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Journal of Biotechnology 154 (2011) 304 311
Contents lists available at ScienceDirect
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: [email protected] (T.
Vanblaere),[email protected] (I. Szankowski), [email protected] (J.
Schaart),@wur.nl (H. Schouten), [email protected]),
[email protected] (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.
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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
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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
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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.,
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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