geneticengineering

ReviewofPlantBiotechnologyandAppliedGenetics

Geneticengineeringinfloriculture

YoshikazuTanaka1,*

,YukihisaKatsumoto1,FilippaBrugliera2&JohnMason2

1InstituteforAdvancedTechnology,SuntoryLtd.,1-1-1Wakayamadai,Shimamoto-cho,Mishima-gun,

Osaka,618-8503,Japan;2FlorigeneLtd.,16GippsStreet,Collingwood,Victoria3066,Australia(*requests

foroffprints:Fax:+81-75-962-8262;E-mail:Yoshikazu_Tanaka@)

Received21October2003;acceptedinrevidform10June2004

Keywords:colour,dia,ethylene,flower,gene,morphogenesis,scent

Abstract

Theglobalflcengineeringisprovidingavaluablemeansof

expandingtheflori-

cialisationofgeneticallyengineeredflowersiscurrentlyconfir,

furtherproductsareexpectedgiventhelevelofactivityinthefiraltermngineeredtraitsare

entonlyconsumertraitsappearabletoprovidea

returncapableosynthesisof

floralpigments,particularlyanthocyanins,hasbeenelucidatedingreatdetailinmodelflowerssuchas

owledgeisnowbeingappliedtoanunderstandingofawiderangeofotherflowersand

providingameansoftargetingcolourmodifiineeringofnoveltraitsina

givenvarietyalsorestsoncapabilitiesinplanttransformationthatarecontinuingtoexpandatarapidrate.

Theexpressionofgenestransferredacrossgeneraisnotalwayspredictableandsorequiresconsiderable

lationofmetabolicpathways,

areflectionofthecomplexity

rstandingofgene

functionisonlyanesntialfiductionofnovelflowercolourhas

beenthefirstsuccessstoryinflraitsthathavereceivedattention

includefloralscent,floralandplantmorphology,nescenceofflowersbothontheplantandpost-harvest

anddiaresistance.

Abbreviations:ACC–1-aminocyclopropane-1-carboxylicacid;AS–aureusidinsyntha;ANS–antho-

cyanidinsyntha;BAMT–S-adenosyl-L-methioninebenzoicacidcarboxylmethyltransfera;bHLH–

basichelix-loop-helix;CaMV35S–cauliflowermosaicvirus35S;C2¢GT–UDP-gluco:2¢,4,4¢,6¢-tetra-

hydroxychalcone2¢-O-glucosyltransfera;CHI–chalconeisomera;CHS–chalconesyntha;DFR–

dihydroflavonol4-reducta;F3H–flavanone3b-hydroxyla;F3¢H–flavonoid3¢-hydroxyla;F3¢5¢H–

flavonoid3¢,5¢-hydroxyla;LIS–S-linaloolsyntha;FLS–flavonolsyntha;FNS–flavonesyntha;

3GT–UDP-gluco:flavonoid3-O-glucosyltransfera;5GT–UDP-gluco:anthocyanin5-O-glucosyl-

transfera;PKR–polyketidereducta;STS–silverthiosulfate;THC–2¢,4,4¢,6¢-tetrahydroxychalcone

Introduction

TheglobalcutflowerindustryexceedsUS$27bil-

lioninannualretailsales(Chandler,2003)and

flowersmakeupabout

one-thirdofthevalueoftheglobalornamental

icationofwildspecies

inconjunctionwithclassicalbreedinghaslong

beentheprincipalroutetogenerationofnoveltyin

centlygeneticengineeringhas

PlantCell,TissueandOrganCulture(2005)80:1–24ÓSpringer2005

openedthedoortofurtherdevelopmentofnov-

bestdemonstratedinthe‘Moon’ries

transgeniccarnationsmarketedinNorthAmerica,

arnationsarethe

world’sfirstgeneticallyengineeredcommercial

flowershavingappearedsome10yearsafterthe

firstreportsofsuccessinthegeneticmanipulation

offlowercolourthroughplanttransformation

(Meyeretal.,1987).Theconsiderableactivityin

thisareaislikelytogeneratemoreproducts,

includingpottedfloweringplants,inthenottoo

modificationofflowercol-

ourisanobviousmeansofcreatingnovelty,

increasingly,asmoreandmoregenesarecharac-

terized,additionaltraitslendthemlvesto

manipulationparticularlythroughtheextensionof

classicalbreedingfollowinggeneticengineering.

Thecurrentglobalflowermarketisprimarily

suppliedbyspecializedgrowingandexporting

countriesthroughpressurebothfromdemandfor

terare

particularlyhighinthemajorflowermarketsof

theUSA,EuropeandJapanalthoughsignificant

localflowerproductionpersistsinJapan.

Traitstargetedformanipulationleadingto

noveltycanbeclassifiedasfortheconsumeror

sbothclasshavebeen

targetedbyclassicalbreeding,itisaconsumer

traitwhich,becauofitsrelativeprofitability,is

reprentedasthefirstsuccessfulattemptbyge-

neticengineerstocreatemarketablenovelcut

flsuchasflowercolour,formand

scentareprimarynoveltymarkersastheyarekey

ehave

reviewedthisfieldpreviously(Tanakaetal.,1998;

Moletal.,1999;Tanakaetal.,1999;Tanakaand

Mason,2003),wefocushereonmorerecent

progress.

Flowercolourmodification

Flowercolourandflavonoidbiosynthesis

Flowercolourispredominantlyduetothreetypes

ofpigment:flavonoids,carotenoidsandbetalains.

Betalainsaretheleastabundantofthethreeand

contributetovarioushuesofivory,yellow,or-

ange,redandviolet(Forkmann,1991).Carote-

noidsareC-40tetraterpenoidsthatarelipid

solubleandarelocatedintheplastidandcon-

tributetothemajorityofyellowhuesinanumber

offlowers(Forkmann,1991).Carotenoids,along

withredormagentaanthocyanins,alsocontribute

totheorange/red,bronzeandbrowncoloursen

infl

flavonoidsarethemostcommonofthethreetypes

ofpigmentandcontributetoarangeofcolours

ewater-soluble

compoundsandoccurinawiderangeofplants.

Theflavonoidmoleculeswhichmakethemajor

contributiontoflowercolouraretheanthocyanins

whichareallO-glycosides(Stafford,1990)andare

usuallylocalizedinthevacuolesofpetalepidermal

cells.

Themodificationofflowercolourviagenetic

engineeringhasgenerallyfocudonmetabolic

engineeringofthefloid

moleculesarecondarymetabolitesofthephe-

flavonoidpathway

leadingtothefirstcolouredanthocyanins,antho-

cyanidin3-O-glucosides,isgenerallyconrved

amongplantspecies(Figure1)andiswellestab-

hepathwayhasbeenrepeatedlyre-

viewedfromvariousperspectives(Forkmannand

Heller,1999;Springobetal.,2003;Tanakaand

Mason,2003)includingflowercolourmodification

bygeneticengineering(DaviesandSchwinn,1997;

Tanakaetal.,1998;Moletal.,1999;Forkmann

andMartens,2001;Martental.,2003a,b),re-

centprogressonlyisthefocusofthisctionand

ncoding

flavonoidpathwayenzymes(Figure1)havebeen

clonedfrommanyplants,includingfloricultural

crops,andcanbeeasilyextractedfrompublic

DNAdatabas(forexample,thewebsiteforthe

NationalCenterforBiotechnologyInformation

/).Thefirstcoloured

anthocyanins,anthocyanidin3-O-glucosidescan

befurthermodifiedwithsugars,aliphaticacids,

reboth

species-andvariety-specificdifferencesintheex-

tentofmodificationandthetypesofglycosyland

acylgroupsattachedtotheanthocyanidincore

r,thefinalvisiblecolourofa

flowerisgenerallyacombinationofanumberof

factorsincludingthetypeofanthocyaninaccu-

mulating,modificationstotheanthocyanidin

molecule,

ofthefactorsisregulatedbyanumberofgenes,

manyofwhichhavenowbeenclonedandchara-

cterid.

2

Modificationofanthocyanins

Anthocyaninscanoccuras3-O-monosides,3-O-

biosidesand3-O-triosidesaswellas3,5-O-digly-

cosidesand3,7-O-diglycosidesassociatedwiththe

sugarsgluco,galacto,rhamno,arabino

andxylo(StrackandWray,1993).Insome

speciessuchasros,theanthocyanidin3-O-glu-

cosidesaregenerallyfurtherglycosylatedatthe

5-positionbyUDP:glucoanthocyanin5-O-glu-

cosyltransfera(5GT)toproduceanthocyanidin

3,ssuchaspetuniaand

pansy,containaUDPrhamno:anthocyanidin3-

O-glucosiderhamnosyltransfera(3RT)which

addsarhamnogrouptothe3-O-boundgluco

oftheanthocyaninmoleculetoproducethe

anthocyanidin3-O-rutinosides.

