Journal of Biotechnology 154 (2011) 304 311

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Journal of Biotechnology

j ourna l ho me pag e: www.elsev ier .com

The development of a cisgenic apple plant

Thalia Va a a b houGiovannia Plant Patholo Switzeb Plant Researc eningec Julius Khn-In arten

a r t i c l

Article history:Received 14 AReceived in reAccepted 25 MAvailable onlin

Keywords:CisgenesisMarker-free pApple scabHcrVf2 resistaMalus domeVenturia inaeq

uenc

1. Introduction

Apple papple scab,(Laurens, 1of chemicalSuch large ctial environpathogen. Nbreeding hagression of1989; MacHand highly hbreeding pr(Gardiner eognized as ae.g. bananaing disease dominated

CorresponE-mail add

iris.szankowskhenk.schouten(H. Flachowskcesare.gessler@

acteristics for producers, storage procedures and consumers thatare difcult to equal. Most of the established cultivars are consid-

0168-1656/$ doi:10.1016/j.

a new the with

we re Hcr

prevsitivee ge

n toeneraroduction worldwide is impaired by diseases such as caused by the fungal pathogen Venturia inaequalis998). Disease control is achieved by a high number

treatments during growing season (MacHardy, 1996).hemical input is under critical scrutiny due to its poten-mental impact and ability to induce resistance in theatural resistance to diseases is known and classicals developed scab resistant cultivars, mostly by intro-

Vf resistance from Malus oribunda 821 (Lespinasse,ardy, 1996). As apple cultivars are self-incompatibleeterozygous, the phenotype of a cultivar is unique andoduces genotypes with new and distinct characteristicst al., 2007). Contrary to most other crops, apples are rec-

cultivar, e.g. Gala, Golden Delicious, and not as a crop,s. Therefore the popularity of the new cultivars carry-resistance genes is limited, as the traditional market iswith older established cultivars that have quality char-

ding author. Tel.: +41 44 632 38 71; fax: +41 44 632 15 72.resses: thalia.vanblaere@agrl.ethz.ch (T. Vanblaere),i@agrl.ethz.ch (I. Szankowski), jan.schaart@wur.nl (J. Schaart),@wur.nl (H. Schouten), henryk.achowsky@jki.bund.dey), giovanni.broggini@agrl.ethz.ch (G.A.L. Broggini),agrl.ethz.ch (C. Gessler).

ered susceptible to apple scab, as they are being intensively grownin monoculture which leads to the selection of adapted pathogenpopulations (MacHardy et al., 2001).

In order to maintain the particular characteristics of a cultivar,single genes coding for enzymes or other proteins, which inhibit orreduce the development of scab and re blight, were introduced byrecombinant DNA technology. A large number of foreign genes cod-ing for lytic enzymes from various sources (e.g. encoding lysozymesfrom bacteria, phages, and animals, chitinases and glucanases fromfungi), have been successfully integrated into the apple genomeand in several cases an increased signicant resistance to the tar-geted pathogen was observed. Pathogen derived genes or pathogeninduced promoters have also been used (reviewed in Gessler andPatocchi, 2007).

In all of these approaches, the incorporated genes and con-trol sequences are exogenous and are coupled with marker genesneeded for the positive selection of the transformed cells on selec-tive media.

A large proportion of the consumers in Europe view geneticallymodied foods as a risk to both health and the environment (Gaskellet al., 2000). To overcome the notorious aversion against transgen-ics by European consumers, Schouten et al. (2006) proposed the userecombinant DNA technology to introduce genes (including intronsand anking regions such as promoter and terminator in a senseorientation) derived from a crossable donor plant. They dened

see front matter 2011 Elsevier B.V. All rights reserved.jbiotec.2011.05.013nblaere , Iris Szankowski , Jan Schaart , Henk Sc A.L. Brogginia, Cesare Gesslera,

