectstheploidy plantlets withoutapicaldominance, arefreque of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to Melon transformationhasbeensuccessfully obtained withreportergenes(Fangand Agrobacterium tumefaciens L. isoneof themain specieswithinthe Comment citer cedocument: l traits.The twomajor pre-requi . 1997)andlongfruitshelf life quency oftetraploidregene Palenius etal.2008). d andwildgenotypes.Itis Solanaceae and they havebeen the efficiency rateandmelon rege et al.2005;Papadopoulou etal. ll genome, with anestimated si Ezura etal.2000;Kintzios

arethe availabilityofarelia ; Castelblanqueetal.2008). fferent melon typesorinto other ance, flavourandsweetne (Guis etal2000). Morphological abnormalities, al. 2003; reviewed by Pech etal.2007;Nunez- al. 2003;reviewedbyPech ntly observed(Gabaetal.1999;LiborioStipp biotechnology canbeusedtointroducegenes squashes. They representthesecondgroupof zation techniques,butdevelopment ofnew ces thatfailtodevel al.2004;Gabaet2004 ; Wu et al2010), or functionalvalidatio level oftheplantsregeneratedfrommelon on iswidespread inthe worldwith alarge e firstreport ofsuccessfultransformation of etal.1993;Gonsalvez1994;Fuchs al.2004)andseveralplanttraits,suchas . Otherspeciesinthe Cucurbitaceae rants isacommonphenomenon sites forgenetictransformation a diploidspecies,withn=12 (Ayub etal.1996;Ezura etal. neration remains difficultand ble plantregenerationsystem al. 2002;Yalcin-Mendiet ze of450Mbp.Melonplant However, severalbiological 2005; Nunez-Palenius etal. subject ofintensebreeding op intonormal shootsor Cucurbitaceae n ofcandidategenes ss isstillrequired. protocols havebeen (Hanet Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study 16-h photoperiodprovided bycool-white fluorescentlamps(60µE/m medium (MurashigeandSkoog1962)at28°Cinth rinsed withsterilewater.Thesterilizedseedswere placedonMurashigeandSkoog (MS) surface-sterilized in2.5 %calciumhypochlor rinsed thrice withsterile water.Seeds were and Italy). Brazil, Russia,China,USA,Turquey irradiance) at26-28°C. chandalak, reticulatus et al.2009)andtenothergenotypesfr line ‘Védrantais’ (Vilmorin release), ahermaphrod Twelve distinctandunrelatedgenotypesof Plant material Materials andMethods exposure. development oftransgeniccellsduring capability of thetransformed plantsand regeneration rate.We studied genotypes ontwoculturemedia inordertode cell transformation and theefficiencyof Essential stepstobeoptimized aretheregenera and routineprocedurestoregeneratetran requirements formelon regenerationcontinue al. 2008)and canreach7%(FangandGrumet Akasaka-Kennedy etal.2004;Yalcin-Mendi al. 2004;Curuket2005;Castelblanque generally rangedfrom 0.7to3%(Gabaetal. al. 2003;Nunez-Paleniuset2006),transfor 1997). Evenifsome exceptionalresultswerere and thetarget explants andfalse positive re of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to In thepresentinvestigation,weevaluatedth , inodorusand Comment citer cedocument: theeffectofdifferent

chate organogenesis from earlystagesafter plant regenerationfrom transformed cells. C. melo om differentbotanicalgroups( ) andgeographicalorigin termine whichcombination providesthehighest manually peeledtoremove theseedcoatand generants are commonly obtained(Guis etal sform allmelon genotypes isstillchallenging. to bepoorlyunderstoodanddevelopingsimple 1990).However,environmental and hormonal ite containing 0.1%tween20for5min and we observed, byhistological analysis the mation efficiency remains relativelylow,and ported withparticulargenotypes(Galperinet 1992; Bordasetal.1997;Guis2000; e regenerationefficiencyofdifferentmelon Seeds weresterilized30sin70°alcoholand tion capability ofexplants,theefficiency wereused inthisstudy: aCharentais type ite line of anOriental type‘Paul’ (Martin e darkfor4daysandthenatlightundera Agrobacterium strains onthe s (France,Bulgaria, 2 per s photon pers Agrobacterium cantalupensis regeneration ,

