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Plant Protect. Sci. Vol. 48, 2012, Special Issue: S43S48

SupportedbytheMinistryofAgricultureoftheCzechRepublic,ProjectsNo.QI101A123andNo.0002700604.
Biotech/GM Crops in Horticulture: Plum cv. HoneySweet
Resistant to Plum Pox Virus
Invosinv POLK
1
, Iinn: KUMAR
1
, Bovis KRKA
2
and Micnri RAVELO^A^DRO
3
1
Departnent of Virology, Division of Plant Health, Crop Research Institute, Prague,
Czech Republic
2
Mendel University inBrno, Faculty of Horticulture, Lednice, Czech Republic
3
I^RA-Bordeaux, Villenave dOrnon, France
Abstract
PoiXJ.,KurvJ.,KvsB.,RvvioovoM.(2012):Biotech/GM crops in horticulture: Plum cv. Honey-
Sweet resistant to Plum pox virus.PlantProtect.Sci.,48 (SpecialIssue):S43S48.
CommercialisationofBiotechiGM(Biotech)cropsstartedin1995.Notonlyfieldcrops,butalsohorticultural
transgeniccropsareunderdevelopmentandarebeginningtobecommercialised.Geneticengineeringhasthe
potentialtorevolutionisefruittreebreeding.Thedevelopmentoftransgenicfruitcultivarsisinprogress.Over
thepast20yearsaninternationalpublicsectorresearchteamhascollaboratedinthedevelopmentofHoneySweet
plumwhichishighlyresistanttoPlun pox virus(PPV)themostdevastatingdiseaseofplumsandotherstone
fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven
years(20022012)inaregulatedfieldtrialintheCzechRepublicfortheresistancetoPPV, Prune dwarf virus
(PDV),andApple chlorotic leaf spot virus(ACLSV),allofthembeingseriousdiseasesofplum.Evenunderthe
highandpermanentinfectionpressureproducedthroughgrafting,PPV hasonlybeendetectedinHoneySweet
treesinseveralleavesandfruitssituatedclosetothepointofinoculumgrafting.Thelackofinfectionspreadin
HoneySweetdemonstratesitshighlevelofPPVresistance.Co-infectionsofPPVwithPDVandiorACLSVhad
practicallynoinfluenceonthequantityandqualityofHoneySweetfruitwhicharelarge,sweet,andofahigh
eatingquality.Inmanyrespects,theyaresuperiortothefruitsofthewell-knowncultivarStanley.Manyfruit
growersandfruittreenurseriesintheCzechRepublicaresupportiveofthederegulationofHoneySweetplum
tohelpimprovetheplumproductionandcontrolthespreadofPPV.
Keywords:geneticmodifications,fruittrees,GMplum,Sharkadisease,resistance
The first BiotechiGM (Biotech) crops, cotton
(Monsanto)andpotato(Syngenta)werecommer-
cialisedin1995.Clearly,theneedforfoodsecurity
andsustainabilityextendstohorticulturalcrops
suchasfruitsandvegetableswhicharethemain
sources of nutrients and healthful compounds
necessaryforhealthandwell-beingoftheworld
population.Biotechzucciniandsquashresistant
toZuccini yellow nosaic virus, Waternelon nosaic
virus, and Cucunber nosaic virus are grown in
theUSA(Dis&Ov:iz2011).Theresearchofthe
transformationofthepotato,cucumber,carrot,egg
plant,sweetcorn,andothervegetablesinmany
countriesoftheworldisaimedattheresistance
toviruses,bacteria,fungi,insects,atthetolerance
to herbicides, at the improvement of economic
properties,prolongationoftheconsumptiontime,
improvementofnutritionvalues,seedlessnessof
fruits.Thedevelopmentoftransgenicfruitcultivars
isinprogress.PapayaresistanttoPapaya nosaic
virus is grown in USA and China ( Jrvs 2011).
Biotechgrapevineresistanttoviral,bacterial,and
fungaldiseaseswithabioticstresstoleranceand
health benefits was developed in South Africa.