gy, Institute of Integrative Biology (IBZ), ETH Zrich, Universittstrasse 2, 8092 Zrich, h International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wagstitut, Bundesforschungsinstitut fr Kulturpanzen, Institut fr Zchtungsforschung an g

e i n f o

pril 2011vised form 17 May 2011ay 2011e 1 June 2011

lants

nce genesticaualis

under its native regulatory seq

a b s t r a c t

Cisgenesis represents a step toward antibiotic or herbicide resistance) infrom organisms sexually compatible increase consumers acceptance. Hereendogenous apple scab resistance genscab susceptible apple cultivar Gala. Atransformation combined with a porecombination machinery allowed thHcrVf2 detailed investigation and were showHcrVf2. This is the rst report of the g/ locate / jb io tec

tenb, Henryk Flachowskyc,

rlandn, The Netherlandsbaulichen Kulturen und Obst, Pillnitzer Platz 3a, 01326 Dresden, Germany

es and no foreign genes. Three cisgenic lines were chosen

2011 Elsevier B.V. All rights reserved.

generation of GM crops. The lack of selectable genes (e.g.nal product and the fact that the inserted gene(s) derivethe target crop should rise less environmental concerns andeport the generation of a cisgenic apple plant by inserting theVf2 under the control of its own regulatory sequences into theiously developed method based on Agrobacterium-mediated

and negative selection system and a chemically inducibleneration of apple cv. Gala carrying the scab resistance gene

for carry a single T-DNA insertion and express the target genetion of a true cisgenic plant.

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311 305

such plants as cisgenics. A less stringent concept is the intragenicapproach, as intragenics (Rommens et al., 2007) are consid-ered transformations with all-native DNA where overexpression,down regulation and silencing by combining genetic elements ofdifferent orcase, the prers such as denition. inition is reby Benjamitant to dowgene nptII, several casefrom a croscorrectly asregulatory 2010; Joshirelevant ste

Several species/plabeen isolate(Belfanti etgene was fous lengthsof resistanAll, howev(nptII) and inserted todevelop mnase systemapplied to apple linesfaciens carrAgrobacterion a kanamthe excisionically activarecombinaning 5-uoroHcrVf2 alonsequences aing the R recodA, allow5-uoro-cyrst scienti

2. Method

2.1. Gene is

The ent(220 bp) ofamplied frHotStarTaqHcrVf2-F antion sites Pproduct werestriction ed (WizardWI, USA) anthe binary vusing 200UMassachusePCR producgene was ex

chematic representation of the pMF1 vector containing the HcrVf2 geneed by its native regulatory sequences. The segment between the left (LB)right border (RB) is transferred into the plant cell, and the segment betweenmbination sites (RS) is then removed on recombinase-mediated deletion,

exception of one of the RS. HcrVf2, apple scab resistance gene from apple cv. fusion marker gene codA/nptII, hybrid gene for positive (nptII) and negativelection. RecLBD, translational fusion of recombinase R-LBD; Rk2 and ColE1,f replication. trfA; replication gene; nptIII, kanamycin resistance gene.

used for vector construction, PCR, RT-PCR and Southern blot Primere (5 3).

-Fa ATACGTATTTAATTAACTAGCTAGTCCTAAATAGCCG-Terma ATCTAGATGGCGCGCCGGGGAGAACATAAACCTTACCCr CGA TTC CGC ATT TTG AAT TT

ev TAC GCC CCG TTA TAG GAG TGtIIfor CCA CAG TCG ATG AAT CCA GAtIIrev AGC ACG TAC TCG GAT GGA AG

TAC TGG AAC ATC ACA GGC TGA C TGG ACC TCT CAT CAT GTT GT

CAA TGC CTT ACG TGG TGA AAv CAG GGA TTC CAG CCA ATC TA

2 GCG AGG AAC TAT GAC GAC CAV1 CCA CAC CAG TTC GTC ATC GTor CCG GTA TAA AGG GAC CAC CTev GGA GTG AAA GAG CCT GAT GCr AAT ATC ACG GGT AGC CAA CGv GAA TGA ACT CCA GGA CGA GG