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study OD centrifugation (10min at5,000rpm at4°C)the supplemented with50mg/lampicillin (pH 7.5)with50mg/lkanamycin, 50mg/l The different and theselectablekanamycinresistance TiAch5 strain(LBA4404withthebina explants: two GV2260 C58strains(AGL-1and Different Agrobacterium 26-28°C. regeneration medium me (MS explants wererinsed thrice insterilewate acetosyringone fora48-hco-cultivation peri to regenerationmedium (MSmedium with were blotted ontosterilefilterpaper toremove Agrobacterium thiamin and39mg/l acetosyringone. Cotyledon explants wereimmersed inthe photoperiod providedbycool-whi min). Plantregenerationand Bacto agar(DifcoLaboratories) medium freeofhormones for rooting.Allmedia with 0.2mg/l BAPand0.1mg/l gibberellicacid(GA medium. Regeneratedshoots we (ABA) (Yalcin-Mendietal. benzylaminopurine (BAP),0.88mg/l 0.2 mg/l dimethyl-allylaminopurine (2-iP)(Guiset two differentregenerationmedia: MSmedium with0.2mg/l explants forshootregeneration.Theywerecuttr Cotyledons andyoungleaves,from413days-oldseedlingrespectively,wereusedas Plant regeneration 100 mg/l kanamycin) andculturedunder a16-h of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to 600 : 0.3(10 A. tumefaciens Agrobacterium inoculationandplanttransformation suspensionfor20-30min withorbital 6 -10 8 bacteria/ml) withMS Comment citer cedocument: strainswereusedforthetransformation ofmelon cotyledon development wereconductedin strainsweregrownovernight dium with0.2mg/l BAP,0.2 mg 2004). Explantsweresub-culturedevery2weeksonfresh and thepHwasadjustedto5.7 re excised,transferredon te fluorescentlamps (60µE/m

indole-3-acetic acid NptII ry vectorpBI121)harboringthe (for AGL-1pBI101andGV2260pBI101).After medium containing200mg/l KH 0.2 mg/lBAP, 2-iP)containing100µM geneundercontroloftheCaMV35Spromoter. theexcessofbacterialsuspension, transferred r, blotteddryandtran od at28°Cinthedark. Subsequently, the ansversely infourequalpiecesandplacedon Agrobacterium photoperiod at26-28°C.Explantswere sub- were supplemented with30g/lsucrose,7 containing the binary vector pBI101) and a containing thebinaryvectorpBI101)anda al.2000)orMSmedium with1.12mg/l 6- rifampycin and (forLBA4404pBI121) 3 )] during2-3weeks shaking (50rpm)at28°C.Explants (IAA)and0.26mg/l abscisicacid elongation medium medium[MS 6-benzylaminopurine (BAP) and in Luria-Bertani(LB) medium a growthchamber undera16-h /l 2-iP,225mg/l timentinand before autoclaving(120°C,20 suspensions were adjusted at suspensionswereadjusted at 2 persphotonirradiance)at sferred toselective