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Vol. 48, 2012, Special Issue: S43S48 Plant Protect. Sci.
Biotechbanana,apple,pear,andstrawberrycul-
tivarsareunderthedevelopment.
Theresultoftheinternationalresearchdoneover
thepast20yearsisthedevelopmentofHoneySweet
plumhighlyresistanttoPPV.GMplumHoneySweet
resistanttoPlun pox virus(PPV)wasderegulated
inUSAin2010.Plums(Prunus donestica)arean
importantsourceofvitamins,minerals,andphy-
tonutrientsandcontainspecificcompoundsthat
supportgooddigestivefunctionandbonehealth.
Sharkadiseaseisthemostdevastatingdiseaseof
plumandisresponsibleforthereductionorloss
oftheplumproductioninmanyareasofEurope
(Crnvet al.2006).Inthepast22yearsofresearch
byaninternationalteamofpublicsectorscientists,
aGMplumhighlyresistanttoPPVwasdeveloped
andthoroughlytestedinthegreenhouseandfield,in
theUSAandEuropetheCzechRepublic,France,
Poland,Romania,andSpainfortheresistanceto
PPVandforenvironmentalsafety(Scovzet al.
1994, Rvvioovo et al. 1997, 2000, 2002,
Hiivet al.2004,2007,PoiXet al.2005,2008a,b,
Miiosiet al.2006,Cvo:vet al.2007,2008,
Zoviet al.2008a,b,2010).Anoriginaltrialofa
highandpermanentinfectionpressureofPPV-Rec
aloneandincombinationswithPrune dwarf virus
(PDV)andApple chlorotic leafspot virus(ACLSV)
wasinitiatedintheCzechRepublic(PoiXet al.
2008a,b).Thetransgenicplumtreeswereevalu-
atedduringtheyears20022012.Here,wepresent
asummaryoftheresultsofelevenyeartestingof
HoneySweet plum (clone C5) and the results of
threeyeartestingofthefruitsquality(20102012)
underthehighandpermanentinfectionpressure
comingbothfromthegraftinoculationandnatural
aphidvectors,anddiscusstheimplicationsofthe
workwithHoneySweetintermsofitspotentialfor
utilisationintheEU.
MATERIAL AND METHODS
Field trial, transgenic plum trees, virus inocu-
lations.OriginalplumcloneC5budsfromUSA
(USDA-ARS, Kearneysville) were grafted onto
the virus-free rootstocks of St. Julien in 2002,
and 55 grafted P. donestica clone C5iSt. Julien
treeswereobtained.Eachinoculationtreatment
PPV-Rec, PPV-Rec + ACLSV, PPV-Rec + PDV,
PPV-Rec + ACLSV + PDV, consisted of 11 C5
trees.TheinoculatednonGMcontrolsandnon-
inoculatedcontrolandC5treeswereincludedand
aplantationwasestablished(Figure1).PPV-Rec,
ACLSV,andPDVinfectedbudswereallowedto
growthroughouttheelevenyearsperiodofevalu-
ation.ThetransgeniccloneC5partofeachtree
remainedelevenyearsunderaverystronggraft
inoculationpressure.
Evaluation of leaf and fruit symptoms, qual-
ity of fruits.Alltreeswereevaluatedeveryyear
in the period from May to September (2002
2012) for the presence of viral symptoms in
leaves. Fruit symptoms were evaluated in July
andAugust20102012(thefirstfewfruitswere
produced in 2009) a short time before ripening
whenfruitswerestillfirmandatfullripening.In
20102012 were included the overall fruit uni-
formity,attractiveness,weight,length,diameter,
flesh thickeness, fruit shape, skin colour, flesh
colour, flesh firmness, flavour, freeness of flesh
fromthestone,totalsolublesolids,totaltitratable
acidity, stone size, weight and stoneiflesh ratio,
anddryweightoffruitsharvestedfromthetrees
ofcloneC5inoculatedwithPPV-Rec,PPV-Rec+
ACLSV, PPV-Rec + PDV, PPV-Rec + ACLSV +
PDV,andfromthenon-inoculatedcontroltrees
ofcloneC5,Stanley,andDomcvestka.