AUDP-gluco:anthocyanin3¢-glucosyltrans-

feraactivityisfoundingentianthatspecifically

transfersglucotothe3¢positionofdelphinidin

oidbiosynthesispathwayrelevanttoflhwaytotheproductionofthefirstcolouredanthocyanins,

anthocyanidin3-O-glucosidescanbefurthermodifiedwithglycosyl,acylormethylgroupsinaspecies-specifi-

propanoids,anotherclassofplantcondarymetabolites,iationsinclude:C2¢GT–

UDP-gluco:tetrahydroxychalcone2¢-O-glucosyltransfera;CHS–chalconesyntha;CHI–chalconeisomera;AS–aureusidin

syntha;F3H–flavanone3b-hydroxyla;F3¢H–flavonoid3¢-hydroxyla;F3¢5¢H–flavonoid3¢,5¢-hydroxyla;DFR–di-

hydroflavonol4-reducta;ANS–anthocyanidinsyntha;FNS–flavonesyntha;FLS–flavonolsyntha;3GT–UDP-glu-

co:anthocyanidin3-O-glucosyltransfera.

3

3,ecDNAencodingthe

enzymewasco-expresdwithatorenia5GT

cDNAinapetunialinethatnormallyaccumulated

delphinidin3-O-glucosidepigments,delphinidin

3,5,3¢-rthe

effectonflowercolourwasnotreadilyobrvable

possiblyduetothelowabundanceoftheresulting

delphinidin3,5,3¢-O-triglucoside(Fukuchi-Mizu-

tanietal.,2003).AgeneencodingUDP-glu-

co:anthocyanin3¢,5¢-O-glucosyltransferahas

beenclonedfromClioriaternatea(butterflypea).

Theenzymecatalyzesquentialglucosylationsat

3¢-and5¢-noacidquence

wassurprisinglyverycloto3GTquences

(Nodaetal.,2004).

Manyanthocyanidinglycosidexistinthe

lgroupsthat

modifytheanthocyanidinglycosidescanbedi-

videdintotwomajorclassbadupontheir

phaticacylgroupsincludema-

lonicacidorsuccinicacidandthearomatic

classincludesthehydroxycinnamicacidssuchas

p-coumaricacid,caffeicacidandferulicacid.

AromaticacyltransferacDNAscatalyzingthe

transferofanaromaticacylgrouptothe3or5-O-

glucoofanthocyaninshavebeenisolatedfrom

perilla(Yonekura-Sakakibaraetal.,2000)and

lavender(Tanakaetal.,unpublishedresults),and

gentian(Fujiwaraetal.,1998)andtorenia(Ta-

nakaetal.,unpublishedresults),respectively.A

petuniageneencodinganaromaticacyltransfer-

acatalyzingthetransferofanaromaticgroupto

the3-rutinosideofanthocyanin3-O-glucosidehas

alsobeenisolated(BruglieraandKoes,unpub-

lishedresults).Theenzymestransferhydroxy-

cinnamoicacid(p-coumaricacidorcinnamicacid)

tospecifi-

maticacylationisthoughttocontributetobluing

andstabilizationofanthocyaninsandthusflower

colour(GotoandKondo,1991;HondaandSaito,

2002).

AcDNAcloneencodingmalonylCoA:antho-

cyanidin3-O-glucoside-600-O-malonyltransfera

wasrecentlyclonedfromdahliapetalsandex-

presdinapetunialinethataccumulatedcyani-

ghupto60%ofthe

anthocyaninwasmalonylated,nosignificantcol-

ourchangeswereobrved(Suzukietal.,2002).

Thisresultisnotentirelysurprisingasaliphatic

acylationdoesnotchangeanthocyaninspectrabut

contributestothestabilizationandsolubilization

rtothisamalonylCoA:

anthocyanidin5-O-glucoside-600-O-malonyltrans-

feracDNAwasclonedfromscarletsage(Salvia

splendens)(Suzukietal.,2001).Itngineered

expressioninaplanthasnotbeenreportedyetbut

theexpectationwouldbethattherewouldbeno

significanteffectonflowercolour.

Methylationatthe3¢and5¢positionsoftheB-

ringofanthocyanidinglycosidescanalsooccur.

Methylationofcyanidin-badpigmentsleadsto

ationofthe3¢

positionofdelphinidin-badpigmentsresultsin

theproductionofpetunidin,whilstmethylationof

the3¢and5¢positionsresultsinmalvidinpro-

rtothis,methylationofmalvidin

atthe5-Oand7-Opositionstoproducecapensinin

(5-O-methylmalvidin)(Harborne,1962;Har-

borne,1967)and5,7-di-O-methylmalvidincan

alsooccurinsomeplants(,unpublished

results).Alargegroupofmethyltransferagenes

havebeenisolated(IbrahimandMuzac,2000)

howeverthospecifictoanthocyaninmodifica-

tionhavebeenclonedfrompetunia(Quattrochio

etal.,1993;Bruglieraetal.,unpublishedresults)

andtorenia(Tanakaetal.,unpublishedresults).

Copigments

Flavonolsandflavonesarecommoncopigments

thatstabilizeandlendbluingtoanthocyaninsby

formingcomplexeswiththem(GotoandKondo,

1991).Flavonolsarederivedfromdihydroflavo-

nolsbytheactivityofflavonolsyntha(FLS).

ThegenencodingFLShavebeenclonedfrom

esaresynthesizedfrom

flavanonesbyflavonesyntha(FNS).Interest-

inglytherearetwokindsofFNS,adioxygena

type(FNSI)andacytochromeP-450type

(FNSII).TheFNSIIgenehasbeenclonedfrom

torenia,snapdragon(Akashietal.,1999),gerbera

(MartensandForkmann,1999)andperilla(Kit-

adaetal.,2001).AparsleyFNSIgenehasalso

beencloned(Martental.,2003a,b).

VacuolarpH

VacuolarpH,thatismostoftenmaintainedas

weaklyacidic,iscriticaltoanthocyaninstability

ghhigher(neutral)pHgener-

allyyieldsbluerflowercolours,anthocyaninsare

lessstableathigherpHandmustbestabilizedwith

4

morethanoneglycosylandaromaticacylgroup

(GotoandKondo,1991;HondaandSaito,2002).

GeneticcontrolofpetalvacuolarpHisknownin

ystructural

geneshowntoregulatevacuolarpHtodateen-

codesaNa+/H+antiporter(Purple)inmorning

glory(Fukada-Tanakaetal.,2000).Thegeneis

highlyexpresdjustbeforefloweropening,which

elevatespHfrom6.5to7.5andchangesthecolour

gueshavebeeniso-

latedfrompetunia,toreniaandNierembergia

(Yamaguchietal.,2001)buttheirfunctioninvivo

vacuolarpHhasbeenshownto

bespecifictoepidermalcellswhereanthocyanins

accumulate(Yoshidaetal.,1995).Giventhat

cytoplasmichomeostasisisalikelydriverofvac-

uolarpHgrossperturbationofvacuolarpHwould

ion

ofvacuolarpHusinganantiportergeneintrans-

genicplantsisyettobereported.

AlargenumberofcDNAclonencodingen-

zymesintheflavonoidandanthocyaninpathways

havebeenclonedandareavailabletothemolecular

engineerinordertomanipulateflowercolour.

Antinandco-suppression(nsuppression)

strategieshavecommonlybeenudtodown-regu-

dofthetwo

namelyRNAi(RNAinterference)hasrecentlybeen

developedasapowerfultoolfordown-regulation

ofatargetquence(WangandWaterhou,2001)

casthefrequencyofphenotypic

changesistypicallymorethan50%andthephe-

notypeismorestablethanthatobtainedusing

antinorco-suppression(Mizutanietal.,2003).

Regulatorsofflavonoidgenesandtheirapplication

tomodificationofflowercolour

Expressionprofilesofstructuralgenesorenzymes

offlavonoidbiosynthesisinflowers,leavesand

edlingshavebeenstudiedinmanyplantssuchas

petunia,snapdragon,gerbera,carnation,ro,li-

sianthus,eggplant,Arabidopsis,grape,perilla,as

reviewedpreviously(Tanakaetal.,1998;Tanaka

andMason,2003).Expressionofthegenesis

bothspatiallyanddevelopmentallyregulatedat

thetranscriptionallevelinacoordinatedwaythat

parallelsflavonoidbiosynthesis(Moletal.,1998).

Twogenefamilies,basichelix-loop-helix

(bHLH)andMyb-typetranscriptionalfactors

predominantlyregulatetheexpressionofthe

structuralgenesinthepathway(Moletal.,1998;

Springobetal.,2003).InvolvementofaWD40

proteinintheregulatorypathwaymaybealso

rewellcharacterizedinsnap-

dragon,petunia,arabidopsis,maizeandperillafor

example(Springobetal.,2003).Regulatorygenes

associatedwiththeanthocyaninpathwayare

functionallyconrvedamongplantspeciesbut

theyhavedistincttsoftargetgenes,whichex-

plainssomespecies-specificdiversityatleast.