uid and placedonMS A reportergene 2 PO 4 , 0.9mg/l Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study µL reactionvolume contai meltingGCGATAGAAGGCGATGCG-3’; temperat NptII meltingCACCGAAGTTCATGCCAGTC-3’; temperat andreverseprimer: 5’- AACTGGACAAGGCACTAGCG-3’ PCR usingspecificprimers ofthe following themethod ofFultonetal(1995).Thepresencethetransgenewascheckedby Genomic DNAwasextractedfrom Detection ofthe were capturedusinganautomatic camera. colored withtoluidineblue thickness) wereserially cutwitharetraction embedded inmethacrylate resin(KitTechnovi 2008), rinsedindistilledwater, dehydratedin fixative (formalin-acetic acid-ethanol at1:1:8 V/V/V)during48hat and5non-inoculatede GV2260pBI101 and 28days)bycytologicalobservations on 5GUS-positiveexplantsinfected by Histological analysesoftheorganogenesisproce Histological studies as describedbyJeffers transformation withthedifferent GUS expressionwasassessedonexplantsor Histochemical GUSassay confined greenhouse. of hormones forrooting.GUS-positive plantswere the same antibiotics during2-3weeks.Devel elongation medium (MSmedium with0.2mg/l BAPand0.1mg/l GA cultured onfreshmedium every2weeksan dNTP mixture, 0.4µM ofeachprimer, 0.5un of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to gene(forwardprimer: 5’-CCGGCTACC uid A and on etal.(1987). Comment citer cedocument: NptII ning 2ngDNA,1xPCR reac and observedwithalightmicr reporter genesbypolymerase chainreaction Agrobacterium

youngleaftissueofputative xplants ascontrol.Explants oped plantletswereplaced onMSmedium free it of Taq DNA polymerase (Go Taq Promega) it ofTaqDNA polymerase (GoTaqPromega) t 7100,Wehrheim, Sections(3µm Germany). ethanolseries(25-50-70-85and100%) microtome, collectedon microscope slides, d shootswere excisedandtransferredto strainsbyperforming histochemical assays TGCCCATTC-3’ and reverse primer: 5’- ss wererealizedatdifferentdates(2,7,14 youngleafoftheplantsregeneratedafter transferred intosoil uid ure 53°C).PCRwereperformed in25 A gene(forwardprimer: 5’- ure 62°C) andspecific primers ofthe tion buffer,1.5mM MgCl oscope. The photographic images oscope. Thephotographicimages transgenic melon plantlets wereimmersed inacold and cultivatedundera 3 ) supplementedwith 4°C (Aarrouf etal. 2 , 0.08 mM , 0.08mM Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study mg/l BAP,0.88 mg/l IAA and0.26mg/lABA(Table different explantssources ofbothgenotypes regeneration wasscored after75 explants foreachgenotype/explants source/medi described byGuisetal.(2000)andYalcin-M explants fromthetwogenotypes‘Védrantais’ genotype, explantssourceand Independent experiments ofre explantssourceand Effect ofgenotype, Results flowers, protrudingstigmas,lowfertility,thicke phenotype oftetraploidmelon plantsischarac cultured inaconfinedgreenhous Ploidy levelofthetransgenicplantswasconf lowest peakcorrespondingto2C ethidium bromide solution. isolated populationsofplant seed. Thenoisesignalsderivedfrom subce cytometer (Partec)calibratedfrom thenuclei Galbraith etal.(1991).TheDNAc acclimatized plantschopped in 1ml ofDA phenotypic characteristics.Crudesamples of The ploidylevelofregeneratedplantswasde Ploidy estimation pH 8.0)for45min at100voltsandvisualized NptII 72°C for10min. Theamplified melting temperature for1min, and adopting thethermal profile:4min at94°Ca and distilledwater.Themixtures weretransf of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to gene)weresubjectedtoelectr Comment citer cedocument: nuclei gavecharacteristicp culture medium onre PCR products (1,200 pb for the PCR products(1,200pbforthe -90 days.Verylowregeneration generation were completed toinvestigate theeffect of e. AccordingtoEzuraetal nuclei, andtheotherpeaksrepr synthesis at 72°C for 45 s, followedbyafinalextensionat synthesis at72°Cfor45s,

