N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl

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PlantScience175(2008)356–363

ContentslistsavailableatScienceDirect

PlantScience

journalhomepage:http://wendang.chazidian.com/locate/plantsci

N,N0-bis-(2,3-Methylenedioxyphenyl)ureaand

N,N0-bis-(3,4-methylenedioxyphenyl)ureaenhanceadventitiousrootinginPinusradiataandaffectexpressionofgenesinducedduringadventitiousrootinginthepresenceofexogenousauxin

AdaRiccia,*,EnricoRollia,LuciaDramisa,CarmenDiaz-Salab

ab

`diParma,http://wendang.chazidian.comberti11/A,43100Parma,ItalyDipartimentodiBiologiaEvolutivaeFunzionale,Universita

´,28871,Alcala´deHenares,Madrid,SpainDepartamentodeBiologiaVegetal,UniversidaddeAlcala

ARTICLEINFOABSTRACT

Articlehistory:

Received28December2007

Receivedinrevisedform28March2008Accepted13May2008

Availableonline21May2008Keywords:

DiphenylureaderivativesMaturation

SCARECROW-LIKESHORT-ROOT

WehaveanalyzedtheeffectofN,N0-bis-(2,3-methylenedioxyphenyl)urea(2,3-MDPU)andN,N0-bis-(3,4-methylenedioxyphenyl)urea(3,4-MDPU),twosymmetricallysubstituteddiphenylureaderivativeswithnoauxinorcytokinin-likeactivity,ontherootingcapacityofPinusradiatastemcuttings.Resultsindicatethatbothdiphenylureaderivativesenhanceadventitiousrootinginthepresenceofexogenousauxin(indole-3-butyricacid,IBA),evenatlowauxinconcentration,inrooting-competentcuttings,buthavenoeffectontheadventitiousrootingoflowornullcompetent-to-rootcuttings.Histologicalanalysesshowthat,inthesimultaneouspresenceofMDPUsandlowconcentrationofexogenousauxin,adventitiousrootformationisinducedinthecelltypesthatretainintrinsiccompetencetoformadventitiousrootsinresponsetoauxin.Thetimecourseofcellulareventsleadingtorootformationandthetimeofrootemergencearecloselysimilartothatobservedincuttingstreatedonlywithhigherauxinconcentration.Inaddition,themRNAlevelofaP.radiataSCARECROW-LIKEgene,whichissigni?cantlyinducedinthepresenceoftheoptimalconcentration(10mM)ofexogenousauxinneededforcuttingstoroot,isincreasedinthepresenceofMDPUsandlowconcentrationofexogenousauxin(1mM).TheexpressionofaP.radiataSHORT-ROOTgeneinrooting-competentcuttingsduringadventitiousrootingisalsoaffectedbythepresenceofMDPUswhencombinedwithauxin.AsMDPUsdonotaffecttheexpressionofeithergeneintheabsenceofexogenousauxin,butonlyinitspresence,wesuggestthatMDPUscouldinteract,directlyorindirectly,withtheauxin-signallingpathwaysinrooting-competentcuttingsduringadventitiousrooting.

ß2008ElsevierIrelandLtd.Allrightsreserved.

1.Introduction

Theinductionofadventitiousrootsisacrucialstepforinvivoorinvitrovegetativepropagationofmanyherbaceousandwoodyspecies.Therootingcapacityvarieswithgenotypesand,fre-quently,dependsontheageoftheplantusedassourceofcuttings.Foryears,manyeffortshavebeenmadetoimproveadventitiousrooting,notonlybytestingthecombinedeffectofdifferentplant

*Correspondingauthor.Tel.:+390521906056;fax:+390521905403.E-mailaddress:ada.ricci@unipr.it(A.Ricci).Abbreviations:Ct,thresholdcycle;IBA,indole-3-butyricacid;2,3-MDPU,N,N0-bis-(2,3-methylenedioxyphenyl)urea;3,4-MDPU,N,N0-bis-(3,4-methylene-dioxyphenyl)urea;qRT-PCR,quantitativereversetranscription-polymerasechainreaction;RT-PCR,reversetranscription-polymerasechainreaction;SCL,SCARE-CROW-LIKE;SCR,SCARECROW;SHR,short-root.

0168-9452/$–seefrontmatterß2008ElsevierIrelandLtd.Allrightsreserved.doi:10.1016/j.plantsci.2008.05.009

growthregulators,butalsobystudyingthein?uenceofnon-hormonalbioactivecompounds,amongothers[1–6].