ATG CGC ATG AGG CTC GTC TTC GAGGAC GCA ACG CAT CCT CGA TCA GCT

b3 ATA AGT GCC CTG CGG TAT TGb4 GCA GCC CTG GTT AAA AAC AA

SEQ1 TTG CTC ATA CAC ATC ACC TGC SEQ2 GTT TCT TTG GTT CTA TGA CAA G SEQ3 TCC GAT TCC CAA ATT GTT GT SEQ4 ACT AAG CTT GTC TGG TAC AGG AA SEQ5 GTA CCC GAT TGT TGG ATG AG SEQ6 CCG AGA TGC TTC CAC AAT TT SEQ7 CCA TGG AGC ATT CTT CTT TC SEQ8 GCT GCA ATT CTT GTT GAG A SEQ9 GTA AGT CCA GAC GCA ACC SEQ10 TTG GGA CAT TCC CAG TTA GG SEQ11 CGT TAG CAT TTT GAG TTG ACC A SEQ12 CCC CGA GAT TAA GAG TTG TAA GASEQ13 TGC TTT AAA CTG AGC AAA GAA GG

SEQ14 TGG TTG CAA TGG CTA GAA AC

tion sites PacI and AscI are underlined.igin, always from a crossable donor, are accepted. In anyesence of selectable marker genes, e.g. nptII or promot-the CaMV S35 promoter does not correspond to eitherCurrently no cisgenic plant corresponding to the def-ported in the literature. The transformants describedn et al. (2009) and declared as cisgenic melon resis-n mildew, contain the S35 promoter and the selectiontherefore they cannot be seen as cisgenic plants. Ins the target genes and regulatory elements originate

sable donor (cisgenes) however the plants are declared transgenics as selectable marker genes and/or theirsequences are from a not-crossable donor (Han et al.,, 2010; Szankowski et al., 2009), however they are thep toward creating cisgenic plants.endogenous resistance genes are known in applents, but only one resistance encoding gene, HcrVf2, hasd and proven functional to date in cvs. Gala and Elstar

al., 2004; Szankowski et al., 2009; Joshi, 2010). Theunctional under the CAMV 35S promoter and vari-

of native 5UTR sequences, giving an identical typece to that observed in classically bred Vf-cultivars.er, produced transgenic plants as the marker geneother regulatory sequences (Joshi, 2010) were also

recognize successfully transformed cells. In order toarker-free plants, the chemically inducible recombi-

reported by Schaart et al. (2004) in strawberry wasapple. In this paper we represent the development of

of the cv. Gala, transformed by Agrobacterium tume-ying the vector pMF1. The method is based on anum-mediated transformation followed by regenerationycin selective medium. The recombinase, and therefore

of the cassette carrying the transgenes, is then chem-ted by addition of dexamethasone to the medium andts selected on a negative selection medium contain-cytosine. The T-DNA of this vector carried the cisgeneg with its native up- and downstream regulatorynd a cassette anked by recombination sites contain-combinase gene and a fusion of marker genes nptII anding positive and negative selection on kanamycin- andtosine selective medium respectively (Fig. 1). This is thec report of the generation of a cisgenic crop.

s and materials

olation and vector construction

ire ORF (2943 bp) with 5 UTR (242 bp) and 3 UTR HcrVf2 (Gene bank accession number AJ297740) wasom the BAC clone M18-5 (Vinatzer et al., 2001) using

DNA Polymerase (Qiagen, Hilden, Germany). Primersd HcrVf2-Term (Table 1), elongated to carry the restric-acI and AscI for cloning, were used. 500 ng of the PCRre digested overnight at 37 C with 5 units each ofenzymes PacI and AscI in 20 l 1X NEB buffer 4, puri-