Anincreainanthocyaninlevelshasbeen

achievedviaoverexpressionofgenencoding

mple,themaize

Lcallelegene(bHLH)underthecontrolofan

esntiallyconstitutive(CaMV35S)promoterled

toanincreaintheamountofanthocyaninsin

tobaccoflowers(Lloydetal.,1992).Expressionof

thesamegenesalsoresultedinincreadantho-

cyaninslevelsinfloralandvegetativetissues,

leaveswerepurpleduetoaccumulationofantho-

cyanins,andmayreprentanovelornamental

plantofcommercialvalue(Bradleyetal.,1998).

However,similarattemptstoenhanceanthocyanin

biosynthesisincarnationandrousingthesame

geneitherfailedtoproduceflowerswithsignifi-

cantlyenhancedanthocyaninbiosynthesisorre-

sultedinreducedanthocyaninbiosynthesis

(FlorigeneLtd.,unpublishedresults).Theresults

indicatelimitationstothebroadapplicationofthis

ericinterac-

tionsbetweentranscriptionfactorsmayproduce

unpredictedoutcomesasfactorscompetewith

eachotherforassociationwithgeneticelements.

Generatingwhiteflowersbygenesuppression

Downregulationofananthocyaninbiosynthesis

structuralgenehasbeenachievedinmanyplant

ybridizationandmutationalbreed-

ingalsoreadilyleadtodevelopmentofwhiteflower

varieties,someoftheresultsaremodelexperi-

mentsonlyastransformationtechnologyisgener-

allymoreexpensivethantraditionalbreeding.

Nevertheless,molecularbreedingofawhitevariety

canbecommerciallyviableasonlytheflowercol-

ourismodifiedpresumablywithoutsacrificingany

icular,when

thehostsaresterileortheresultanttransgenic

plantshavenovelcolourationpatterns,genetic

engineeringcancomplementtraditionalbreeding.

5

Itshouldbepossibletoobtainwhiteflowersfrom

anthocyaninproducingflowersbydown-regulating

theexpressionofoneofmanystructuralorregu-

sfulreductionof

anthocyaninbiosynthesishasbeenreportedin

petunia(vanderKroletal.,1988),gerbera(Elomaa

etal.,1993),chrysanthemum(Courtney-Gutterson

etal.,1994),ro(Gutterson,1995),carnation

(Gutterson,1995),lisianthus(Deroletal.,1998;

Katoetal.,2001)andtorenia(Aidaetal.,2000a;

Suzukietal.,2000;Mizutanietal.,2003,Fig-

ure2A).Morerecently,Nishiharaetal.(2003)

transformedabluegentian(Gentiantriflora)using

anantingentianCHSgeneandsuccessfully

obtainedtransgenicgentianplantswhoflower

(Japan)hasalsogen-

eratedtransgeniccyclamenwithdown-regulated

flowercoloursobtainedwere

white,red,pinkandamixtureofredandwhite.

TheCHSgeneisthemostcommontargetfor

down-regulationofanthocyaninbiosynthesis.

However,sinceblockageofCHScanresultin

flavonoid-freetransgenicplantsandflavonoids

havebeenfoundtoplayanimportantroleinUV

protection,generalplantdefenandsignaling

(Winkel-Shirley,2002),downregulationofthe

CHSgenemaynotreprentanidealstrategyto

,wehaveobrved

thatplantswhoCHSgeneissuppresdare

gu-

lationofothergenesinthepathway,suchasDFR

orF3H,maybeamoreviablealternativetogen-

eratingwhitefloweredvarietieswithoutdeleterious

sideeffr,asZukeretal.(2002)

unexpectedlyfoundwhentheydownregulated

carnationF3H,anthocyaninlevelswerenotthe

onlychangeobrvedinthetransgeniccarnations

nationswerealsomorefragrant

duetoanincreainmethylbenzoate,whichmay

beconsideredamorepositiveoutcomewhen

commercialisingtheflnetal.

(2002)discoveredthatco-suppressionofF3¢5¢Hor

DFRinpetuniaresultedinfemaleinfertility,pre-

sumablyduetotheaccumulationofdihydrofl-

avonolsintheedcoat.

Generatingblueflowers

Mostblueflowerscontainaromaticallyacylated

,chrysanthemumand

carnationmakeupover50%oftheworldcut

flowermarketbutonlyaccumulatepelargonidin

andcyanidinderivativesthatarenotmodifiedwith

eyhavebecome

targetsforattemptsatengineeringthesynthesisof

delphinidinderivativeswiththehopeofeventually

generatingblueflorbanceof

anthocyaninshiftstowardslongerwavelengths

(blue)byabout10nmwitheachhydroxylationof

theBringandby4nmfollowinganaromatic

acylation(GotoandKondo,1991).

Asdescribedpreviously,thekeyenzymeinthe

biosynthesisofdelphinidinisF3¢5¢H.F3¢5¢Hgenes

frompetuniaandlisianthushavebeenshownto

directproductionofabluehueinflowersof

petuniaandtobacco(Holtonetal.,1993a,Shi-

madaetal.,1999).IntroductionofaF3¢5¢Hgene,

isolatedfromCanterburybells(Campanulamed-

ium)resultedinflowerswithagreaterpercentage

ofdelphinidin(99%delphinidin)thanwhenthe

petuniaorlisianthusF3¢5¢Hgeneswereintroduced

(Okinakaetal.,2003).Thisispresumablydueto

moreefficientenzymeactivityoftheCampanula

F3¢5¢H.

TransformationofapinkLobeliaerinuswitha

lisianthusF3¢5¢Hgeneunderthecontrolofa

CaMV35Spromoterproducedbluecoloured

flhetransgenicplantsisshownin

aappearstobeaufulmodel

plantforthestudyofcolourmodificationasitis

easytotransformandflowersaslittleas3–

4monthsafterco-cultivationoftissuewithAgro-

bacteriumcarryingbinarytransformationvectors

(Kannoetal.,2003).

ExpressionofapetuniaF3¢5¢Hinacarnation

linethataccumulatedcyanidin-badpigments

resultedinverylowlevelsofdelphinidinproduc-

tionandnodramaticeffectonflowercolour

(Bruglieraetal.,2000b).Itappearsthatthe

introducedpetuniaF3¢5¢Hcouldnotefficiently

competewiththeendogenouscarnationF3¢Hand

r,whenapetuniacyto-

chromeb5genealongwiththepetuniaF3¢5¢Hgene

wereexpresdinthesamecarnationlinetheresult

wasadramaticimprovementinthelevelofdel-

phinidinproductionandashiftintheflowercol-

ourfromavariegatedpinkandredtovariegated

mauveandpurple(Figure2C).

ccess-

fullydevelopedarangeoftransgenicvioletcar-

nationsbyintroductionofaF3¢5¢Hgenetogether

6

modifiedflowers.(A).Colourmodifitivarissterileand

gene;thehost,middle;atransgeniclinewithaco-suppresd

DFRgene,right;atransgeniclinewithco-suppresdCHSgene(Suzukietal.,2000).(B)ColourmodificationofLobeliaerinus,Left:

thehost,right;atransgeniclobeliaexpressinglisianthusF3¢5¢H(Kannoetal.,2003).ofAomoriGreenBioCenter,Japan,

kindlyprovidedthephotos.(C)CarnationcultivarExquisite(left)accumulatespredominantlycyanidin-badpigments,transgenic

ExquisiteflowerexpressingpetuniaF3¢5¢Handcytochromeb5genes(right)accumulatespredominantlydelphinidin-badpigments

(Bruglieraetal.,2000b).(D)TransgeniccarnationexpressingF3¢5¢andard(upper)andtwospray

(lower)varietiesaresoldinUSA,AustraliaandJapan.(E)Orangepetuniaproducingpelargonidinwasmadefromaredoneproducing

cyanidinbydownregulationoftheF3¢HgeneandexpressionofroDFRgene(Mizutanietal.,2003).(F)Co-suppressionofthe

F3¢5¢waveBlueproducedpinkfl-expressionofatoreniaF3¢Hgeneinthelinegenerated

darkerpinkflowers(Ueyamaetal.,2002).(G)ApaleyellowpetuniaexpressingaLotusjaponicaPKRgenethatwaskindlyprovided

fNihonUniversity,Japan.(H)Morninggloryflapurpurea(CHS-D::Tip100),

Ipomoeanil(DFR-B::Tpn1).oandIidakindlyprovidedthephotos.