Inadditiontothecrucialeffectofauxininpromotingadventitiousrooting[3,7,8],someauthorsreportedthatsmallamountsofcytokininsarerequiredforimprovingrootformationinvitro[9],andthatadventitiousrootingcouldalsobeenhancedbyweakadenine-orurea-typecytokinins[10,11].Ureaderivativesarealargegroupofsyntheticcompounds,includingphenylureaanddiphenylureaderivatives.Somephenylureaderivatives,suchastheN-phenyl-N0-(2-chloro-4-pyridyl)urea(forchlorofenuron,CPPU;[12]),theN-phenyl-N0-1,2,3-thiadiazol-5-ylurea(thidia-zuron,TDZ;[13]),ortheN-phenyl-N0-benzothiazol-6-ylurea[14],showacytokinin-likeactivityexceedingthatofnaturallyoccurringadeninecompounds.Theirhighcytokininactivitycouldbeduetotheirextremestabilityinplanttissueculture,astheyarenotdegradedbycytokininoxidases[15].Onthecontrary,dipheny-lureaandsomediphenylureaderivativesareweakcytokinin-like

A.Riccietal./PlantScience175(2008)356–363357

Fig.1.Molecularstructureofthediphenylureaderivativesusedinthisstudy.

compounds,astheirhighestactivityislessthanahundredthofthatfornaturallyoccurringadeninecompounds[16].However,Riccietal.[17],basedontheresultsofaspeci?cstructure–activityrelationshipstudy,reportedthattwosymmetricallysubstituteddiphenylureaderivatives,theN,N0-bis-(2,3-methylenedioxyphe-nyl)urea(2,3-MDPU)andtheN,N’-bis-(3,4-methylenedioxyphe-nyl)urea(3,4-MDPU)(Fig.1),enhancetheformationofadventitiousrootswhensupplementedtoMaluspumilarootstockM26microcuttings.Rootsemergedatthebaseofthecuttingsintheabsenceofexogenousauxinandwithoutcallusformation.Alltherootedmicrocuttingssurvivedwhentransferredtogreenhouseconditions.Ithasbeendemonstratedthatbothcompoundsdidnotshowanyauxin-likeactivity,and,althoughstructurallyveryclosetourea-typecytokinins,theyshowedneitherweakcytokinin-likeactivity,http://wendang.chazidian.comteron,Riccietal.[18]reportedthatthesecompoundsalsointeractwithexogenousauxinpromotingadventitiousrootinginapplestemslices,anendogenousgrowthregulators-freesystem,fromtheveryearlystagesofadventitiousrootformation.So,ithasbeenhypothizedthattheadventitiousrootingenhancementpreviouslydescribedinapplemicrocuttings[17]couldbeduetoaninteractionbetweenMDPUsandendogenousauxin.Otherdiphenylureaderivativeswithdifferentsubstituentsindifferentpositionsofthephenylringshavebeensynthesizedandtested,butnoneofthemenhancedadventitiousrootingtoabetterextentthan2,3-and3,4-MDPUdidinallthebioassaystested[19,20].

InordertobettercharacterizetheactionspectrumofMDPUswehaveextendedourinitialinvestigationscarriedoutinappletothestudyoftheeffectofthesecompoundsontherootingcapacityofdistantlyrelatedwoodyplantsystem,suchasconifers.Inmanyforestspecies,therateandextentofrootingcapacityisage-limited[1,3]andspecies-dependent[21,http://wendang.chazidian.cominganexperimentalsystemwhichisbasedonthedifferentrootingcapacityofhypocotylandepicotylcuttingsfrom21-and56-day-oldseedlingsofPinusradiata,wehavetestedtheeffectofdifferentcombinationsofauxinandthepreviouslyreporteddiphenylureaderivativesontheiradventitiousrootingcapacity.Inaddition,toapproachtheMDPU-signallingpathwaysregulatingadventitiousrootformationandtheirpossibleinteractionwithauxin,theexpressionofgenesinducedinrooting-competentcuttingsduringadventitiousrootingwasanalyzedinthepresenceofauxin(indole-3-aceticacid,IBA)andMDPUs.