ophoresis ina1% ontent oftheisolatednucleiwasanalyzedwithaflow culture mediumplant regeneration on placed onMSmedium supplemented with1.12 of youngleavesdiploi terized byhavinglargemale andhermaphrodite llular debriswereeliminated bygating.The termined byflowcytometry andconfirmed by PI andfilteredaccording tothemethodof by ultravioletlightafte ned leaves,shortinternod nd 30cyclesat94°Cfor 30s,annealingat endi etal.(2004)re and‘Paul’werecultivatedontwomedia erred toathermo cyclerforamplification irmed byphenotypicobservationsofplants nuclei werepreparedfrom theleavesof um combination.Thepercentage ofplant generation efficienc agarosegel( 1). Thehighest rege eaks offluorescenceemission, the . (1992)andNugent(1994),the uid rates were obtained withthe esenting 4Cand8Cnuclei. A geneand400pbforthe Tris-acetate-EDTA spectively, with70-220 r immersion 20min in d plantsobtainedfrom y. Leafandcotyledon es androundseeds. neration rates were buffer buffer Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study assessed by flowcytometry andconfirmed transferred tosoilandcultivat results wereobtainedwith GV2260pBI101(3.0%± 1.10) (Table3).Transgenic plantswere transformation efficiencieswere comparison withtheuninfected significantly reduced(0.075±0.02withLB4404pBI121 to0.22±0.5withAGL-1pBI101)in obtained from cotyledonexplants protuberances andshootswasobservedat28days(Fig.1). transient GUSactivity14daysafter tested byhistochemical GUSassays(Fig.2a). 70to90%oftested cultures werefollowedduring90daysandsome e explants incomparison withcontrolexperiment strains (AGL-1pBI101,GV2260pBI101andLB Independent experiments oftransformati Effect of regeneration rate. 1.12 plantsregeneratedperexplan showed thattheregeneration and 2-iP),theregenerationcapacityof11 arisen from eitherdipl diploid, whereasonly47%of tetraploid cells).Itwasfoundthat87.8%ofregeneratedplantsissued fromleafexplantswere from leafwerediploid,whereasexplants to determine theploidylevelof susceptible toanincrease inploidy level.Ther plantlets perexplant).Melon this medium from ‘Paul’cotyledon shoots.Lowresultswerealsoobtainedon per explant,withonly13-15%ofexplantsgiving ‘Védrantais’ leafexplantsregenerationwa final mean rate of1.13elongate 0.2 mg/l BAPand0.2mg/l2-iP.Inthiscondition, obtained from‘Védrantais’cotyledonexplants culturedonMSmedi of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to

Using thebestexperimental conditions(cot Agrobacterium oid ortetraploid cells). Comment citer cedocument: infectiononregenerationa response ishighlygenotypede plants regeneratedfrom cotyle ed inaconfinedgreenhouse (F observedwiththedifferent explantsandregeneratedplan plants regeneratedthroughpl controlexplants d plantletsregeneratedperexplant.On thesame medium, after exposuretothedifferent explants(0.15plantletspere t (Table2).The ’Védrantais’

Agrobacterium on wererealized withthree different s 10times lower andrangedaround0.11plantlets

from cotyledonweremixoploid (diploidand efore, flowcytometry analyseswereperformed melon genotypeswasevaluated.Theresults by phenotypic observations:themajority of s onuninfectedexplants xplants andalltheregeneratedplantswere yledon explantsonMSmedium withBAP 4404pBI121) on‘Vedrantais’cotyledon (1.81 ±0.88).Signifi nd transformation efficiency 50-80 %ofexplantsgaveshootswitha exposure and the presence of blue exposureandthepresenceofblue Agrobacterium pendent andrangedfrom 0.05to don explants(shootscouldhave ig. 2c).The ploidylevelwas ts. Alltheexplantsproviding The plantregenerationrates ant tissueculturearevery xplant) orleafexplants(0.06 genotypegivesthehighest Agrobacterium um supplemented with explants exhibiteda cant differencesof (Table 3). In vitro vitro In 3). (Table strainsandhigher Agrobacterium strains were