7

withapetuniaDFRgeneintoaDFR-deficient

whitecarnation(unpublished,Moletal.,1999).

Thepetalsoftheengineeredcarnationscontain

predominantlydelphinidinthatnativecarnations

luishhueinthetrans-

genicflowershasneverbeenachievedbytradi-

tionalbreedingofcarnation(Figure2D).The

transgenicvioletcarnationsnamedFlorigene

MoondusteandFlorigeneMoonshadowehave

beenmarketedinAustralia,Japan,NorthAmerica

andUKafterbeinggrantedgeneralreleaper-

estrategyhasbeenutilizedto

generatesimilarandevendarkerviolet-purple

coloursindifferentcarnationvarietiesbearing

differentflionflowersaretyp-

icallyavailableinthreeforms,spray,midiand

rigeneMoondusteandFlori-

geneMoonshadowecarnationsareofthemidi

type,FlorigeneMoonvistae,FlorigeneMoonac-

quae,FlorigeneMoonliteeandFlorigene

Moonshadee(Figure2D)arestandardcarnations

thathavebeendevelopedusingthesamestrategy

andarecurrentlysoldinNorthAmerica,Australia

andJapanarebeingtrialled,preparatorytoek-

ingapprovalforreleafromregulatoryauthori-

ties,inadditionalkeyproductionandmarketing

tionsgoverningthereleaof

geneticallymodifiedcropsarechangingworldwide

andinsomecasnewapprovalsforflowersal-

readyonthemarketarerequirednecessitating

extensivemolecularanalysisofthetransgenic

plantsfurtherraisingthecostofdevelopmentof

newproductsuchthatsomesmallmarketsareno

longerattractive.

TheflavonoidsofFlorigeneMoonshadowe

petalswereanalyzedindetail(Fukuietal.,2003).

Nativecarnationpetalsmainlycontainpelargon-

idinorcyanidin3,5-O-diglucoside-600-O-4,6¢¢¢-O-

nsgenicflowers

containeddelphinidin3,5-O-diglucoside-600-O-

4,6¢¢¢-O-1-cyclic-malyldiesterasthemajorpig-

esultsindicatethatcarnation

anthocyaninbiosyntheticenzymesareflexibleen-

petalsalsocontainedanapigenin6-C-glucosyl-7-

O-glucoside-6¢¢¢-malylesterthatisthoughttohave

exhibitedastrongco-pigmenteffuolar

pHoftheMoonshadowflowerwastimatedtobe

around5.5bymeasuringthepHofpetalcrush.

Thus,thebluecolourcanbeaccountedforbythe

accumulationofthedelphinidintypeanthocyanins

throughF3¢5¢Hgeneexpression,theprenceof

theflavone,astrongco-pigment,andtherelatively

onditions

shouldbefavourableforengineeringblueflowers

inotherspecies.

Copigmentlevelscanbemodifiedviagenetic

engineering,asFLSandFNSgeneshavealsobeen

rasflavonolsandflavonesshare

commonprecursorswithanthocyanins,thelevels

ofcopigmentandanthocyaninaregenerallyneg-

nsuppressionofthe

FLSgeneintobaccoresultedinadecreainthe

levelsofflavonolsanduptoa3-foldincreain

thelevelofanthocyanins(Holtonetal.,1993a,b).

However,down-regulationofthepetuniaFLSina

purplepetuniaresultedindecreadlevelsof

fl

flowerswereredderincolourpresumablydueto

thereducedco-pigmentlevels(Holtonetal.,

1993b).Nielnetal.(2001)downregulatedFLS

inlisianthus(Eustomagrandiflorum)usingan

sfromthetransgenic

plantsaccumulateddihydroflavonolsatthe

expenofflavonolsandasaresultwereredderin

edpigmenta-

tionwasalsoprentinearlystagebudsandthe

notypewas

stableandwasinheritedincondgeneration

plants.

Aidaetal.(2000b)obrvedthattheflowersof

toreniaharbouringanantinDFRgenewere

bluerthanthoharbouringanantinCHS

genebecauincompletedown-regulationofDFR

leadtoanaccumulationofflavonesandthe

resultingcopigmentationeffectwiththeremaining

anthocyaninsshiftedtheflowercolourtowards

ybeaufulstrategyfortheengi-

neeringofbluefleFNSIIgenein

bluetoreniawasdown-regulated,thelevelsof

flavonesweredecreadandthoofitsprecursor,

theflavanones,r,unex-

pectedly,thelevelsofanthocyaninswerereduced

andtheresultantflowercolourwaspaleblue

(Ueyamaetal.,2002).

Generatingredtoorangeflowers

PetuniaDFRisunabletoreducedihydroka-

empferolandsopetuniaflowersrarelycontain

pelargonidin-typeanthocyaninsandthereforedo

-

8

genicbrick-redpetuniasaccumulatingpelargoni-

din-typeanthocyaninshavebeenobtainedby

expressionofDFRgenesfromheterologousspe-

ciessuchasmaize,gerberaandroinamutant

petunialinethataccumulateddihydrokaempferol

(deficientinF3¢5¢H,F3¢HandFLS).Identification

ofasimilardihydrokaempferolaccumulatingline

incommerciallyimportantspecies(orcultivars)

canbediffinietal.(2003)wereableto

engineeraredpetunialinethatnormallyaccu-

mulatescyanidin-badpigmentstoproducepe-

largonidin-badpigments(orange)bydown

regulationoftheF3¢Hgeneandexpressionofa

roDFRgene(Figure2E).Manyimportantflo-

riculturalspeciesincludingcyclamen,delphinium,

iris,gentianandCymbidiumarepresumednotto

accumulatepelargonidinduetothesubstrate

specifir

strategiescouldthereforebeemployedtogenerate

orangecolouredflowersinthespecies.

Ueyamaetal.(2002)udatwo-steptrans-

formationprocesstoproducedarkpinkflowers

fromanormallyblueflllythe

F3¢5¢Hgenewasdownregulatedsothatapink

flrtransfor-

mationofthistoreniawithaCaMV35Spromoter

drivingatoreniaF3¢Hgeneanddifferentlection

markerresultedindarkpinktoreniaflowers.

(Figure2F).

Effortstogenerateyellowflowers

Chalconesandauronescontributetotheyellow

coloursobrvedinsomefltcom-

monchalcone,THC,isyellowbutisspontane-

ouslyisomerizedtonaringenininvitroandrapidly

isomerizedinvivobyCHI(Jezetal.,2000).In

yellowflowersofcarnation,peonyandperiwinkle,

THCaccumulatesasa2¢-glucoside(isosalipurpo-

side).AccumulationofTHC2¢-glucosideis

attributedtoadefit

study(Itohetal.,2002)showedthatbothCHIand

DFRgenesaredisruptedbyatransposoninv-

eralofthecarnationcultivarsthatbearyellow

flowersvariegatedwithwhiteflecksandctors.

Paleyellowcyclamenhasbeenalsoshowntobe

defioreitappearsthat

alackofCHIacitivityandprenceofaUDP-

gluco:THC2¢-glucosyltransfera(C2¢GT)

activityarerequiredfortheaccumulationofthe

iongenes

encodingC2¢GTactivityhaverecentlybeeniso-

lated(Ishidaetal.,2003;Okuharaetal.,2004).

Thereforegeneticengineeringofisosalipurposide

inflowersshouldnowbepossible.

Auronesarebrightyellowflavonoidsand

thereforeprovideyetanothertemptingtargetfor

sarefoundinyellow

flowersofdistantlyrelatedspeciesincluding

snapdragon,dahlia,limonium,zinniaandmorn-

synthesisofauroneswas,until

recently,oneofthelastunsolvedmysteriesoffla-

syntha,morespe-

cificallyaureusidinsyntha(AS),waspurified

fromyellowsnapdragonpetalsandthecDNA

encodingtheenzymewascloned(Nakayamaetal.,

2000).

InyellowvarietiesofsomeAsteraceaeplants

suchascosmosanddahlia,6¢-deoxychalconesare

themainpigments(DaviesandSchwinn,1997).

Deoxylationatthe6¢-positionofTHCiscatalyzed

bypolyketidereducta(PKR)(formallycalled

chalconereducta)whichstabilizesthechalcone

etal.(1998)

expresdaPKRcDNAfromMedicagosativaina

whitepetunialineandobtainedpaleyellowflow-

ersthataccumulatedthechalconesbutein3-O-

unately

thecolourwasonlyvisibleinflowerbudsandnot

intenenoughtoreprentanewyellowvariety

rresultswereob-

tainedbytransformingpetuniawithalicorice

PKRgene(Figure2G,Tanakaetal.,unpublished

results).Joungetal.(2001)reportedthatexpres-

sionofaPKRgenefromPuerariamontanain

tobaccochangedtheflowercolourfrompinkto

whiteastheresultofadecreainanthocyanins

andthenovelproductionoftheflavonoidliquiri-

tigenin.