Serological detection of viruses.ELISAtesting
of the leaves was performed every year in June.
FruitswereevaluatedinAugust20102012.Poly-
clonalantibodiesraisedagainstPPV,ACLSV,and
PDV (Bioreba, Reinach, Switzerland) were used
inDAS-ELISA(Civ&Aors1977).Theleaf
samples were extracted in phosphate-buffered
saline.TherelativeconcentrationofPPV-Recwas
determinedbysemiquantitativeDAS-ELISAinthe
samplespreparedfromthesymptomaticleavesin
June2005and2007.Therelativeconcentrationof
PPVproteinwasestablishedbydeterminingthe
Figure1. PlantationofHoneySweettreesinCzechRe-
public(Orig.J.Polk)
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Plant Protect. Sci. Vol. 48, 2012, Special Issue: S43S48
lowest dilution of leaf or fruit samples with the
positivereactioninsemiquantitativeDAS-ELISA
(Ainvvcn:ovXet al.1986).
Detection of viruses by reverse transcription-
polymerase chain reaction (RT-PCR). 100 mg
ofgroundleaforfruittissuewereusedfortotal
RNAextractionbyusingRNeasyPlantMiniKit
(QIAGENGmbH,Hilden,Germany)accordingto
theprocedurerecommendedbythemanufacturer.
PPV-RecwasdetectedbyRT-PCRusingtheprimer
pairmD5imM3asdescribedbyunvet al.(2004).
For PDV and ACLSV, the primers were used as
describedbyJvosovXandKuou(2010).
RESULTS AND DISCUSSION
NoPPVsymptomsappearedintheleavesofthe
transgenicplumcloneC5HoneySweettreesinthe
firstyearafterthegraft-inoculationwithPPV-Rec.
PPV symptoms appeared only in the leaves that
emergedfromtheinfectedbuds(IB).Milddiffuse
spotsandringsappearedtwoyearsaftertheinocu-
lationinsomebasalleavesofHoneySweettrees
inoculatedwithPPV-Rec,andinthoseinoculated
withtheviruscombinationsPPV-Rec+ACLSV,
PPV-Rec+PDV,andPPV-Rec+ACLSV+PDV
(PoiXet al.2005).PPVpresenceinthesebasal
leaves was confirmed by ELISA and RT-PCR. A
reductionofsymptomswasobservedbeginning
inthethirdyearafterthevirusinoculation.PPV
symptoms were observed only in several basal
leaves and the symptoms were milder in each
followingyear(PoiXet al.2008a).
FurtherreductionofPPVsymptomswasobserved
in years 20092012, and during the vegetation
periodfromJunetoSeptember.NoPPVsymptoms
were found in leaves in 2012, and the presence
ofPPVwasnotprovedbyELISA.Nodifferences
in the intensity of PPV leaf symptoms between
different virus combinations were observed in
theyears20042012.NosymptomsofPDVand
ACLSVappearedduringthevegetativeperiodsof
20022012. PDV was not detected by ELISA in
transgenicpartsoftreesinoculatedwithPPV-Rec +
PDVandPPV-Rec+PDV+ACLSV.Thepresence
ofPDVwasdubiousbyRT-PCR.PDVwasdetected
byELISAandRT-PCRonlyinleavesgrowingfrom
theIB.ACLSVwasdetectedbyELISAandRT-PCR
inleavesoftransgenicpartsofthetreesinoculated
with PPV-Rec + ACLSV and PPV-Rec + PDV +
ACLSV.NosymptomsofPPV,PDV,andACLSV
appearedintheleavesofthenon-inoculatedcontrol
treesofHoneySweetthroughouttheexperiment,
andPPV,PDV,andACLSVwerenotdetectedby
DAS-ELISAandRT-PCR.Thegrowthofthenon-
inoculated HoneySweet control trees was more
vigorousincomparisonwiththoseinoculatedwith
PPVandthecombinationswithPDVandACLSV.