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study histological analysesandcytolo tomato orpotatoforinstance, forwhichregeneration frequenciesfrom 40to98%with3-12 (Nunez-Palenius etal.2008). Thes percentage ofexplantsdeveloping shoots explant (Guisetal.2000; Yalc reported inliteraturewithregenerationrate achieve forsome plantspeciesorgenotypes.Successesonmel determining theconditions for invitro plant cult were made tobetterunderstandingthemetabol development ofanefficientin characterization ofcandidategenesinvolve The development ofabreedingprogram associated Discussion explain theimportantreduction dislocation (Fig.3e,f).The an abnormal development ofthemeristematic of GUSactivityat14days(Fig.3d).However, observed at7days(Fig. 3c)an occurred inepidermal andsub-epidermal cells. obtained in5-6weeks.For the regenerationmedium (Fig.3a involved. These cellsformed meristematic struct epidermal cells arethesourceoforganogene and onuninfectedexplantsascontrol.Observa vitro culture(at2,7,14and28days)onexplan In ordertoevaluate Histological analysisoftheregenerationprocess pollination of thediploid plants. Segregant transgenicplantsandhomozygous were diploid.Flowersexpresse transgenic plantsobtainedfrom cotyledonexpl of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to theinfluenceof Comment citer cedocument: vitro plant regeneration and tr and regeneration plant vitro disorganization of the transf d GUSactivitybothinovule(Fi d reached the meristematic stru meristematic the d reached of plantregenerationafter Agrobacterium gical observationswererealized in-Mendi etal.2004;reviewed ) anddevelopedshootsat28da e ratesarerelativelylow compar

Agrobacterium ranging from 0to44%depending oncultivars s rangingfrom0.3to2plantselongatedper -inoculated explants,genetictransformation tions ofnon-transformed showedthat explants after 28dayscytologicalobservationsshowed ic processescorrelated d inagriculturaltraitsdependuponthe The presenceofcellswithGUSactivitywas sis andmany small independent areasare ants weretetraploid ts inoculatedwiththeGV2260pBI101strain ures between7and14daysaftertransferon transgeniclineswereobtainedbyself ures isstillempirical structureswithcellvacuolizationand tobiotechnological infectiononplantregeneration, Agrobacterium infection. ormed meristematic areas may may areas meristematic ormed ansformation system. Advances ctures expressing ahigh level g. 2d,e)andpollen(Fig.2f). byPechetal.2007)with a during different ys (Fig. 3b);plantletswere ed to other plants, such as ed tootherplants,such as on organogenesiswere and only 15% of them and only15%ofthem and canbedifficult to with regeneration,but ols orthefunctional phases ofin Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study Melon genetictransformation mediated by genotype. different accessionstestedconfirmtheneedto most important factordetermining regenerati regeneration abilityunder thesame invitroconditions. The greatvariability inmelon isthe cantalupensis, inodorus, melon tissuecultures(Adelbergetal.1994; source toaffecttheregenerati tetraploid cells(Gonsalvez etal.1994). These providing from cotyledonweremixoploid andshootscouldhavear leaf explantsand47%ofthepl developing shoots)thanfrom frequency ofdenovoshootswas rates were obtained from theoriental melon ‘Paul’.Using‘Védrantais’ahigherregeneration ‘Védrantais’ (1.13±0.02plantsregeneratedper explant). Incontrast, shown tobewelladaptedtheregenerationfrom cotyledonexplantsofthecharentaismelon diversity usingthemost successfulprotocol. compared the regeneration rateof 11accessi explants sources (leafandcotyledon) oftw for themelon regenerationprocess.Inthis hormonal balance, theenvironmental conditions responses ofplanttissuesareaffectedbydifferent factors.Theorigin shoots perexplantwerereported(Yeeetal. transformation andwe showeditsadvantage (Hellens etal.2000). TheC58chromosomal backgroundisoftenusedforplant transformation aredefinedbytheir chromo and improved transformation selectable markerandgene construct, whichc result ofacombination offactorssuchas morphogenetic alteredcharacteri recovered transgenicplantshad somaclona (Fang andGrumet 1990)or12%(Nunez-Palenius al. 2000;Yalcin-Mendi etal.2004; Castelblanqu rates obtainedgenerally of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to