Othermodificationstoflowercolour

Variegationpatternsinflowersandleavesareof-

tenhighlyvaluedinornamentalplantsandhave

beenstudiedinmorninggloryflowersformany

ationintheflowersiscaudbya

transposon(Figure2H,Iidaetal.,1999).Inrtion

ofatransposonintoaflavonoidbiosyntheticgene

oraregulatorygeneofthebiosyntheticpathway

hasresultedinwhitectorsinacolouredback-

onofsuchatransposonfroma

particulargeneoftenleadstocolouredctionson

9

l.(2001)engineered

variegatedflowersbytransformingtobaccousing

abinaryvectorcontainingtheArabidopsistrans-

posonTag1thatwasinrtedbetweenaCaMV35S

esulting

transgenicpetunias,theRgene,adominantposi-

tiveregulatorofanthocyaninbiosyntheticgenesis

the

transgenicplantxhibitedvariegatedflowerpat-

nehadadifferentpattern,with

rtheengi-

neeringoffloriculturecropswithtransposonswill

produceflowersofcommercialvalueisyettobe

determined,astheindustrytendstopreferstable

lines.

Generatinglonglifeflowers

Thepost-harvestlifeofflowersisinfluencedpri-

marilybynutrition,microbialcolonizationand

ethylene,acommonplanthormoneassociated

with,amongstotherrespons,

mostpopularcutflowersontheglobalmarketare

ro,e

endogenouthyleneproductiontriggersflower

ropinro

can,insomevarieties,bepromotedbyexposureto

exogenouthylenetypically,associatedwith

transportandstorageoffl

flowersaresusceptible,invaryingdegrees,to

microbialgrowthinvawaterleadingtoblockage

ofvasculartissuepreventingmovementofwaterin

thestem(xylem)andthuswiltingtypicallyleading

crobesaretypically

associatedwithflowersinproductionandclean

practicethroughallstagesofthepostharvest

treatment,includingpreparationofvawater,

nutrients,pri-

marilysugars,

suchdeficienciescanbeamelioratedthrough

applicationofnutrientadditivestovawater.

Carnationsaretypicallytreatedpostharvest

withsilverthiosulfate(STS)(orlesffective

alternativechemicals).Thistreatmenthasvarying

efficacydependingonthetimingoftreatment,

concentrationofthesolutionandflowertype.

Silvereffectivelyinterfereswiththeperceptionof

ethylenebytheflower(viabindingtothemem-

brane-associatedethylenereceptor)thusrender-

ingtheflowersinnsitivetoendogenousand

onsdepletedinsilver

willbelesffectiveincompleteknockoutofeth-

ytypecarnationswith

multipleflowersoneachstem,oftenatdifferent

stagesofdevelopment,therearisariskthatsome

flowerswillbeexpodtolesssilverthanothers

oodpracticecan

lasdasa

toxicchemicalandtheincreasingpressureonthe

industrytoreducetheusageofsuchchemicals

togetherwithadvancesinunderstandingoffloral

nescencehaveprovidedanopportunityforge-

neticengineerstoaddressprolonged,chemical-

free,r,commer-

cialisationofsuchaproductisyettooccurdue

primarilytothecostofengineeringthetraitina

rangeofdifferentcoloursandvarietiesbutalso

becau,asthereislittleknowledgeoftoxicityis-

sueswithconsumers,thereisreluctancetopay

extraforwhatisalreadyperceivedasalongva-

lifeflower(whenproperlytreated).Thuscom-

mercialisationhasstalled.

Anumberofdifferentbutrelatedstrategies

havebeenudtoengineerprolongedva-lifein

carnationswithouttheneedforchemicaltreat-

firstinvolveddownregulationof

ethyleneproductionincarnationflowersviapost-

transcriptionalfloral-specificgenesilencingofa

geneencodingACCOxida(ACO)(Savinetal.,

1995)orACCSyntha(ACS)(FlorigeneLtd.,

unpublishedresults,Figure3)theenzymescata-

lyzingthetwopenultimatestepsinethylenebio-

fttheflowersnsitiveto

exogenouthylenealbeitwithavalifecompa-

rabletothatobrvedwhenstemsaretreatedwith

ghexogenouthylene

levelshavenotbeenshowntobeanissueinthe

carnationtransportchaintheperceptionremains

thattheproductislessattractivethanchemically

ationoftheethylenetrans-

ductionpathwayinArabidopsis(Bleeckerand

Schaller,1996;Fluhr,1998)ledtotheisolationof

ageneencodingtheethylenereceptorfromAra-

bidopsis(Etr1)whichsubquentlyenabledpro-

ductionofcarnationflowerswithchemical-free

prolongedvalifeandinnsitivetoboth

endogenousandexogenouthylenethroughthe

introductionofamutatedArabidopsithylene

receptorgene(Etr1-1).Transgeniccarnation

plantsharboringtheEtr1-1geneunderthecontrol

ofitsownpromoter,aconstitutiveCaMV35S

10

promoter,oranFBP1(floralbindingprotein)

alfofthemhad

delayednescenceatleastby6days,witha

maximumdelayof16days,athree-foldincreain

elifewavenlongerthanin

flowerspretreatedwithchemicalsthatinhibiteth-

ylenebiosynthesisortheethylenerespon(STS)

(Bovyetal.,1999).Similarresultswereobtained

usingEtr1-1drivenbyaCMB2promoter(CMB2

isacarnationMADSboxgene(Baudinetteetal.,

2000,FlorigeneLtd.,unpublishedresults).

ConfinementofEtr1-1expressiontoflowers

hasbeendemonstratedtoproducethesame

phenotypeandhasanadvantagebadonthe

theoreticalconcernthatinterferencewithethyl-

eneperceptionthroughouttheplantwouldim-

pairtheplant’ot

beenshownwhetherthisisaproblemforfloral-

specificEtr1-1expressingplantsbutitdoes

appearlesslikely.

Anumberofpottedplantxhibitpetal

abscissioninrespontoethyleneandastrategy

aimedatengineeringameliorationofthiffecthas

-specificexpressionofthe

mutatedgenewoulddoubtlessbeadvantageous.

Aidaetal.(1998)havereporteddownregula-

tionofACOintwocultivarsofToreniafournieri.

Theaverageflowerlongevityofthetransgenic

toreniawithdown-regulatedACOwas2.7–

7.1days,whilethatofwild-typeplantswas

rmore,transgenicto-

reniaplantswiththeextendedflowerlifeproduced

moreflowerssimultaneouslyperstemthandidthe

racteristicofextended

flowerlifewasinheritedtotheprogenieslinkingto

theexistenceofthegene.

WhenEtr1-1wasintroducedintopetuniaun-

derthecontrolofanenhancedCaMV35Spro-

moter,thetransgenicpetuniaflowershadextended

flower-life(twotofourtimeslonger)anddelayed

abscissionrelativetothenon-transgeniccontrols.

Theywerealsoinnsitivetoexogenouthylene

butproducedmoreethylene(Wilkinsonetal.,

1997).Howeverconstitutiveethyleneinnsitivity

Etr1-1geneunderthecontrolofafloralbinding

protein(FBP1)oranapetala(AP3)promoterwas

60transgenicpetu-

70%and

30%oftheplants,respectively,hadflowerlifetwo

timesthatofthenon-transgenicpetuniaflowers.

SomeoftheplantshavingtheEtr1-1generegu-

latedbyaFBP1promoterhadfullyopenflowers

for14dayswhilenon-transgenichadfor3days

(Cobbetal.,2002).

Whenpetuniawastransformedwithamutated

ERS(anethylenereceptorgene)ofBrassicaoler-

acea,flowersofthetransgenicplantsretained

turgidityandpigmentationlongerthanthoof

non-transgenicplantsandwereinnsitiveto

ormedplantsproduced

largerflowersbuthadhighermortalitysuggesting

thattheethyleneinnsitivepetuniasweremore

susceptibletodia(Shawetal.,2002).

-lifecarnationwithdownregulatedpetalACCsyntha(FlorigeneLtd.).Thenativecarnations(left)nescedafter

2weeksofharvestwhilethetransgeniccarnations(right)havecomparableva-lifetoSTStreatedones(center).

11

Morphologicalmodification

Manypotentiallyufulgenesthatareinvolvedin

thepathwaysassociatedwithflowerandplant

riptional

factorsregulatingplantdevelopmentandbiosyn-

theticorregulatorygenesinvolvedinplanthor-

r,onlya

fewofthegeneshavebeenactuallyappliedto

flthefewcas,con-

stitutivepromoterswereusuallyemployed,which

sophisticatedregulationofexpressionofthe

genesmayproducefloriculturalcropswithnovel

ribeafew

exampleshere.