This may have been due in the whole or part to
the competition by the extensive growth of IB
shootsgrowingfromtheinoculatedHoneySweet
trees(Figure2).TheseverePPVsymptomswhich
appearedfirstin2003inIBleavesgrowingfrom
the buds infected with PPV-Rec appeared again
everyyear(20042012)withthesameintensity.
The relative concentration of PPV-Rec in the
symptomaticleavesofHoneySweetdeterminedby
semiquantitativeDAS-ELISAfluctuatedfrom1.56 x
10
2
to9.76x10
4
in2005andfrom5.0 x 10
1
to
7.81x10
3
in2007.Therewerenosignificantdiffer-
encesintherelativeconcentrationofPPVbetween
thecombinationsoftheinoculatedviruses.The
relativeconcentrationofPPVintheleavesofIB
shootswasatleastthirtytimeshigherascompared
tothesymptomaticleavesofHoneySweet.
Pomologicalevaluationoftheexternalandinternal
characteristicsofthefruits(Figure3)harvestedfrom
non-graft-inoculatedHoneySweettrees,Stanley,
andDomcvestkatrees,andfromHoneySweet
treesgrowingelevenyearsunderthehighandper-
manentinfectionpressurebyPPV-Rec,PPV-Rec+
PDV,PPV-Rec+ACLSV,PPV-Rec+ACLSV+PDV
demonstratedthehighqualityofHoneySweetfruits.
ThePPVpresencewasprovedbyELISAinseveral
fruitssituatedclosetotheplaceofIBgraftingin
2010and2011only.AllthefruitswerePPVfreeas
foundbyELISAin2012.Three-yearresultsindicate
thatthecharacteristicsofHoneySweetfruitshar-
vestedfromthecontrolvirusnon-inoculatedtrees
arewellwithintherangeofthecharacteristicsof
controlcultivarsStanleyandDomcvestkaand
areofhigherqualityinsomecharacteristics.The
fruitsharvestedfromHoneySweettreesinoculated
withPPV-Rec+ACLSV +PDV,PPV-Rec+PDV,
PPV-Rec+ACLSV,andPPV-Recwerecomparable
with the fruits from the control healthy Honey-
Sweettreesindicatingthattherewaslittle,ifany,
effectofthevirusinoculationsonthefruitquality
ofHoneySweet.
HoneySweet plum trees resistant to PPV re-
mainedvirus-freeunderthenaturalaphid-vectored
infection pressure throughout this eleven-year
study.Thisisinagreementwiththeresultsobtained
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in France and Romania (Rvvioovo et al.
1997,2002),andinSpainandPoland(Miiosi
et al.2006).Originalresultswereobtainedwhen
graftinoculatedtreeswereexposedtoaveryhigh
infectionpressurewithIBbeingallowedtoreach
thesizeof2030ofthesupportingHoneySweet
tree (Figure 2). Under this high and permanent
virus pressure, HoneySweet trees showed PPV
symptomsandpositiveserologicalandmolecular
tests on some basal leaves only, and even these
symptoms subsided during the growing season.
Most trees were without any leaf symptoms in
2010 and 2011, and no symptoms appeared in
leaves in 2012. ACLSV infection did not appear
toaffectPPVsymptomsandPDVinfectioncould
not be detected in HoneySweet throughout the
courseofthestudydespitethegraftinoculation.
The evaluations of the fruit quality of the graft
inoculatedandnon-inoculatedHoneySweettrees,
maintained for eleven years under the high and
permanent infection pressure by PPV, ACLSV,
andPDV,confirmednotonlythehighresistance
of HoneySweet to PPV, but also suggested that
HoneySweet fruits maintain their quality and
healthful properties when exposed not only to
PPVbutalsotoACLSVandPDV.