Genesrelatedtotheinductionofadventitiousrootsinforestspecieshavebeendescribed[23–29],butthereislittleunder-standingabouttheinteractionbetweenhormonesandotherbioactivecompoundsatthemolecularlevelduringadventitiousrootformationinstemcuttings.Recently,ourresearchgroup

characterizedtwogenesinpine,aP.radiataSCARECROW-LIKE1gene(PrSCL1)[29],homologoustotheLiliumlongi?orumSCARE-CROW-LIKEgene[30]andspeci?cmembersoftheArabidopsisthalianaSCARECROW-LIKEgenefamily[31],andaP.radiataSHORT-ROOT(PrSHR)[Sole

´etal.,unpublisheddata],homologoustotheArabidopsisthalianaSHORT-ROOTgene[32],putativetranscriptionfactorsoftheGRASfamily,someofwhichhavebeeninvolvedinrootmeristemformation[33].Bothgenesareinducedinthecambialandrooting-competentcellsofP.radiatahypocotylsasearlyas8h,andtheinductionisextendedduringthe?rst24hoftherootinductionprocess,aperiodinwhichcellreorganizationtakesplace,butbeforetheresumptionofcelldivisionandtheappearanceofadventitiousrootprimordia,indicatingthatbothgenesmayplayaroleduringtheearlieststagesofadventitiousroot

formation([29],Sole

´etal.,unpublisheddata).PrSCL1issigni?cantlyinducedinthepresenceoftheoptimalconcentration(10mM)ofexogenousauxinneededforcuttingstoroot[29].AlthoughPrSHRinductionduringadventitiousrootingisbarelyaffectedbyexogenousauxin,anditsinvolvementintheauxin-signallingpathwayshouldbekeptinmind,thepresenceofexogenousauxinisnotthemainregulationpathwayofitsinductionduringtheearly

stagesofadventitiousrootformationinP.radiatahypocotyls[Sole

´etal.,unpublisheddata].ThedifferentregulationofthegeneinductionduringadventitiousrootingprovidedtheopportunitytoanalyzetheeffectofMDPUsatmolecularlevelduringtheearlystagesofadventitiousrootformation.2.Materialsandmethods2.1.Plantmaterial

P.radiataD.DonseedswereprovidedbyOihanberri(Vitoria,Spain)andkeptat48Cuntilused.Theseedsweresoakedinrunningtapwaterfor12handthensownin?newetvermiculite.Germinationandseedlinggrowthoccurredinagrowthchamberatlightintensityof27mmolmÀ2sÀ1undera16hphotoperiod.Light/darktemperatureswere25and218C,respectively.Theseedlingswerewatereddailywithdistilledwater.After21days,theseedlingswerewateredweeklywith2g/lofacommercialsolublefertilizer(NPK20–7–19[w/w/w]).

2.2.PreparationofN,N0-bis-(methylenedioxyphenyl)ureasolutionsTheN,N0-bis-(2,3-methylenedioxyphenyl)urea(2,3-MDPU)andtheN,N0-bis-(3,4-methylenedioxyphenyl)urea(3,4-MDPU)weredissolvedindimethylsulfoxide(DMSO)andthe?nalconcentrationofDMSOinthemedium(0.06%)didnotexceedtheoneconsideredtoxic(0.2%)[34].ThetwoMDPUs,synthesizedaspreviouslyreported[17],wereofanalyticalgrade.2.3.Adventitiousrootinduction

P.radiatacuttingsforadventitiousrootinductionwerepreparedaccordingto[29].Brie?y,hypocotylandepicotylcuttingsfrom21-and56-day-oldseedlingswerepreparedbyseveringthehypocotylatitsbaseandtheepicotyljustabovethecotyledons.Allbutoneapicaltuftofneedleswereremovedfromtheepicotylstoobtainafoliarsurfacesimilartothatofthehypocotyls.Hypocotylsandepicotylsweretrimmedatthebasetoalengthof2.5cm.

Groupsof?vehypocotylcuttingsfrom21-day-oldseedlingswereculturedinvialscontaining20mlofaqueoussolutionsofindole-3-butyricacid(IBA)at0.1,1or10mM,asexogenousauxin,orIBA(0.1,1or10mM)incombinationwithMDPUs(0.1,1or10mM).Similarly,groupsof?vehypocotylorepicotylcuttingsfrom56-day-oldseedlingswereculturedinthepresenceof10m

M

358A.Riccietal./PlantScience175(2008)356–363

IBAalone,orincombinationwithMDPUs(0.1,1and10mM).Distilledwater,solutionsofMDPUsaloneatthedifferentconcentrations(0.1,1or10mM)andasolutionwithDMSOalone(0.06%)wereusedascontrols.Thevialswereincubatedatlightintensityof27mmolmÀ2sÀ1at248Cundera16hphotoperiod,andtheywerere?lleddailywithdistilledwatertoreplacewaterlosttoevapotranspiration.