Controlofbranchingisconsideredtobeone

r-

expressionofapetuniazinc-fingertypetranscrip-

tionfactor,Lateral-shootInducingFactor(LIF),

inpetuniaunderthecontrolofaCaMV35Spro-

moterresultedinadramaticincreainthenumber

-orderbranches;which

rarelyforminwild-typepetuniaswerecommonin

ntshadadecread

numberofenlargedcellsinthestem,leafand

fleloffreecytokininswaslowerwhile

theirnucleosideandnucleotideformswerehigher

instemandleaves(Nakagawaetal.,submitted).

Indeedcytokininregulationisimportantifthegoal

istoincreathenumberofflenic

tobaccotransformedwithanAgrobacteriumipt

(isopentenyltransfera)geneunderthecontrolof

anArabidopsisleafnescencepromoterproduced

moreflowersduetodelayedleafnescence(Gan

andAmasino,1995).Delayofcorollanescencein

petuniahasbeenreportedwithanidenticalgene

construct(Changetal.,2003).

TherolCgenefromAgrobacteriumrhizogenes

nsgenic

plantsofPetuniacvMitchellexpressingrolCdri-

venbyaCaMV35Spromoterexhibitedvarious

morphologicalchangessuchasreducedplant

height,leafandflowersizeandincreadbranch-

ing(Winefieldetal.,1999).IntroductionofrolA,B

ay

reportedlyresultedinimprovedrootingcharac-

teristics(vanderSalmetal.,1997).

TheCENTRORADIALIS(CEN)geneof

snapdragonencodesaphosphatidylethanolamine

bindingproteinhomologueandisrequiredfor

ther

hand,tobaccoisadeterminatespeciesinwhich

shootmeristemsterminatebyconversiontofloral

oplantsover-expressingCEN

haveanextendedvegetativephawithmore

leavesandatallergrowthhabit,delayingthe

switchtofloweringformorethan10months

(Amayaetal.,1999).Modernrossuchashybrid

teaandfloribundavarietiesareusuallydetermi-

natebutindeterminatemutantsoftenariresult-

(2003)identifieda

transposonwhichwasinrtedinto‘aterminal

flower’homologueinthegenomeofdeterminate

onofthetransposonleadtoreversion

ofthedeterminaterostoindeterminateclimbing

esultssuggestthatupordown-reg-

ulationofCENanditsorthologuescouldbeud

toalterplantheightandarchitecture.

Chemicalssuchasuniconazolearewidelyud

nesinvolving

gibberellinbiosynthesisandsignalinghavebeen

isolated(Olszewskietal.,2002).Amongthem,a

mi-dominantmutationalleleofGAI,gai-1,

greatlyreducesgibberellicacidresponsiveness

necanbe

udtogeneratedwarfplantsasSuntoryLtdhas

successfullyinduceddwarfingbyintroducing

genomicgai-1quencesintopetunia(Figure4).

Modificationoffloralscent

Floralscentplaysanimportantroleinattracting

soimportanttoconsumer

choiceinflowerpurchaduetoitsnsualasso-

scentismadeupofvariouscom-

700ofthehavebeenidentifiedin

60familiesofplants(Knudnetal.,1993).They

arefattyacidderivativessuchasbenzenoids,

phenylpropanoidsandterpenoids(monoterpenes

andsquiterpenes).Thestructuresofhundredsof

thescentcompoundshavebeendetermined.

Althoughthenumberofclonedgenesinvolvedin

thebiosynthesisoffloralscentcompoundsis

steadilyincreasing,biochemicalandmolecular

biologicalknowledgeofthebiosynthesisofscent

compoundsisstilllimited(DudarevaandPicher-

sky,2002).Reportsofthemodificationoffloral

scentusinggeneticengineeringareevenrarer.

Generallyspeaking,floralscentcompoundsare

ymesin-

12

volvedinbiosynthesisoffloralscentxpress

stronglyinpetalepidermalcellsandtheirexpres-

sionisregulatedatthetranscriptionallevel

dependentonthestageofpetaldevelopment.

Maturepetuniaflowersmainlyreleaben-

issionhasacircadianrhythm

lesarenot

storedduringperiodsoflowemissionbutrather

-microarrayanalysishas

revealedthatgenesofthepathwayleadingtothe

productionofbenzoidsareupregulatedduringthe

dayprecedinganincreaintheiremission(Ver-

donketal.,2003).Thesynthesisofcarnation

flowervolatilesisdevelopmentallyregulatedandit

hasbeensuggestedthatsynthesisismembrane-

associatedandpartitioningintothecytosoloccurs

inaccordancewithpartitioncoefficients(Schadeet

al.,2001).Snapdragonpetalcuticlesprovide

hardlyanydiffusiveresistancetovolatilesandso

permitrapidemissionofthecompounds

(Goodwinetal.,2003).Theresultsimplythat

expressionofascentbiosyntheticgeneina

transgenicplantcanleadtoamodificationof

floralscent.

Thefirststructuralgeneisolatedencodinga

floralscentbiosyntheticenzymewasS-linalool

syntha(LIS)fromClarkiabreweri,aplantnative

toCaliforniathatemitsastrongsweetscentof

whichS-Linaloolisamajorcomponent.S-Linal-

oolisbiosynthesizedfromgeranylpyrophosphate,

anintermediateofvariousterpenoids,

LISgeneishighlyexpresdintheepidermalcells

ofpetalsandincellsofthetransmittingtractof

thestigmaandstyleandthelevelofproteinpro-

lso

beenshownthatspecializedscentglandsandre-

latedorgansarenonesntialtotheproductionof

floralscent(Dudarevaetal.,1996)whichsuggests

thatfloralscentcanbemodified,withoutthe

structuresbeingprent.

PetuniahybridaW115wastransformedwith

theClarkiaLIScDNAunderthecontrolofa

appropriateenzymaticactivitywasdetectedin

iaplanttransformedwithArabidopsisgai-1(right).Geneticmodificationmayreplacechemicalgrowthretardantsin

yobtainedthegaigenefgene

asHarberdattheJohnInnesCenter,Norwich,oraholdscommercialrightsforgaifor

ornamentalsthroughanagreementwiththepatentowners.

13

r,

onlyatraceofS-linaloolwasdetectedinflowers.

ItappearsthatmostofthesynthesizedS-linalool

wasconvertedintonon-volatileS-linalyl-b-D-

glucopyroanosidepresumablybytheactionofan

amountofS-linaloolanditsglycosideemedto

bedependentmoreontheavailabilityofthesub-

strateGDPthantheexpressionleveloftheLIS

gene(Luckeretal.,2001).Thesamegeneunder

thecontrolofaCaMV35Spromoterwasalso

hich

normallyproducesbenzoicacidderivativesand

ul-

tanttransgenicplantsproducedlinaloolandits

derivatives,r,

theemissionoflinaloolwasnotatalevelthatled

todetectablechangesinflowerscentasmeasured

bythehumanno(Lavyetal.,2002).Whenthe

samegenewaxpresdintomatofruit,enough

S-linaloolwasproducedtoenabledetectionbythe

humanno(Lewinsohnetal.,2001).There-

portsindicatethatmodificationofscentispossible

byintroducingageneencodingabiosynthetic

enzymeandthatanactivepathway,toprovide

sufficientlevelsoftheprecursor(geranylpyro-

phosphateinthisca)isnecessary.

Othermoleculartoolsformodificationoffloral

encod-

ingacetylCoA:benzylalcoholacetyltransfera

andbenzylCoA:benzylalcoholbenzoyltransfera

thatareresponsibleforproductionofbenzylace-

tateandbenzylbenzoate,respectively,andarein-

volvedinscentbiosynthesishavebeenclonedfrom

iandweresubquentlycharacterized

(Dudarevaetal.,1998).Thegeneencodingan

acetylCoA:alcoholacetyltransferafromstraw-

berrythatplaysacrucialroleinbiogenesisof

flavourduringfruitripening(Aharonietal.,2000)

andanacetylCoA:geraniolacetyltransferafrom

rothatalsoacceptsalcoholssuchascitronellol

and1-octanolassubstrate(Shalitetal.,2003)have

alsobeencloned.

Snapdragonflowermitamajorphenylprop-

anoidfloralscentcomponent,methylbenzoate.S-

Adenosyl-L-methionine:benzoicacidcarboxyl

methyltransfera(BAMT)catalyzesthefinalstep

inthebiosynthesisofmethylbenzoateandthe

correspondingcDNAhasbeencloned(Dudareva

etal.,2000).Emissionofmethylbenzoatein

snapdragonflowersoccursinarhythmicmanner

amountofbenzoicacidratherthanthelevelof

armolecu-

larmechanismisinvolvedintheproductionof

scentinnocturnallyemittingplants(tobaccoand

petunia)(Kolosovaetal.,2001).Theresults

indicatethatsuccessfulmodificationoffloral

scentscouldbeachievedbyoptimizingboth

expressionofdenovobiosyntheticgenesand

agonalso

emitsthemonoterpenes,myrceneand(E)-b-

ocimene,whicharebiosynthesizedfromgeranyl

lolyrelatedcDNAsof

twomyrcenesynthasandan(E)-b-ocimenehave

rmanew

-

natedregulationofphenylpropanoidandiso-

prenoidscentproductionhasbeenobrvedin

snapdragonflowers(Dudarevaetal.,2003).Even

thepetalsofarabidopsis,alf-pollinatedplant,

producesmallamountsofterpenesandcontain

terpenesyntha(Chenetal.,2003).