SV Gel and PCR Clean-Up System, Promega, Madison,d ligated overnight between the PacI and AscI sites ofector pMF1 with an insert/vector ratio of 20:1 at 16 C

of T4 DNA Ligase (New England Biolabs Inc., Beverly,tts, USA) (Fig. 1). To verify the correct insertion of thet in the vector, the pMF1 vector containing the HcrVf2tracted from transformed E. coli clones (GenElute Plas-

Fig. 1. Scontrolland the the recowith theFlorina;(codA) seorigins o

Table 1PrimersSequenc

HcrVf2HcrVf2codAfocodAr167np367npEF1forEF1revRT1forRT2retrfA FWtrfA REnptIIIfnptIIIrnptIIfonptIIrepicA1 picA2pmf bpmf bHcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2HcrVf2

aRestric

306 T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311

mid Miniprep Kit, SigmaAldrich, St. Louis, Mo., USA) and the insertwas sequenced using BigDye terminator kit 3.1 (Applied Biosys-tems, Foster City, CA) (primers sequences are listed in Table 1).The plasmids were then transferred for apple transformation intoAgrobacteriution.

2.2. Plant trplants

Agrobactapple cv. G(2003). In sin vitro shoand the topa suspensiountil a nal25 C in thevitamins (MTDZ, 2.6 M

After thonto a regecefotaxime

Explantsthey were mEvery two wappeared omedium comyo-inositocillin, 250 magar; pH (5to fresh me

The procbination wafrom three-leaf strips wtaining 10 mMo., USA) (amethasonewere cultu(described continue th(5-FC) (Sigmcells still haation proceof transgenTwo differegenic shootwas added ture in DEXwhere the 5weeks cultuovernight Dby regeneramedium wi

Cisgenicpropagatedgrafted on Msion in the irootstock asland, USA) the rootstoparent plasday on cuttually reducon the bags

graftings were grown in a greenhouse (day/night temperature of20/15, photoperiod of 16/8 h, 70% rH).

2.3. Presence of cisgenes and copy number determination

presmarkriedny). or de001eciGalaons

postIII akboncteris picd witing tGENolumacturles aion aed order

T7.1,9, Ccribed bygenis 16he Tr in

alita

wa Paisl

ands. Gan TA) to1revgenesed actiopliresisl RN

riptaga, anu

F1-stedr and

abov

ults

nera

indeultem tumefaciens (Hood et al., 1993) through electropora-

ansformation and recombination to obtain cisgenic

erium tumefaciens EHA105 was used to transform theala, using the protocol described by Szankowski et al.ummary, the top four youngest leaves of four week oldots from the cv. Gala were cut, leaving out the basal

part of the leaf. The leaf strips were inoculated withn of Agrobacterium (resuspended in liquid MS medium

OD620nm of 0.8) and co-cultivated during three days at dark on co-culture medium containing MS salts andurashige and Skoog, 1962), 3% (w/v) sorbitol, 22 M

NAA and 0.8% (w/v) plant agar; pH (5.65.8).e three day co-culture the explants were transferredneration medium where 150 mg/l ticarcillin, 250 mg/l

and 50 mg/l kanamycin were added. were kept in the dark for two weeks at 25 C beforeoved to an environment with a photoperiod of 16/8 h.eeks the explants were subcultured and when shoots

n the callus they were transferred onto elongationntaining: MS salts and vitamins, 3% (w/v) sucrose, 1%l, 3.1 M BAP, 0.5 M NAA, 2.8 m GA3, 150 mg/l ticar-g/l cefotaxime, 50 mg/l kanamycin and 0.8% (w/v) plant.65.8). The resulting transgenic lines were transferreddium every 45 weeks.edure to regenerate marker-free shoots through recom-s carried out by cutting the top four youngest leavesweek old in vitro transgenic mother lines in strips. Theere embedded overnight in a liquid MS solution con-M dexamethasone (DEX) (SigmaAldrich, St. Louis,