Alltheexperimentswerecarriedoutintriplicateandrepeatedtwice.Rootingwasanalyzedaspercentageofrootedcuttingsandasthenumberofrootspercutting.Resultsweretakenafter28daysofculture.

2.4.Anatomyofrooting

ForhistologicalanalysisofadventitiousrootformationinthepresenceofIBAand2,3-MDPU,basal5mmsegmentsofhypocotylcuttingsfrom21-day-oldseedlingstreatedwith1mMIBA,10mMIBAor1mMIBAplus1mM2,3-MDPUwere?xedinformalin-aceticacid-alcohol(FAA),dehydratedinatertiary-butyl-alcoholseries,graduallyembeddedinparaf?n,transverselysectionedat10mmthicknesswitharotarymicrotome(Reichert-Jung2040)andstainedwithsafranin-fastgreen[35].Hypocotylcuttingsweresampledatthebeginningoftherootingexperiment(time0),after1,2,4,6,10days,and?nallywhenrootsemerged(13or15days),foreachtreatment.

2.5.RNAextraction,quanti?cationandcDNAsynthesis

Thirtybasal1cmsegmentsofthehypocotylcuttingsfrom21-day-oldseedlingswerepooledforeachtreatmentandtimepoint,asspeci?edineachexperiment,immediatelyfrozeninliquidnitrogenandstoredatÀ708CuntilusedforRNAisolation.TotalRNAisolation,quanti?cationandcDNAsynthesishavebeenpreviouslydescribed[29].RNAwaspreparedfromthreebiologicalreplicates.

2.6.QuantitativeRT-PCR(qRT-PCR)

Primerdesignandpolymerasechainreactionswerecarriedoutaspreviouslydescribed[29].A18SrRNAgene(Ri18S)wasusedascontrol.PrimersweredesignedbasedonthesequenceofPinuswallichiana18SrRNA(gi:403026),andcon?rmedinpine.PrSCL1(accessionnumberDQ683567)andPrSHR(accessionnumberEU044786)speci?cprimersweredesignedbasedonthepineRACEsequencesobtainedinourlab.

Expressionratiostotime0wereobtainedfromtheequation2ÀDDCT(AppliedBiosystems,TechnicalBulletin#2P/N4303859B).ResultsareexpressedasmeanvaluesÆstandarderrorfromthreebiologicalreplicates.

Primers:

18SrRNA

Ri18StrFor50-GCGAAAGCATTTGCCAAGG-30Ri18StrRev50-ATTCCTGGTCGGCATCGTTTA-30SCARECROW-LIKEgene

PrSCL1trFor50-TCAATGTCTGGCAAATCGTCC-30PrSCL1trRev50-GCGCCCAGTCTCTTCAATTCT-30SHORT-ROOTgene

PrSHRtrFor150-GAACCAGTGCAAGGAGCATTG-30PrSHRtrRev150-AAATCCTGCCTCCTTGAGCCT-302.7.Statisticalanalysis

ThedifferencesamongthepercentagesofrootedcuttingswereexaminedwiththeKolmogorov–Smirnovnon-parametrictest

(p<0.05);thedifferencesamongthemeanrootnumberswereexaminedwiththeStudent’stparametrictest(p=0.1).Fortheexpressionresults,comparisonbetweentwogroupsweredonebyStudent’sttest,andcomparisonamongmultiplegroupsweredonebyANOVA.Resultswereconsideredtobestatisticallysigni?cantatp 0.05.3.Results

3.1.MDPUsenhanceadventitiousrootformationinrooting-competentcuttings

InordertodeterminetheeffectofMDPUsonthecapacityofpinestemcuttingstoformadventitiousroots,therootingresponseofhypocotylandepicotylcuttingsfrom21-and56-day-oldP.radiataseedlingswasanalyzedinthepresenceofdifferentauxinandMDPUconcentrations.

Table1showstheeffectofIBAplus2,3-MDPU(Table1A)or3,4-MDPU(Table1B)onthepercentageofrootedhypocotylsfrom21-day-oldseedlingsandonthenumberofrootsperrootedcutting(totalinducedrootsdividedbythenumberofrootedhypocotyls).