TheregulatoryprocessintheUSAforHoney-
Sweet was successfully completed in 2010. The
stronginternationalcooperationbetweenpublic
sectorscientistsinEuropeandtheUSAandthe
approvalofHoneySweetintheUSAwarrantthe
submissionofHoneySweetforregulatoryconsid-
erationintheEU.TheabilitytogrowHoneySweet
Figure2. HoneySweettreewith(a)non-transgenicand(b)cuttedawaynon-transgenicPPVinfectedbottom
part(Orig.J.Polk)
(a) (b)
Figure3.FruitsofplumHoneySweet(Orig.J.Polk)
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Plant Protect. Sci. Vol. 48, 2012, Special Issue: S43S48
plumintheCzechRepublicwouldcontributetothe
viabilityoftheplumproductionbyCzechgrowers
and support the producers of the products that
dependuponasupplyofplumsincludingproducers
ofplumbrandy.ThecultivationofHoneySweetin
theCzechRepublicandotherEuropeancountries
wouldrepresentauniqueopportunitytoestablish
PPVfreeorchardsandtogrowhighqualityfruits
forthebenefitofgrowersandconsumers.
Acknowledgements.AuthorsareindebttoMrsM.Du-
cnXovXandJ.PIviovXfortechnicalassistance.
Ref erences
Ainvvcn:ovXL.,KvvsovXR.,PiunZ.,BicvovXE.
(1986):ELISAmethodusedfortheevaluationofresist-
anceofplumcultivarstoplumpoxvirus.In:Proceedings
10
th
ConferencePlantProtection,Brno,CzechRepublic:
203204.
Crnv M., Cvo:v N., Mvv: A., Licvv G. (2006):
Plun pox virusandtheestimatedcostsassociatedwith
sharkadisease.OEPPiEPPOBulletin,36:202204.
Cvo:vN.,MozoC.,UvnviA.,Pvvvz-PovsJ.,
CvnoviiE.,RvvioovoM.,ScovzR.,Cr-
nv M. (2007): Risk assessment of the field release of
transgenicEuropeanplumssusceptibleandresistantto
Plun pox virus.ITEA,103:156167.
Cvo:vN.,Pvvvz-PovsJ.,MozoC.,CvnoviiE.,
UvnviA.,ScovzR.,RvvioovoM.,Crnv
M.(2008):Assessmentofthediversityanddynamicsof
Plun pox virusandaphidpopulationsintransgenicEuro-
peanplumsunderMediterraneanconditions.Transgenic
Research,17:367377.
Civ M.F., Aors A.N. (1977): Characteristic of the
microplate method of enzyme-linked immunosorbent
assayforthedetectionofplantvirus.JournalofGenetic
Virology,34:5157.
DisA.S.,Ov:izR.(2011):Transgenicvegetablesfor21
st

centuryhorticulture.In:BookofAbstracts:Genetically
Modified Organisms in Horticulture Symposium. Sep-
tember1216,2011,Nelspruit,SouthAfrica:27(Abstr.)
Hiiv J. M., Scovz R., Miiosi T., Zoz B.,
RvvioovoM.(2004):Stabilityofgenesilencing-
based resistance toPlun pox virus in transgenic plum
(Prunus donesticaL.)underfieldconditions.Transgenic
Research,13:427436.
HiivJ.M.,RvvioovoM.,Drs:vvo:V.,Bssv::
C.,Pv:viC.,LiuZ.,ScovzR.(2007):Plun pox virus
coat protein gene intron-hairpin-RNA (ihpRNA) con-
structsprovideresistancetoPlun pox virusin^icotiana
benthanianaandPrunus donestica.JournalofAmerican
SocietyofHorticulturalSciences,132:850858.
JrvsC.(2011):GlobalStatusofCommercializedBiotechi
GMCrops:2011.ISAAABriefNo.43.TheInternational
ServicefortheAcquisitionofAgri-biotechApplications
(ISAAA),Ithaca,USA.
JvosovX J., Kuou J.K. (2010): Simultaneous detection
ofstonefruittreevirusesbyone-stepmultiplexRT-PCR.
ScientiaHorticulture,125:6872.
Miiosi, T., Crnv M., Cvo:v N., Zoz,
B.,GovvisM.T.,ScovzR.,RvvioovoM.(2006):
Field trials of plum clones transformed with thePlun
pox virus coat protein (PPV-CP) gene. Plant Disease,
90:10121018.