MS salts and vitamins, 3% (w/v) sucrose, 10 M dex-, pH 5.65.8). After overnight induction the explantsred on a regeneration medium without kanamycinabove) supplemented with 1 M dexamethasone toe recombination activity and 150 mg/l 5-uorocytosineaAldrich, St. Louis, Mo., USA) to select against the

rboring the fusion marker gene. The following regener-ss was identical as described above for the regenerationic plants after Agrobacterium-mediated transformation.nt strategies were applied to regenerate putative cis-s, a normal negative selection strategy where the 5-FCto the regeneration medium after an overnight cul--medium and a delayed negative selection strategy-FC was added to the regeneration medium after twore on regeneration medium containing only DEX and anEX-culture. An additional control test was performedting the transgenic explants immediately on a DEX-th 5-FC and omitting the overnight DEX-culture.

shoots and in vitro control shoots were micro- on elongation medium and six weeks old shoots were9 rootstocks. Grafting was carried out through a V inci-

n vitro shoot and a vertical incision in a shoot of the M9 described by (Joshi, 2010). A latex tape (Sealtex, Mary-was used to hold the in vitro shoot in the incision ofck. Subsequently the graftings were covered by trans-tic bags to maintain a high humidity and from the 3rdings were made every three days in the bags to grad-e the humidity level until no condensation was visible. After about two weeks the bags were removed and the

Thefusion was veGermaused fet al., 2non-spin cv. to deming thetrfA, npify bacAgrobaprimerisolatefollow1X QIAtotal vmanuf35 cycextensidenti

In olines ((C7.1.4as desdigestedigoxyprimerber of tnumbe

2.4. Qu

RNArogen,in vitrotrol cvAmbioCity, Cand EFfactor were uPCR reThe amtropho

10 Transc(Promeing to mgene Ewere tecodAfotioned

3. Res

3.1. Ge

Six and resence/absence of the genes HcrVf2, trfA, nptIII and theer gene nptII/codA in the in vitro and greenhouse plants

by PCR using Qiagen Multiplex PCR kit (Qiagen, Hilden,The primers used are listed in Table 1. The primerstection of HcrVf2 were RT1for and RT2rev (Vinatzer) and give a specic band at 856 bp and an additionalc band at 1050 bp, this additional band is also visible. Primers for the marker gene, nptII/codA, were usedtrate the successful excision of the cassette contain-itive/negative fusion marker gene, whereas primers fornd the primer pair pmf bb3/pmf bb4 were used to ver-e integration or absence in the lines. The presence ofum was tested to avoid false positive results (using theA1/picA2 listed in Table 1) The genomic plant DNA wash the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)he manufacturers protocol. PCR reactions consisted of

Multiplex PCR Master Mix, 0.2 M of each primer in ae of 15 l. PCR reactions were performed applying theers protocol conditions (95 C for 15 min, followed byt 94 C for 30 s, 60 C for 90 s, 72 C for 90 s, and a nalt 72 C for 10 min). The amplicons were separated andn agarose gel (1%).

to assess the copy number, the transgenic mother T11.1, T12.1) with their respective cisgenic sublines11.1.53, C12.1.49) were analyzed by Southern blotd by Szankowski et al. (2009). Genomic DNA was

XhoI and the blotted membrane was hybridized usingn-labeled PCR probes on nptII (amplied using the7nptIIfor/367nptIIrev listed in Table 1). The copy num--DNA insert in the cisgenic sublines is based on the copythe transgenic lines.

tive expression of cisgenes

s isolated using PureLinkTM Plant RNA Reagent (Invit-ey, Scotland) using 100 mg of fresh young leaves from

greenhouse-grown trans- and cisgenic lines and con-la and Florina. The RNA was subsequently treated withURBO DNA-freeTM DNase (Applied Biosystems, Foster

eliminate genomic DNA contamination. Primers EF1for (Table 1) that amplify a fragment of the elongation