Thecombinationsof0.1mMIBAplus0.1,1,or10mM2,3-MDPU(Table1A)wereineffectiveandthepercentagesofrootedhypocotylsdidnotdifferfromthatofthesameIBAconcentrationalone.However,thecombinationsof1mMIBAplus0.1,1,or10mM2,3-MDPUenhancedtheadventitiousrootformationasallthepercentagesofrootedcuttingsweresigni?cantlyhigher(Kolmogorov–Smirnovtest,p<0.05)thanthatofthesameIBAconcentrationalone.Amaximumwasreachedinthepresenceof1mMIBAplus1mM2,3-MDPU(73.2%vs46.6%without2,3-MDPU).Nocallusformationwasobservedatthebaseofthecuttings.Thecombinationof10mMIBAplus0.1mM2,3-MDPUenhancedadventitiousrooting,while10mMIBAplus10mM2,3-MDPUinhibitedit.Callusinductionwasobservedwhencuttingswereculturedinthepresenceofhighauxinconcentrationand2,3-MDPU.Onlyinthepresenceof1mMIBAplus0.1mM2,3-MDPUand1mMIBAplus1mM2,3-MDPUthenumberofrootsperrootedcuttingwasenhanced,asitwassigni?cantlyhigher(Student’sttest,p=0.1)thanthatofthesameIBAconcentrationalone(4.1vs3.1,respectively)(Fig.2).

InthepresenceofallthecombinationsofIBAplus3,4-MDPUthepercentagesofrootedhypocotylswerealsosigni?cantlyhigher(Kolmogorov–Smirnovtest,p<0.05)thanthatofthesameIBAconcentrationalone(Table1B),exceptforthecombination10mMIBAplus10mM3,4-MDPU.Adventitiousrootingwassigni?cantly(Kolmogorov–Smirnovtest,p<0.05)inhibitedunderthiscondi-tion.Themeanrootnumberperrootedcuttingwasonlyenhancedinthepresenceof10mMIBAplus1mM3,4-MDPU,butalarge-sizedcalluswasalsoinduced.

Basedonthepercentageofrootedcuttings,themeanrootnumberperrootedcuttingandespeciallytheabsenceofcallus,thecombinationof1mMIBAplus1mM2,3-MDPUwaschosenasanappropriaterootingconditionforthefurtherstudies.

Similarexperimentswereconductedusinghypocotylsandepicotylsfrom56-day-oldseedlings,whichshowedalowornullcompetencetoroot.Noneofthetestedcombinationswereabletoenhancetheformationofadventitiousrootsineitherhypocotylorepicotylcuttings(datanotshown).

Astothecontrolconditions,norootswereformedwhenthecuttingswereculturedindistilledwater,orinthepresenceofdifferentMDPUconcentrationsalone.NeitherrootsnortoxiceffectswereevidentwhentheywereculturedinthepresenceofDMSOalone(datanotshown).

A.Riccietal./PlantScience175(2008)356–363

Table1

EffectofIBAplus2,3-MDPU(A)or3,4-MDPU(B)onadventitiousrootingofhypocotylsfrom21-day-oldpineseedlings

359

mMIBA

0%Rootedcuttings

Meanrootnumber

0.1%Rootedcuttings

Meanrootnumber

1%Rootedcuttings

Meanrootnumber

10%Rootedcuttings

Meanrootnumber

(A)mM2,3-MDPU000.1010100(B)mM3,4-MDPU000.1010100

0000000

3.33.33.33.33.316.6*29.9*20.0*

1Æ0.11Æ0.11Æ0.12Æ0.21Æ0.11.4Æ0.11.9Æ0.11.8Æ0.2

46.666.673.251.0*46.653.3*73.3*64.4*

3.1Æ0.24.1*Æ0.24.1*Æ0.13.2*Æ0.23.1Æ0.22.9Æ0.13.8Æ0.24Æ0.2

43.366.6*43.319.9*43.359.9*79.9*25.3*

9.0Æ1.711.9Æ1.212.8Æ1.68.8Æ0.99Æ1.710.2Æ0.714Æ1.19.4Æ0.8

Resultsareexpressedaspercentageofrootedcuttings(*signi?cantlydifferent,Kolmogorov–Smirnovtest,p<0.05)andmeanrootnumberperrootedcuttings(ÆS.E.;*

signi?cantlydifferent,Student’st-test,p=0.1).Foreachtreatment15hypocotylswereusedandtheexperimentswererepeatedtwice.