Comment citer cedocument: rangefrom 0.7to3%(Dongetal. chate on rateandinducesomaclonal varia leaf explants(14-15%).87.8%of etc.) andcommercialcultivars ants obtainedfrom cotyledonexplantswerediploid.Explants stics (Gonsalvezet efficiencies. Strainsof

obtained fromcotyledonexplants(50-84%of A. tumefaciens o geneticallydistantmelon accessions. We then experiment, wecompared twoprotocolson Guisetal.2000).Melonvarieties( The MSmedium addedwithBAPand2-iPwas Agrobacterium ould bemodified toprovide optimal conditions on potentialandtheresultsobtainedwith 2001; Abu-El-Hebaetal.2008).Theinvitro for melon transformation. Inour experiments results confirm theimpor somal backgroundandresidentTi-plasmid adapt theregenerationprotocoltoeachmelon l variation especially ons representativeoftheworldwidemelon and thegenotypewereshowntobecritical e et al. 2008) and exceptionally reach 7% e etal.2008) andexceptionallyreach7% etal.2006).Inmany casesmostofthe al. 1994).Genetictransformation isthe remains difficultandthe efficiency 1991; Bordasetal.1997;Guis Agrobacterium strain,binaryve theplantsregeneratedfrom exhibit differencesintheir tions inplants isen from eitherdiploidor very lowregeneration ploidychangesor oftheexplants, tance oftheexplants usedforplant ctor structure, structure, ctor issuedfrom reticulatus, Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study higher transformationefficien Ti-plasmid orbinaryvect Further investigationsshould berealizedtofindother Agrobacterium disorganization ofmeristematic structures Agrobacterium gene reduc co-introduction oftheACCdeaminase 2000). Genetictransformation melon of“Egusi” ( stress response, inhibits been demonstrated thatethylene, whichregu on recalcitrantspecieslikemaize andbanana(H Gelvin 2003).Presenceofnucl (Lippincott etal.1977)orthe reported and attributed todifferent causes as th 2007). Variationinresponseofplanttissuesfollowing DNA integrationintotheplantgenome (Veena suppresses plantdefenceresponses,andhijacksthemeta Agrobacterium of recalcitrantspecies(VeenaJiangetal power areimportantfactorsinfl used forgene transferwere “disarmed” (lack hypotheses. uninfected explants(more than transformation andtheimportantfall inthere which may explainthedifficult Agrobacterium organogenesis. cytological observations thatepidermal ands watermelon andpepper(Yalcin-Mendietal be competitive both for transformation and lower regenerationabilityofthetransformedcel exhibiting aGUSactivity14daysafter transient transformation of cotyledon explants 4.5%forGV2260pBI101)werestillrelativelylowalthoughthe (ranged between1.8and and AGL-1pBI101thanwiththeLBA4404pBI121st of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to Agrobacterium tumefaciens remains themajor obstacletodevelopefficient transformation technology. infection(Ntuietal.2010).Forrecalcitrant specieslike melon,the inducestheexpressionofhostgenes brought tolightadisorganizationof

The histologicalanalysisof Comment citer cedocument: Agrobacterium or) allowing goodcell transforma cies wereobtainedwithth efficiency oftheT-DNAtransfermachinery (Nam al.1997; et ear DNA fragmentation andapoptot ear DNAfragmentation 10 times less).Thisphenomenonc uencing thefrequencyoftran ies encounteredtoregeneratemelon plants aftergenetic

-mediated genetransfer(Dav isasoil-bornephytopathogenand,evenifthestrains Agrobacterium ing oncogenes),theirnatureandvirulence . 2003).The infection ofplanttissuesby following theexposu lates many aspectsofplantdevelopment and generation rateobservedbetweeninfectedand . 2003; Wolf et al. 2001), we showed by . 2003;Wolfetal.2001),weshowed was highlyefficientwith70-90%ofexplants e ability of the bacterium toattach plant cells ub-epidermal cells are thesourceofmelon

