Journal of Biotechnology 154 (2011) 304311

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

The development of a cisgenic apple plant

Thalia Vanblaere a , Iris Szankowski a , Jan Schaart b , Henk Schouten b , Henryk Flachowsky c ,
Giovanni A.L. Broggini a , Cesare Gessler a,
a
b
c

Plant Pathology, Institute of Integrative Biology (IBZ), ETH Zrich, Universittstrasse 2, 8092 Zrich, Switzerland
Plant Research International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, The Netherlands
Julius Khn-Institut, Bundesforschungsinstitut fr Kulturpanzen, Institut fr Zchtungsforschung an gartenbaulichen Kulturen und Obst, Pillnitzer Platz 3a, 01326 Dresden, Germany

a r t i c l e

i n f o

Article history:
Received 14 April 2011
Received in revised form 17 May 2011
Accepted 25 May 2011
Available online 1 June 2011
Keywords:
Cisgenesis
Marker-free plants
Apple scab
HcrVf2 resistance gene
Malus domestica
Venturia inaequalis

a b s t r a c t
Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g.
antibiotic or herbicide resistance) in the nal product and the fact that the inserted gene(s) derive
from organisms sexually compatible with the target crop should rise less environmental concerns and
increase consumers acceptance. Here we report the generation of a cisgenic apple plant by inserting the
endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the
scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated
transformation combined with a positive and negative selection system and a chemically inducible
recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene
HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for
detailed investigation and were shown to carry a single T-DNA insertion and express the target gene
HcrVf2. This is the rst report of the generation of a true cisgenic plant.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Apple production worldwide is impaired by diseases such as
apple scab, caused by the fungal pathogen Venturia inaequalis
(Laurens, 1998). Disease control is achieved by a high number
of chemical treatments during growing season (MacHardy, 1996).
Such large chemical input is under critical scrutiny due to its potential environmental impact and ability to induce resistance in the
pathogen. Natural resistance to diseases is known and classical
breeding has developed scab resistant cultivars, mostly by introgression of Vf resistance from Malus oribunda 821 (Lespinasse,
1989; MacHardy, 1996). As apple cultivars are self-incompatible
and highly heterozygous, the phenotype of a cultivar is unique and
breeding produces genotypes with new and distinct characteristics
(Gardiner et al., 2007). Contrary to most other crops, apples are recognized as a cultivar, e.g. Gala, Golden Delicious, and not as a crop,
e.g. bananas. Therefore the popularity of the new cultivars carrying disease resistance genes is limited, as the traditional market is
dominated with older established cultivars that have quality char-

Corresponding author. Tel.: +41 44 632 38 71; fax: +41 44 632 15 72.
E-mail addresses: thalia.vanblaere@agrl.ethz.ch (T. Vanblaere),
iris.szankowski@agrl.ethz.ch (I. Szankowski), jan.schaart@wur.nl (J. Schaart),
henk.schouten@wur.nl (H. Schouten), henryk.achowsky@jki.bund.de
(H. Flachowsky), giovanni.broggini@agrl.ethz.ch (G.A.L. Broggini),
cesare.gessler@agrl.ethz.ch (C. Gessler).
0168-1656/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.05.013

acteristics for producers, storage procedures and consumers that

are difcult to equal. Most of the established cultivars are considered susceptible to apple scab, as they are being intensively grown
in monoculture which leads to the selection of adapted pathogen
populations (MacHardy et al., 2001).
In order to maintain the particular characteristics of a cultivar,
single genes coding for enzymes or other proteins, which inhibit or
reduce the development of scab and re blight, were introduced by
recombinant DNA technology. A large number of foreign genes coding for lytic enzymes from various sources (e.g. encoding lysozymes
from bacteria, phages, and animals, chitinases and glucanases from
fungi), have been successfully integrated into the apple genome
and in several cases an increased signicant resistance to the targeted pathogen was observed. Pathogen derived genes or pathogen
induced promoters have also been used (reviewed in Gessler and
Patocchi, 2007).
In all of these approaches, the incorporated genes and control sequences are exogenous and are coupled with marker genes
needed for the positive selection of the transformed cells on selective media.
A large proportion of the consumers in Europe view genetically
modied foods as a risk to both health and the environment (Gaskell
et al., 2000). To overcome the notorious aversion against transgenics by European consumers, Schouten et al. (2006) proposed the use
recombinant DNA technology to introduce genes (including introns
and anking regions such as promoter and terminator in a sense
orientation) derived from a crossable donor plant. They dened