3.2.Rootprimordiaareinducedfromnaturallyrooting-competentcellsinthepresenceofMDPUs

ThetimecourseofcellulareventsleadingtotheformationofadventitiousrootsinresponsetoIBAandIBAplus2,3-MDPUwasanalyzedincrosssectionsofthebaseofIBA-andIBAplus2,3MDPU-treatedhypocotylcuttingsfrom21-day-oldseedlings(Fig.3).ResultsindicatethatrootsalwaysformeddirectlyfromvascularparenchymaandcambialderivateslocatedcentrifugaltotheresincanalsinthepresenceofIBAandIBAplus2,3-MDPU.Hypocotylsfrom21-day-oldP.radiataseedlingsshowedawellde?nedprimarystructure(Fig.3A),consistingin5–6polesofprimaryxylemandphloemmarkedbyacentrifugalresincanal.After1dayoftreatment,nochangeswereobserved.Ahighrateofcelldivisions,showedbyanincreasednumberofcondensednuclei,wasobservedinthevascularparenchymaafter6daysinalltestedconditions,howeverdivisionsweremorelocalizedtothecellscloselypositionedtotheresincanalinthepresenceofIBAplus2,3-MDPU(Fig.3BandC).Norootprimordiawerevisibleatthistime.After10daysoftreatment,celldivisionsreorientingintoanewplanewerevisibleinthecellslocatedcentrifugaltotheresincanalinallthetestedconditions(Fig.3D–F).Rootprimordiawereclearlydetectedafter13daysincuttingstreatedwith10mMIBAorwith1mMIBAplus1mM2,3-MDPU(Fig.3GandH,respectively).Norootprimordiawereinducedincuttingstreatedonlywith1mMIBAbefore15daysoftreatment(Fig.3I).

3.3.MDPUsaffecttheexpressionofgenesinthepresenceofexogenousauxin

InordertoapproachthemodeofactionofMDPUsandtheirpossibleinteractionwithauxinatthemolecularlevel,weanalyzedtheexpressionoftwogenes,aP.radiataSCARECROW-LIKE1gene(PrSCL1),andaP.radiataSHORT-ROOTgene(PrSHR),in1mMIBAplus1mM2,3-MDPU-treatedhypocotylcuttingsfrom21-day-oldseedlingsduringtheveryearlystagesofadventitiousrootformation.QuantitativeRT-PCRwasperformedusingRNAisolatedfromhypocotylcuttingsat0and24hoftherootinductionprocess,

´whichcorrespondstothetimeofhighestinduction([29],Sole

etal.,unpublisheddata).Non-treatedcuttingsandcuttingsexposedto1mMIBA,10mMIBAand1mM2,3-MDPUalonewereusedascontrols.Resultsareexpressedasrelativevaluestotime0.Asexpected,PrSCL1mRNAlevelsincreasedsigni?cantly(p<0.01)after24hinresponsetotheapplicationof10mMIBA(Fig.4A).2,3-MDPUalonedidnothaveanyeffectontheexpressionofthegene.However,inthepresenceof1mMIBAand1mM2,3-MDPU,therewasasigni?cantincrease(p<0.01)ofPrSCL1transcriptlevels,

withregardtotime0andto1mMIBA,althoughthemRNAlevelsdidnotreachthosemeasuredinthepresenceof10mMIBA.TheexpressionofanotherGRASfamilygene,aP.radiataSHORT-ROOTgene(PrSHR),wasalsoanalyzed(Fig.4B).PrSHRwassigni?cantlyinducedoverthe?rst24hoftherootinductionprocesswithregardtotime0inalltheconditionstested(p<0.01)(Fig.4B).Theinductionofthegenewasbarelyaffectedbythepresenceoflowauxinconcentration.Inthepresenceof2,3-MDPUandlowauxinconcentration,adecreaseofPrSHRgeneinductionwasmeasuredwithregardto1mMIBA(p=0.05).4.Discussion

4.1.MDPUsenhanceadventitiousrootingandmagnifytheresponsetoauxinofcompetentcuttings