ls suggestinganincapacityformelon cellsto Jiang etal.2003;Gelv ansen 2000;Khannaet regeneration. Asreported previously on explants 10daysaftertheinfection by Colocynthis citrullus ing ethyleneproductionofexplantsafter rain. However,thesetransformation rates involved inmediating transformation, Agrobacterium the transformed meristematic areas, exposure.Theseresultsshoweda e twoC58strainsGV2260pBI101 Agrobacterium tion butless damage on melon sformation andtheregeneration bolic machinery towardstheT- ould berelatedtodifferent ic celldeathwerereported is etal.1992;Ezura strains (with different different (with strains L.)wasenhancedby in 2003;Anandetal. re of explants to re ofexplants al. 2007).Ithasalso exposurehasbeen Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study for melon genetic transformation. al. 1998;Yanget2009;Hao2010)yet al. 1995;Schulzeet1995)orpollen-tubepathway(Zhou infection. Alternativetransforma plant regenerationandtolimit th of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to Comment citer cedocument: tion methods asbiolistic such e ethyleneproductionofexplants

applied onmelon,couldalsobeendeveloped (Gonsalvez etal.1994;Gray al. 1983;Wen-Shaw Chenet inresponsetothebacterial Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Culture media Culture 2000) (Guis etal. medium Regeneration et al2004) (Yalcin-Mendi medium Regeneration Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study b a 187 URS Carosello Italy 637 Kirkagac B. Golden Casaba perfect Smith 51 China Chandaljak 1 Gaucho Koravci Vidrinski Gynadou France France Védrantais Genotype Tabl c b a Table 1Effectofculturemedium, genotype Cotyledon explants on MS medium MS medium with 0.2 mg/l BAP and 0.2 mg/l 2-iP rate: numberplants/ Regeneration ofregenerated Regeneration rate: number of regenerated MS mediumwith mg/lBAP, 1.12 mg/l 0.88 IAA, mg/l0.26 ABA of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to c

e b 2Regenerationofdiffe

Genotype Genotype Paul cotyledon Paul cotyledon cotyledon cotyledon Védrantais cotyledon cotyledon Védrantais Russia Russia Bulgaria Bulgaria USA USA China Russia Brasil Botanical Origin group Turquey Turquey Comment citer cedocument: source Explant leaf 70 leaf 70 70 leaf 70 leaf rent melongenotypes with 0.2 mg/l BAP and 0.2 mg/l 2-iP mg/l0.2 0.2 BAPand 2-iP with Unknown Unknown Reticulatus Reticulatus Chandalak Cantalupensis Cchate Iinodorus Cantalupensis Cantalupensis Inodorus Cantalupensis plants/number of explants in in culture of explants plants/number

200 200 70 70 150 150 100 100 culture culture in explants Number of 70 220 70 100 70 70 number of explants in culture; ± standard error andexplantssourceonmelon regeneration 82 82 108 5 108 72 24 78 44 82 23 118 11 65 72 15 75 84 explants 118 22 82 43 Number Number of 0 0 126 126 106 2 3 with shoots shoots with explants Number of 12 10 30 11 12 14 4 2 a

10 10 5 regenerated plants Number of 0 0 171 171 223 2 5 plants regenerated Number of 5 3 25 8 11 10 3 0

0.12 0.12 rate Regeneration 0.05 0.05 0.33 0.56 0.28 0.09 0.08 0.21 1.12 0.19 0.52 - - 47 Not tested plants diploid % of Not tested 87.8 0.11± 0.001 Not tested Not Not tested b

1.13 ± 0.02 rate Regeneration 0 0 0.041± 0.04 0.06± 0.02 0.15 ±0.01 0.15 0.018± 0.02 a

strains Mean Mean Mean Mean Experiment Number 5 4 3 2 1 2 1 2 3 2 1 Comment citer cedocument: - explants of 250 250 270 270 270 200 170 155 250 300 150 150 150 - - -

Agrobacterium - plants regenerated Number of 49 39 53 55 30 45 29 23 19 223 171 422 - - - tyledon explantsusingthreedifferent strains 1.81 ± 0.88 ±0.88 1.81 rate Regeneration 0.2 0.14 0.20 0.20 0.15 0.26 0.19 0.09 0.06 1.49 1.14 2.81 0.19 0.04 ± 0.075 ±0.02 0.22 ± 0.5

b containing the

uidA - plants transgenic Number of 7 10 6 5 9 4 1 1 1 ------uid and A and NptII c