PoiX J., PIviovX J., Jovs M., Svonoo J., Scovz
R., Rvvioovo M. (2005): Preliminary results of
interactionsofPlun pox virus(PPV),Prune dwarf virus
(PDV),andApple chlorotic leafspot virus(ACLSV)with
transgenic plants of plum Prunus donestica, clone C5
grown in an open field. Phytopathologia Polonica, 36:
115122.
PoiXJ.,PIviovXJ.,Kurv-KuouJ.,JovsM.,Scov-
zR.,RvvioovoM.(2008a):Behaviouroftrans-
genicPlun pox virus-resistantPrunus donesticaL.clone
C5grownintheopenfieldunderahighandpermanent
infectionpressureofthePPV-Recstrain.JournalofPlant
Pathology,90(Suppl.1):S1.33S1.36.
PoiX J., Kurv-Kuou J., PIviovX J., Scovz R.,
Rvvioovo M. (2008b): Interactions ofPlun pox
virusstrainRecwithApple chlorotic leaf spotandPrune
dwarf viruses in field growing transgenic plum Prunus
donesticaL.,cloneC.PlantProtectionScience,44:15
Rvvioovo M., Scovz R., Bcnviivv J.C., Ln-
nov G., Lvvv L., Drs:vvo: V., Ciin A.M.,
DuvzJ.(1997):Resistanceoftransgenicplums(Prunus
donesticaL.)toPlun pox virusinfection.PlantDisease,
81:12311235.
RvvioovoM.,ScovzR.,CiinA.,LvvvL.,
JcQuv:C.,MosioM.,Drs:vvo:V.(2000):The
use of transgenic fruit trees as a resistance strategy for
virus epidemics: the plum pox (sharka) model. Virus
Research,71:6369.
Rvvioovo M., Scovz R., Mioiu N., Zovi I.,
Pi:oI.(2002):FieldtestsoftransgenicplumsinRo-
mania.SanatateaPlantelor(SpecialEdition):1618.
ScovzR.,RvvioovoM.,CiinA.M.,Covo:s
J.M.,FucnsM.,DuvzJ.,GosivvzD.(1994):Trans-
genicplums(Prunus donesticaL.)expresstheplumpox
viruscoatproteingene.PlantCellReports,14:1822.
unv Z., Pi::vvovX S., Gis M. (2004): A simplified
RT-PCR-baseddetectionofrecombinantPlun pox virus
isolates.ActaVirologica,48:173176.
S48
Vol. 48, 2012, Special Issue: S43S48 Plant Protect. Sci.
ZoviI.,RvvioovoM.,GnovvuI.,Fvvvcz
B.,ScovzR.,ZoviL.,KvivrvB.
,
PrriiD.,Povv-
scu O. (2010): Transgenic plums expressing the Plun
pox virus (PPV)coatproteingenedonotassistthede-
velopmentofPPVrecombinantsunderfieldconditions.
JournalofPlantPathology,93:159165.
Zovi I., Rvvioovo M., Scovz R., Mioiu N.,
ZoviL.(2008a):Fieldreleaseoftransgenicplumsin
Romania.BulletinofUniversityofAgriculturalSciences
andVeterinaryMedicineCluj-Napoca.AnimalScience
andBiotechnologies,65:358365.
ZoviI.,ZoviL.,RvvioovoM.,GnovvuI.,
PrriiD.,FvvvczB.,PovvscuO.,ScovzR.,Cvo-
:v N. (2008b): Environmental impact assessment of
transgenic plums on the diversity of Plun pox virus
populations.ActaHorticulturae(ISHS)781:309318.
ReceivedforpublicationMay23,2012
AcceptedaftercorrectionsOctober30,2012
Corresponding author
Doc.Ing.JvosivPoiX,DrSc.,Vzkumnstavrostlinnevroby,v.v.i.,odborrostlinolekastv,
oddlenvirologie,16106Praha6-Ruzyn,eskrepublika
tel.+420 233 022 315,e-mail:polak@vurv.cz