EF1 alpha (EF1-, GenBank accession no. DQ341381)to check DNA contamination in the RNA samples. Then was performed as described above using 1 l of RNA.ed fragments were analyzed through agarose gel elec-.A was converted into cDNA using the M-MLV Reversese, RNase H Minus Kit with oligo(dT)18 primersMadison, WI, USA). All steps were performed accord-facturers instructions. The expression of the reference, target gene HcrVf2 and fusion marker gene codA/nptII

with the primers EF1for and EF1rev, RT1for and RT2rev, codArev, using 2 l cDNA and the conditions as men-e.

tion of transgenic lines

pendent transformation experiments were performedd in the regeneration of a total of ten transgenic (T) lines

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311 307

Fig. 2. Backbone integration. Polymerase chain reaction analysis using primers specic for nptIII to detpositive control and untransformed cv. Gala and water were used as negative control.

out of 1635 explants (T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3, T11.1,T11.2, T12.1). Two transformation experiments failed to regeneratetransgenic shoots. The overall transformation efciency was 0.6%(Table 2). A control transformation experiment that was performedwith an em(data not sh

In all traproven by Pgenic lines nptIII insert

3.2. Genera

All ten tsubsequentbinase by camended mand a maxitransgenic whereas alltransgenic (T11.1, T12regeneratedegy with aT7.1 and 26test where was omittewith overntransgenic tion; therefExcept in pcodA/nptII mnation wasT8.3 still cothese sublin

Three cisfor more exmother line

Table 2Data of the traused for eachtransformatio

Transformat

7 8 9 10 11 12

Total

genic mothHcrVf2 and(HcrVf2 andthe DNA is

, iso DEXsful r

beting

sformnd foed

g thnd thnsgecisgeh thees ba

us bisgerans-es th

loss mo

(C73/pm

that ine, tnotywith .1.17sentther11.1ted, ines

chogreen

cussi

outepty pMF1 vector had a transformation efciency of 1%own).nsgenic lines the presence of HcrVf2 and codA/nptII wasCR and RT-PCR (data not shown). Eight out of ten trans-(T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3, T11.2) showedion, conrming backbone integration (Fig. 2).

tion of cisgenic lines

ransgenic lines were subjected to recombination and regeneration through an induction step of the R recom-ulturing the transgenic explants on a dexamethasoneedium. For each transgenic line 200 explants were usedmum of 36 putative cisgenic sublines from which themotherlines contain backbone were kept for analyses,

the putative cisgenic sublines were kept from the twomotherlines that are free from backbone integration.1) (Table 3). The delayed negative selection medium

more shoots than the normal negative selection strat-n increase of 8% for putative cisgenic sublines from% for T7.2 regenerated cisgenic sublines. The controlthe overnight culture in liquid MS medium with DEXd resulted in efciencies comparable to the strategyight DEX-culture (data not shown). The regenerants ofline 7.1 suffered great losses due to fungal contamina-ore no quantitative data are reported for these sublines.utative cisgenic sublines of T8.1, T11.2 and T12.1, noarker gene was found, conrming that the recombi-

efcient. All the putative cisgenic sublines of T8.2 andntained backbone along with the HcrVf2 gene, thereforees are not cisgenic (Table 3).genic lines, C7.1.49, C11.1.53 and C12.1.49 were chosentensive research, based on the fact that their transgenics T11.1 and T12.1 are backbone-free and the trans-

nsformation experiments with their respective amount of explants experiment, the amount of regenerated transgenic shoots and then efciency.

of DNAvatingsucceslocatedconrmuntrancic baperformshowinlines athe train the througindicatshowsin its cother tindicat

Thetigatedof T7.1pmf bbshow genic lrst gealong and C7was ab

Sou(T7.1, Toriginathree l

Thein the

4. Dis

Schion # Explants # Transgenic shoots Transformationefciency (%)