NptII NptII genes) - efficiency Transformation 2.8 3.7 2.2 1.8 4.5 2.0 0.6 0.4 0.3 - - - 0.35±0.07 3.0 ±1.10 1.3 ± 0.99 genes d (%) Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study Bar presence ofblueprotuberances(Pr Agrobacterium Fig. 1GUShistochemical analysisonmelon of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to 0.5cm exposure(GV2260pBI101)andculture Comment citer cedocument: ) andshoots(

Sh) expressingGUSactivitywasobserved. cotyledon explants14and28daysafter on theregenerationmedium. The

c genebyPCR (amplified fragment 400pb):( Transgenicplantscu GV2260pBI101andAGL-1pBI101strains; ( ; (4-14)melon plantletsobtainedafter a GUShistochemical analysisonleafof ltivated ingreenhouse.

d,e 1 and2) GUS GUS 3) Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study and celldislocation( transformation withGV2260pBI101. Transformed explants: at 14daysofcultureand Non-transformed explants: regeneration medium. Fig. 3Histologicalandmorphologi of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to Cd Comment citer cedocument: ) 28daysaftertransformation. c,d b shootsdevelopment ( a development ofthe GUSactivity inmeristematic structure 7and14days after

cal structuresofmelon coty e,f meristematic structurewithcellvacuolization ( Sh) after28days. meristematic zone( Bar 100µm ledon explantscultivatedon Mz ) from epidermis (

Ep Cv ) ) Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript obtain transgenic plants?. PlantCell Reports, 30 (11),2001-2011. DOI :10.1007/s00299-011-1108-9 Chovelon, V.,Restier, V.,Giovinazzo, N.,Dogimont, C.,Aarrouf,J. (2011).Histological study Akasaka-Kennedy Y,Tomita K,EzuraH Adelberg JW,RhodesBB,SkorupskaHT,BridgesWC (1994) Explantoriginaffectsthe Abu-El-Heba GA,HusseinGM,AbdallaNA Aarrouf J,GarcinA,Li References Fang GW, Grumet R(1993)Genetic-engineering Fang GW, Grumet R(1990) Ezura H,YuhashiK-I,YasutaT,Mina Ezura H,Hitomi A,HigashiK,SatoT, Ezura H,Amagai H,YoshiokaK,Oosawa Dong JZ,YangMZ,JiaSRChuaNH Davis WE, MillerAR,Linerberger RD(1992) Curuk S,CetinerElman C,XiaX,Wang Y, Chen WS,HY,LeeTL,Cheng ChiuC,Liu Castelblanque L,MarfaV,ClaveriaE,Martin Bordas M,MontesinosC,DabauzaSalv Ayub R,GuisM,BenAmor M,GillotL,Rousta Anand of organogenesis inCucumis meloL. aftergenetictransformation: whyisitdifficult to (2007) Agrobacterium melon.frequency oftetraploidplantsfrom tissue-culturesof regeneration andtransformation system. AgricForRes58:103-110 leaf andfruittissuesofpeach(Prunus pers histocytopathological effectsof Interact 6:358–367 derived fromthezucchini yellowmosaicvi T-DNA integrationinplants.PlantCell19:1695-1708 regeneration ofmuskemelon pl tumefaciens-mediated genetransferto Abstract Buyukalaca S(eds)FirstinternationalSy antisense gene tomuskmelon ( ( tetraploidy inregeneratedplants,aunivers plants. Bio-Technology9:858-863 and expressionfrom thecauliflowermosaic Agrobacterium tumefaciens transformation oftomato cotyledons( cultivars isfacilitated Watad AA,GabaV( Biol Int46:1201-1209 transfer via pollen-tube pathwayfor anti- (France), May21-24th2008,pp627-631 EUCARPIA meeting on geneticsand bree Sapo’ via Improving thegenetictransf evaluation of salttolerance. Transfer oftheyeastsalttolerance geneHAL1to Nat Biotechnol.14:862-866 Expression ofACCoxidaseantis melo Cucumis melo

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