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

such plants as cisgenics. A less stringent concept is the intragenic

approach, as intragenics (Rommens et al., 2007) are considered transformations with all-native DNA where overexpression,
down regulation and silencing by combining genetic elements of
different origin, always from a crossable donor, are accepted. In any
case, the presence of selectable marker genes, e.g. nptII or promoters such as the CaMV S35 promoter does not correspond to either
denition. Currently no cisgenic plant corresponding to the definition is reported in the literature. The transformants described
by Benjamin et al. (2009) and declared as cisgenic melon resistant to down mildew, contain the S35 promoter and the selection
gene nptII, therefore they cannot be seen as cisgenic plants. In
several cases the target genes and regulatory elements originate
from a crossable donor (cisgenes) however the plants are declared
correctly as transgenics as selectable marker genes and/or their
regulatory sequences are from a not-crossable donor (Han et al.,
2010; Joshi, 2010; Szankowski et al., 2009), however they are the
relevant step toward creating cisgenic plants.
Several endogenous resistance genes are known in apple
species/plants, but only one resistance encoding gene, HcrVf2, has
been isolated and proven functional to date in cvs. Gala and Elstar
(Belfanti et al., 2004; Szankowski et al., 2009; Joshi, 2010). The
gene was functional under the CAMV 35S promoter and various lengths of native 5 UTR sequences, giving an identical type
of resistance to that observed in classically bred Vf-cultivars.
All, however, produced transgenic plants as the marker gene
(nptII) and other regulatory sequences (Joshi, 2010) were also
inserted to recognize successfully transformed cells. In order to
develop marker-free plants, the chemically inducible recombinase system reported by Schaart et al. (2004) in strawberry was
applied to apple. In this paper we represent the development of
apple lines of the cv. Gala, transformed by Agrobacterium tumefaciens carrying the vector pMF1. The method is based on an
Agrobacterium-mediated transformation followed by regeneration
on a kanamycin selective medium. The recombinase, and therefore
the excision of the cassette carrying the transgenes, is then chemically activated by addition of dexamethasone to the medium and
recombinants selected on a negative selection medium containing 5-uorocytosine. The T-DNA of this vector carried the cisgene
HcrVf2 along with its native up- and downstream regulatory
sequences and a cassette anked by recombination sites containing the R recombinase gene and a fusion of marker genes nptII and
codA, allowing positive and negative selection on kanamycin- and
5-uoro-cytosine selective medium respectively (Fig. 1). This is the
rst scientic report of the generation of a cisgenic crop.

Fig. 1. Schematic representation of the pMF1 vector containing the HcrVf2 gene
controlled by its native regulatory sequences. The segment between the left (LB)
and the right border (RB) is transferred into the plant cell, and the segment between
the recombination sites (RS) is then removed on recombinase-mediated deletion,
with the exception of one of the RS. HcrVf2, apple scab resistance gene from apple cv.
Florina; fusion marker gene codA/nptII, hybrid gene for positive (nptII) and negative
(codA) selection. RecLBD, translational fusion of recombinase R-LBD; Rk2 and ColE1,
origins of replication. trfA; replication gene; nptIII, kanamycin resistance gene.