250 4 1.6380 3 0.8250 0 0250 0 0250 2 0.8255 1 0.4

1635 10 0.6

whereby alfrom a croing debate Williams, 2and SchoutMachray, 20ing with pldirectly concultivar. Cisuse in potatect backbone integration. pMF1 (plasmid with HcrVf2) was used as a

er line T7.1 was chosen as a transgenic line carrying backbone DNA. Expected fragments of the two genes

codA) were detected in the plasmid (pMF1) and inolated from the transgenic mother lines. PCR analysislated from shoots obtained after the recombinase acti-

treatment (C7.1.49, C11.1.53, C12.1.49), indicated theemoval of the fusion marker gene codA/nptII, which isween the recombination sites. HcrVf2 was still present,

the cisgenic character of the regenerated plants. In theed in vitro cv. Gala, neither the transgenes nor the spe-r the cisgene HcrVf2 were detected (Fig. 3). RT-PCR wasfor the HcrVf2 gene and fusion marker gene codA/nptII,at the HcrVf2 gene is expressed in trans- and cisgenice expression of the marker gene can only be detected innic lines, no expression of the marker gene is observednic lines (Fig. 4). Backbone presence was investigated

genes trfA and nptIII. The presence of one of these genesckbone integration. PCR analysis of transgenic line T7.1ands for trfA and nptIII, but trfA and nptIII are absentnic line C7.1.49. The lack of amplied products in the

and cisgenic lines (T11.1, C11.1.53, T12.1 and C12.1.49)e absence of trfA and nptIII (Fig. 5).

of the backbone from T7.1 to C7.1.49 has been inves-re in detail by testing other putative cisgenic sublines.1.17a, C7.1.63 and C7.1.3 g) with the primer pair,f bb4, located on the backbone close to the RB. Results

during the recombination step toward a putative cis-wo different recombination genotypes occurred. In thepe (C7.1.49 and C7.1.3 g) the backbone has been cut outthe marker gene and in the second genotype (C7.1.63a) the backbone was still present but the marker gene

(Fig. 6).n blot analysis with nptII probe of the transgenic lines

and T12.1), from which the three selected cisgenic linesshowed single integration events of the T-DNA in all(Fig. 7).sen lines were micro-grafted on M9 rootstocks grownhouse for future phenotypic screening.

on

n et al. (2006) dened the concept of cisgenic plants

l introduced genes and control sequences are derivedssable donor plant and sparked a long, still ongo-involving numerous articles or letters (Schubert and006; Van Bueren et al., 2007; Houdebine, 2007; Jacobsenen, 2008; Jacobsen and Nataraja, 2008; Akhond and09). The cisgenic approach found most interest in deal-

ant diseases of crops which are vegetative propagated,sumed and have quality aspects related to a particulargenes are being discussed in articles with its potentialo against potato late blight (Jacobsen, 2007; Kuhl et al.,

308 T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311

Fig. 3. Genomic DNA analysis. Polymerase chain reaction analysis using primers specic for HcrVf2 (a) and codA (b) genes. The fragment visible at 1050 bp, which wasamplied using the RT primers, results from homologues sequences present in cv. Gala. The HcrVf2 specic band has a size of 856 bp. In the PCR reactions we also includedcv. Florina, a natural resistant plant containing HcrVf2, as a positive control for HcrVf2 and as a negative control for codA. Cv. Gala and water were used as negative controlsand pMF1 (plasmid with HcrVf2) was used as a positive control for both genes.

Fig. 4. Qualitative expression analysis. RT-PCR using the elongation factor as a control for genomic DNA contamination (a). Expression of HcrVf2 (b) and codA (c) is analyzed.In the RT-PCR reactions we also included cv. Florina, a natural resistant plant containing HcrVf2, as a positive control and untransformed cv. Gala and water as negativecontrols. pMF1 (plasmid with HcrVf2) was used as positive control for the PCR reaction.