ThankstolargescaleESTquenceprojects,

roisnowanothersourceoffloralscentgenes

(Channeliereetal.,2002;Gutermanetal.,2002).

Rosproducemorethan400volatilecompounds

andappearlikelytobeagoodsourceofscent-

relatedgenessuchasthoencodingS-adenosyl-

methionine:orcinolO-methyltransfera(Lavid

etal.,2002;Scallietetal.,2002)andterpenoid

tentiallyuful

genes,suchaslimonenesynthas,forthealter-

ationoffloralscentshavebeenisolatedfromro

(Bohlmannetal.,1997;Luckeretal.,2002).

Aspreviouslymentionedtheunintentional

modificationfloralscenthasbeenreportedin

gulationoftheF3Hgenere-

sultedinadecreainanthocyaninleveland

resultantpalerflowercolourandanincreain

methylbenzoateandwerethereforemorefragrant

thanflgeofthe

anthocyaninbiosyntheticpathwaymaychangethe

metabolicfluxthroughthephenylpropanoid

pathway(Figure1,Zukeretal.,2002).

Diaresistance

Plantdiasbothlimitthetypeofplantsthatcan

begrowninagivengeographicareaandleadto

signififlori-

14

cultureindustryisthusvulnerabletoarangeof

diasresultinginsignificantannuallossand

dproduction

costsareadirectresultofspecificandbroad-

spectrumdiapreventionormanagementof

asonalorclimate-drivenvariationinpathogen

rofmeasuresareemployedto

-

calsareincommonuagainstboththeagents

andvectorsofdiaimpactingonthefloriculture

industry(domesticandcommercial).Theare

oftenexpensiveandtoxictoawiderangeof

organismsincluding,insomecas,

benefitisgainedbytheapplicationofbroad-

spectrumcompoundsdirectedatcommonpatho-

stocropmanagementcansometimes

beeffectiveagainstpathogensormanagementof

ofhydroponicsincarnation

productionforexamplegreatlyreducesboththe

likelihoodofpathogenattackandtheimpact

shouldanoutbreakoccurasitisreadilylocalized.

Suchregimesarefrequentlymoreexpensiveto

variousstrategiesudagainstpathogens,alarge

proportionofwhicharepathogenicfungi,con-

ventionalplantbreedinghasbeenthebestexploi-

r,thisapproachassumestheexistence

ofadequateresourcesofresistanceinthecropit-

tion,natural

barrierstohybridisationwillpreventtransferof

rproblemisthe

lengthoftimetakentodevelopresistantplants

andthelackofdurabilityoftheresistancedueto

thecomplexnatureofhost-parasiterelationships.

Geneticengineeringhasthepotentialtoovercome

someofthedifficultiesandreducetheimpactof

othersthusdeliveringtothegrowerlowerpro-

ductioncostsandimprovedcompetitiveness.

However,asbroadspectrumchemical-bad

strategiesareincommonuagainstmany

pathogensanyengineeredstrategymusttakethis

intoaccountorproductioncostswillnotbere-

ducedrenderingengineeredvarietiesunprofitable.

Astrongmotivationtolowerproductioncosts

andgrowingconcernabouttheenvironmentare

encouragingthedevelopmentofcropswhichre-

-

ducetheneedforchemicals,cropsneedtobe

developedwhichareresistanttofungal,bacterial,

viralandnematodepathogensandtotheinct

pestswhichbothtransmitdiasandthemlves

cengineeringhasbeenud

tomodifyassociatedtraitsinplantswithlimited

success.

Thereareover100,000differentspeciesof

fungus,morethan8,000ofthemcapableof

causingdiasinplants(Agrios,1988).All

plantsaresusceptibletofungalattackandtypi-

callyonefunguscanattackmorethanoneplant

allfungispendpartoftheirlife

cycleinthesoiloronplantdebrisinthesoil.

Differentsymptomsmayprentthemlvesasa

resultofinfectionofdifferenthostsbythesame

symptomsinplantsinfectedby

fungiarenecroticlesions,rot,wilt,stunting,rust

andmildew.

Whileplantdefenmechanismsarecomplex

theyappeartohavecommonsignalpathways.

Necroticlesionsreprentanattemptbytheplant,

throughlocalizedcelldeath,toproduceanaddi-

-

thermore,abroadrangeofgenesisactivatedina

plantinrespontothegenerationofwoundsand

theinvasionbyapathogen(Heath,2000;Martin

etal.,2003).Someofthearespecifictoapath-

ogenorgroupofpathogenswhileothersarein-

ed

numberofsuchgeneshavebeencharacterized,

someofwhichareimportanttoplant-pathogen

lgenes

havebeenidentifiedwhichencodeeitherenzymes

involvedinthesynthesisofcompoundstoxicto

fungiorproteinswithaninhibitoryeffectonthe

growthoffungi(CornelisnandMelchers,1993).

Centraltosuchdefenstrategiesisthemechanism

bywhichfungipenetrateplanttissuesandthusthe

compositionofbothplantandfungalcellwalls.

Fungalcellwallsarecommonly,thoughnot

always,compodofpolymersofchitinandb-1,3-

glucanandarethusvulnerabletodegradationby

theactionofchitinasorb-1,3-glucanas(Agri-

os,1988).Suchenzymesarecommoninplantsand

havebeenbestdescribedintobacco(Collingue

etal.,1993).Strategiemployedtoenhancethe

resistanceofplantsandmorespecificallyfloral

cropstofungalpathogenshavebeengenerally

limitedtoexpressionofhydrolyticenzymesor

antimicrobialcompounds(reviewedinPunja,

2001).

Fusariumwiltisamajordiaaffectingpro-

eenestimated

thatupto20%(dependingonlocation)ofeach

15

year’um

wiltiscaudbythefungusFusariumoxysporumf.

i(principallyrace2).Thisdiais

knowntobesoilborneandmeanstocontrolits

spreadhavecentredtodateonvarioussoiltreat-

ogicalevidence(Baayen,1998)indi-

catesthatthefungusfirstentersthecarnation

followedbycolonisa-

tionanddegradationofthevascularsystemofthe

heepi-

dermisandcortexofcarnationrootcanbecol-

rum,themainroutetothestem

isthroughthevascularsystem:Thisisprobably

penetratedviarootwoundsorregionsofstructural

weaknesssuchasbranchpoints.

Incarnationengineeringimprovedresistance

tofusariumwilthasmetwithsometechnical

r,thecommercialvalueifanyof

ion

varietiesaretypicallyratedwithregardtotheir

ionplants

typicallystayinthegroundfor2–3yearsanditis

duringthelatterhalfofthisperiodthatinfection

onic

growingsystems,thoughrelativelyexpensive,are

commonlyudtoconfineoutbreaksofinfection

ormationofanumber

ofcarnationvarietieswithageneencodinga

chitina,capableofhydrolyzingfungalcellwalls

invitro,andconstitutivelyexpresdusinga

CaMV35Spromoterproducedsomeeventswith

significantlydelayedontofsymptomsina

eexhibiting

delayedontofsymptomsanddelayedtimeto

death,rum

irace2(Bruglieraetal.,2000a),was

generatedviatransformationwithabacterial

chitinagene(ChiA)fromSerratiamarcens.

Thissuccesshasnotyetbeentranslatedtothe

field.

Gardenrosontheotherhandaresusceptible

toadifferentmixofailmentsincludingdowny

mildew(Peronosporasparsa),powderymildew

(Sphaerothecapannosa)andblackspot(Diplocar-

ponrosae).Adifferentsuiteofdiascallsonthe

uofdifferentstrategiesaimedatcurtailingloss

andspreadofinfection.

dingswastrans-

formedwithabasicclassIchitinageneviabio-

theresulting

transgenicroplantxhibitedreducednsitivity

toblackspotinfection(Marchant,1998).Black-

-

centlyLietal.,(2003)havereportedonenhanced

acvCarefreeBeautyto

powderymildewbyexpressionofanantimicrobial

proteingene(Ace-AMP1).

Transformationoffloriculturalspecies

Geneticallymodifiedplantsarecurrentlyculti-

vatedon58.7millionhectares,byabout5.5–6.0

millionfarmersin16countries(James,2002).