Large-scalevegetativepropagationofherbaceousandwoodyspeciesdependsontheirrootingability,whichisaffectedbygenotypes,physiologicalcharacteristics,ortissuematurity.There-fore,adventitiousrootformationhasbeenextensivelyinvestigatedandmanyeffortshavebeenmadetoimprovetherootingcapacityofstemcuttings.Riccietal.[17,18]havereportedthatN,N0-bis-(2,3-methylenedioxyphenyl)urea(2,3-MDPU)andtheN,N0-bis-(3,4-methylenedioxyphenyl)urea(3,4-MDPU)enhanceadventi-tiousrootformationinangiospermsbyamechanismwhichisunrelatedtoanycytokinin-orauxin-likeactivity,presumablyinteractingwithauxin,andwithoutcallusformation.Basedontheseresults,wedecidedtoanalyzetheeffectsofthesecompoundsontheadventitiousrootingcapacityofotherplantsystemsuchasconifers.

FromtheresultsobtainedbyadventitiousrootinductiontestswecaninferthatP.radiataseedlingsareunabletorootwhenculturedeitherinthepresenceofwaterorinthepresenceofMDPUsalone.Theseresultsareconsistentwiththephysiologicalincapacitytorootwithoutanyexogenousauxinsupplementationand,oncemore,theydemonstratethatMDPUsdonothaveany‘‘rootingactivity’’perse.Inordertostudynotonlytheeffectofthesecompoundsonrootingbutalsoapossibleinteractionwithauxin,MDPUsweretestedincombinationwithdifferentauxinconcentrations.ThepresenceofauxincombinedwithMDPUsenhancesadventitiousrootingofhypocotylsfrom21-day-oldseedlingsofP.radiata(Table1).Inthepresenceofspeci?ccombinationsofIBAplus2,3-MDPUor3,4-MDPU,thepercentagesofrootedcuttingsandthemeanrootnumberperrootedcuttingaresigni?cantlyhigherthanthoseobtainedinthepresenceofthesameIBAconcentrationalone.RootinductioninthepresenceofMDPUsisadirectprocess(Fig.3),andcallusinductionwasnot

360A.Riccietal./PlantScience175(2008)356–363

Fig.2.Inductionofadventitiousrootsinhypocotylscuttingsfrom21-day-oldpineseedlingsinthepresenceof1mMIBA(A),1mMIBAplus1mM2,3-MDPU(B),10mMIBA(C)and10mMIBAplus1mM3,4-MDPU(D).Picturesweretakenafter28daysofculture.

observedinthepresenceoflowauxinconcentrations(0.1and1mMIBA).However,asauxinconcentrationisincreased,callusformationisalsoinducedinthepresenceofMDPUs,and,althoughforspeci?ccombinationssuchas10mMIBAplus0.1mM2,3-MDPUor10mMIBAplus1mM3,4-MDPUthepercentageofrootedcuttingswashigherthanthatof10mMIBAalone,alarge-sizedcallus,undesiredfeatureofhighauxinconcentrationtreatment,wasalsopresent.So,rootscouldhavealsobeeninducedfromthecalluscellswhichcouldresultinaninappropriaterootinductionsystem,notonlyforbasicstudiesbutalsoforpracticalapplica-tions.Inaddition,rootsdevelopedintheseconditionsareshortandstumpyresemblingtheeffectofsupraoptimalauxinconcentra-tionsonrootdevelopment[36].WearetemptedtospeculatethatasupraoptimalthresholdofauxinconcentrationcouldhavebeenreachedinthepresenceofhighauxinconcentrationplusMDPUs.Thispresumptionissupportedbythesigni?cantreductionofthepercentageofrootedcuttingswhenhypocotylsareculturedinthepresenceofthehighestIBAandMDPUsconcentrations.TheseresultswouldsuggesteitherapossibleincreaseofauxinsensitivityorchangesintheauxinmetabolismordistributioninthepresenceofMDPUsincompetentcuttings,whichcouldresultinanenhancementofadventitiousrootingatveryloworlowauxinconcentrations.Anyway,itseemsthatthepresenceofMDPUsmagni?estheresponsetoauxinstimulusofnaturallycompetent-to-rootcuttings.Thisbehaviourhaspositiverelatedeffects:theuseoflowauxinconcentrationtostimulateadventitiousrootformation,theemergenceofrootsdirectlyfromthebaseofthecutting,theabsenceofcallusattheemergencesiteandlast,butnotleast,agoodqualityrootsystem.SimilareffectsonrootinghavebeenalreadydescribedbyRiccietal.[17]inM.pumila

.