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311 309

Fig. 5. Backbo ) and was used as po ed as n

Table 3Frequencies of

Transgenicmother lines

T7.1T7.2 T7.3T7.4 T8.1 T8.2T8.3 T11.1T11.2 T12.1

2007; Haveapple scab in downy m

So far, nature that sexperimentnative geneet al., 2010cisgenic po

Thereforcrop ttingGala lines HcrVf2, whiBelfanti et aing any fore

The aboever, the higmethod cou

From thT12.1) we dgrated into

The DEXimpairing ane integration. Polymerase chain reaction analysis using primers specic for trfA (asitive control. Cvs. Gala and Florina, two untransformed plants and water were us the possible genotypes after recombination for each transgenic mother line; assessed by

# Shootstested

% HcrVf2: +codA: +trfA: +

% HcrVf2: +codA: +trfA:

N/A N/A N/A 36 0 0 36 0 0 18 0 0 18 44 0 36 0 09 0 0 126 0 0 36 6 0 108 0 24

rkort et al., 2008; Park et al., 2009), in apple against(Gessler et al., 2009; Joshi et al., 2009; Joshi, 2010) andildew of melon (Benjamin et al., 2009).o cisgenic crops have been reported in scientic liter-atisfy the denition of Schouten et al. (2006). Lately,s have been performed to evaluate the use of additionals (cisgenes) in poplar to enhance the growth rate (Han; Strauss et al., 2009), however without developing aplar line.e, this is the rst report of the development of a cisgenic

the denition of Schouten et al. (2006). Our cisgenicwere shown to contain the apple scab resistance genech induces resistance to apple scab (Barbieri et al., 2003;l., 2004; Joshi, 2010; Szankowski et al., 2009), and lack-ign genes.ve results proof the functionality of the method. How-h degree of backbone integration (80%) observed in thisld pose a considerable problem.e analysis of the transgenic mother lines (T7.1, T11.1,educe that there is only one copy of the T-DNA inte-the plant genome.

treatment which activates the recombinase gene is notpple explant regeneration. The subsequent elimination

of the foreignase gene, T11.2 and Tter recoverselection stobserved on

Fig. 6. AnalysT7.1 using thepositive contrnptIII (b) to detect backbone integration. pMF1 (plasmid with HcrVf2)egative control. PCR on HcrVf2, codA and trfA.

% HcrVf2: +codA: trfA: +

% HcrVf2: +codA: trfA: cisgenic

% Agro bacteriumcontamination

N/A N/A N/A3 94 322 78 072 22 60 50 6100 0 0100 0 00 81 1988 6 60 74 2

n genes, i.e. the fusion marker gene and the R recombi-occurred as expected, with exception of the lines T8.1,12.1. The negative selection with 5-FC showed a bet-y of putative cisgenic shoots when a delayed negativerategy was used, but overall no negative effects were

the growth of the shoots.

is of two different genotypes after recombination of transgenic line primers pmf bb3/pmf bb4. pMF1 (plasmid with HcrVf2) was used asol. Untransformed cv. Gala and water were used as negative control.

310 T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304 311

Fig. 7. Copy n lines and the blotte plasmcontrol for the

We mennation of liread-througon over theright bordeanother coption sites cabackbone wout during the marker

In this repMF1 vectocisgenic lingene of inteon recombionly one T-The appearin the greeGala. Furtheorder to conthe effectivpromising swithout addengineered

Acknowled

The authSwiss NatioContributio

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The development of a cisgenic apple plant1 Introduction2 Methods and materials2.1 Gene isolation and vector construction2.2 Plant transformation and recombination to obtain cisgenic plants2.3 Presence of cisgenes and copy number determination2.4 Qualitative expression of cisgenes

3 Results3.1 Generation of transgenic lines3.2 Generation of cisgenic lines

4 DiscussionAcknowledgementsReferences