However,applicationandcommercializationof

thetechnologytofloriculturalcropsislimitedin

spiteoftheavailabilityofthemanypotentially

primarily

duetothelackofefficienttransformationsystems

forfloriculturalspeciesandtherathersmallmar-

ketsforeachspeciesincomparisontomajorfood

fficient,reproducible,cultivar-inde-

pendenttransformationsystemiscriticaltogen-

eratetherequisitenumberofelitetransgeniclines.

Successfultransformationshavebeendescribed

forover120speciesin35differentfamilies(Birch,

1997)

isthankstothedevelopmentofarangeofAgro-

bacterium-mediatedanddirectDNAdeliverytech-

niques,alongwithappropriatetissueculture

techniquesforregeneratingwholeplantsfromplant

cellsortissues(reviewedinGalunandBreiman,

1997;HannandWright,1999).However,many

speciesarestilldifficulttotransformefficientlyand

furtherimprovementisntialtoobtaintrans-

genicfloriculturalcropsofcommercialvalue.

Agrobacterium-mediatedtransformation

Themajorityofgenetransfersystemsudinor-

namentalcropsareAgrobacterium-mediated(De-

roletal.,1997).Onlyrecentprogressis

summarizedhere.

Themulti-auto-transformationvectorsystem

(MATVectorÒSystem)isbadonaunique

conceptsuchthatAgrobacteriumoncogenescanbe

udasalectablemarkertoregeneratetrans-

genicplantsandlectmarker-freetransgenic

VectorÒSystemisdesignedto

removetheoncogenesfromtransgenicplantsafter

transformationbyutilizingayeastsite-specific

recombinationsystem,R/dsofMAT

16

Vectors,cytokinin-typewiththeiptgeneofA.

tumefaciensandauxin-typewiththerolgenefrom

enes,areavailable(EbinumaandKom-

amine,2001).Thesystemprovidesanalternate

approachtoregeneratevariousrecalcitrantplant

speciesthroughinternalmanipulationofthe

cytokinin-to-auxinratio,anditalsoenablesto

producemarker-freetransgenicplantswithout

xualcrossandedproductionforapyramid

ofmultiplegenesbyrepeatedtransformation.

TransgenicAntirrhinummajusplantxpressinga

GUSgeneweresuccessfullygeneratedwiththe

auxin-typeMATvectorÒsystem(Cuietal.,2000;

Cuietal.,2001).

Woundingofplanttissueisalsoarate-limiting

stepinAgrobacterium-mediatedtransformation.

Sonication-assistedAgrobacterium-mediatedtrans-

formationgreatlyincreasthepenetrationof

bacteriainthehosttissue(TrickandFiner,1997).

Thetechniqueinvolvesbriefperiodsinwhichthe

targettissueisrepeatedlysubjectedtoultrasound

atment

producessmalluniformfissuresandchannelsin

thecellsallowingthebacteriaeasyaccessintothe

efficientAgrobacterium-mediated

transformationofcarnationwasachievedby

woundingstemexplantsviamicroprojectilebom-

bardmentfollowedbyco-cultivationwithAgro-

dtoovera10-foldincreain

transientGUSexpression(Zukeretal.,1999).

Recently,auniqueandsimplewounding

methodforAgrobacterium-mediatedtransforma-

tal.(2003a,b)

havereportedonatoolwhichinvolvespastinga

waterproof-sandpaperontheinsideofatube.

Targettissuesarethenwoundeduniformlyby

vortexingthetubecontainingculturemediaand

transgeniclilieswereobtained

viasubquentAgrobacterium-mediatedtransfor-

infiltration,

anotherinfectionaid,hasalsobeenshowntoim-

proveAgrobacterium-mediatedtransformation

(Bechtoldetal.,1993)thoughitsapplicationto

floriculturalcropshasnotbeenreportedtoour

floralspraymethodofAgrobac-

teriumcanalsoachievehighefficiencyinplanta

transformationcomparabletovacuum-infiltration

andflfloralspraymethod

opensupthepossibilityofinplantatransforma-

tionofplanttissueswhicharetoolargefordipping

orvacuuminfiltration(Chungetal.,2000).

Identificationofveralplantgenesinvolvedin

Agrobacterium-mediatedtransformation,andtheir

over-expressionincurrentlytransformablespecies,

suggestthatmanipulatingthehostapproachholds

greatpromiforimprovingthetransformationof

recalcitrant,buteconomicallyimportantplants

(Gelvin,2003).Transformationsoffloriculture

cropshavepreviouslybeenreviewed(Deroles

etal.,1997;Tanakaetal.,1999).Recentreportsof

transformationoffloriculturalcropsaredescribed

inTable1.

Tissuecultureandregenerationtechniques

Selectionofgenuinetransformantsandtheirsub-

quentregenerationandpropagationaretime

consumingandoftentherate-limitingstepinge-

neticengineeringofflhof

culturingandregenerationabilityofplantsdiffers

betweenspeciesandoftenbetweenvarietieswithin

nsformationprocessisdependent

ontheabilityofplanttissuetoproducetotiopo-

tentcellsthatcanberegeneratedintoacompletely

y,recipientplantcellsare

regeneratedtoproducewholeplantsthrougha

especies‘inplanta’

transformationhasallowedthetissueculturestep

pmentofregeneration

systemsforpelargonium(Mithilaetal.,2001)and

Catharanthusrous(Leeetal.,2003)haverecently

beenreported.

Continuedrearchshouldbedevotedtoim-

provethevarioustechnologiesandprocedures.

Thiswillallowthecommencementofnovelgenetic

rearchandthedevelopmentofnoveltransgenic

plantxpressingavarietyofphenotypes.

Concludingremarks

Theincreasinglyrapidisolationandidentification

ofplantgenesandthesomewhatslowermovement

towardsunderstandingcomplexgeneandgene

productinteractionsarebroadeningthescopeof

attemptsaimedatengineeringnoveltraitsinflo-

temptsrveelaborationof

modelsystemssuchasinarabidopsis,petuniaand

snapdragonaswellaxplorationsaimedat

developmentofnovelvarietiesforthemarket-

ributionofgenefunctionisusually

olationofthe

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understandingderivedfromsuchstudiesoften

providesacarrotudtoenticecommercial

applicationofgenes,increasinglyasourceof

remunerationforrearchbodieshungryfor

funds,tonotonlypromoteinstitutionalR&Dbut

alsotosupportwhatareoftenheftypatentcosts

associatedwithattemptsatprotectionoftheR&D.

Furthertotheadvancestheincreasingnumberof

transformablefloriculturespecies(andvarieties)is

addingtothestoreofexperimentaldataaswellas

tothepotentialforcommerciallyviableproducts.

Inspiteofallthisgrowththecommercialappli-

cationofgeneticengineeringinfloriculturere-

rofkeyhurdleswillhavea

majorimpactonthefieldfortheforeeablefu-

eredandcommerciallyviabletraitsare

rareandareconfinedtothodirectedatthe

dtimetomarketareinfluenced

mtooperateand

regulatoryapprovalsforwhatisntiallyaglo-

balmarketarekeyhurdlesfortoday’sfloriculture

rtotheandcompoundedbythe

coststheyincuristhepaucityoftraitsinfloricul-

turewhichcanbereadilyengineeredandwhich

cancommandaviablepremiumonagivenprod-

illcomedownasthetechnologyma-

tures(andcomesoffpatent)anddevelopmentcosts

fornew,relatedproductsareamortidagainstthe

sibilityoftraitengi-

neeringrestsnotonlyontheidentificationof

appropriategenesbutalsothefactthatsome

traits,flowercolourbeingoneofthem,involve

manipulationofmetabolicpathwayswhichfre-

quentlyrequiretheintroductionofmultiplegenes.

Theefficientfunctionofmultipletransgenesis

interimatleasttheuoftraditionalbreedingis

onewaytoovercomethislimitationatthepossible

costofsignificantlylongerdevelopmenttimesfor

helessthefloriculture

industrywillbenefitinthelongtermandthe

numberofengineeredcropsonthemarketwill

doubtlessriovertheyearsahead.

Acknowledgementss

WeacknowledgeDrSuzuki(AomoriGreenBio-

Center),DrAyabe(HihonUniversity),Drs

HoshinoandIida(NationalInstituteforBasic

Biology)andDrDobres(NovaFlora)forprovid-

ingphotosorgenes(efigurelegendsfordetails).

WethankDrYamamuraofIwateBiotechnology

RearchCenter(Japan)andDrTakatsujiof

NationalInstituteofAgrobiologicalSciences(Ja-

pan)

gratefultopastandcurrentcolleaguesofSuntory

Ltd.,Japan,FlorigeneEuropeBV,TheNether-

landsandFlorigeneLtd.,Australia.

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