Table 1
Primers used for vector construction, PCR, RT-PCR and Southern blot Primer
Sequence (5 3 ).
HcrVf2-Fa
HcrVf2-Terma
codAfor
codArev
167nptIIfor
367nptIIrev
EF1for
EF1rev
RT1for
RT2rev
trfA FW2
trfA REV1
nptIIIfor
nptIIIrev
nptIIfor
nptIIrev
picA1
picA2
pmf bb3
pmf bb4
HcrVf2 SEQ1
HcrVf2 SEQ2
HcrVf2 SEQ3
HcrVf2 SEQ4
HcrVf2 SEQ5
HcrVf2 SEQ6
HcrVf2 SEQ7
HcrVf2 SEQ8
HcrVf2 SEQ9
HcrVf2 SEQ10
HcrVf2 SEQ11
HcrVf2 SEQ12
HcrVf2 SEQ13
HcrVf2 SEQ14

2. Methods and materials

2.1. Gene isolation and vector construction
The entire ORF (2943 bp) with 5 UTR (242 bp) and 3 UTR
(220 bp) of HcrVf2 (Gene bank accession number AJ297740) was
amplied from the BAC clone M18-5 (Vinatzer et al., 2001) using
HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany). Primers
HcrVf2-F and HcrVf2-Term (Table 1), elongated to carry the restriction sites PacI and AscI for cloning, were used. 500 ng of the PCR
product were digested overnight at 37 C with 5 units each of
restriction enzymes PacI and AscI in 20 l 1X NEB buffer 4, puried (Wizard SV Gel and PCR Clean-Up System, Promega, Madison,
WI, USA) and ligated overnight between the PacI and AscI sites of
the binary vector pMF1 with an insert/vector ratio of 20:1 at 16 C
using 200U of T4 DNA Ligase (New England Biolabs Inc., Beverly,
Massachusetts, USA) (Fig. 1). To verify the correct insertion of the
PCR product in the vector, the pMF1 vector containing the HcrVf2
gene was extracted from transformed E. coli clones (GenElute Plas-

305

ATACGTATTTAATTAACTAGCTAGTCCTAAATAGCCG
ATCTAGATGGCGCGCCGGGGAGAACATAAACCTTACCC
CGA TTC CGC ATT TTG AAT TT
TAC GCC CCG TTA TAG GAG TG
CCA CAG TCG ATG AAT CCA GA
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 AA
CAG GGA TTC CAG CCA ATC TA
GCG AGG AAC TAT GAC GAC CA
CCA CAC CAG TTC GTC ATC GT
CCG GTA TAA AGG GAC CAC CT
GGA GTG AAA GAG CCT GAT GC
AAT ATC ACG GGT AGC CAA CG
GAA TGA ACT CCA GGA CGA GG
ATG CGC ATG AGG CTC GTC TTC GAG
GAC GCA ACG CAT CCT CGA TCA GCT
ATA AGT GCC CTG CGG TAT TG
GCA GCC CTG GTT AAA AAC AA
TTG CTC ATA CAC ATC ACC TGC
GTT TCT TTG GTT CTA TGA CAA G
TCC GAT TCC CAA ATT GTT GT
ACT AAG CTT GTC TGG TAC AGG AA
GTA CCC GAT TGT TGG ATG AG
CCG AGA TGC TTC CAC AAT TT
CCA TGG AGC ATT CTT CTT TC
GCT GCA ATT CTT GTT GAG A
GTA AGT CCA GAC GCA ACC
TTG GGA CAT TCC CAG TTA GG
CGT TAG CAT TTT GAG TTG ACC A
CCC CGA GAT TAA GAG TTG TAA GA
TGC TTT AAA CTG AGC AAA GAA GG
TGG TTG CAA TGG CTA GAA AC

Restriction sites PacI and AscI are underlined.

306

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

mid Miniprep Kit, SigmaAldrich, St. Louis, Mo., USA) and the insert
was sequenced using BigDye terminator kit 3.1 (Applied Biosystems, Foster City, CA) (primers sequences are listed in Table 1).
The plasmids were then transferred for apple transformation into
Agrobacterium tumefaciens (Hood et al., 1993) through electroporation.
2.2. Plant transformation and recombination to obtain cisgenic
plants
Agrobacterium tumefaciens EHA105 was used to transform the
apple cv. Gala, using the protocol described by Szankowski et al.
(2003). In summary, the top four youngest leaves of four week old
in vitro shoots from the cv. Gala were cut, leaving out the basal
and the top part of the leaf. The leaf strips were inoculated with
a suspension of Agrobacterium (resuspended in liquid MS medium
until a nal OD620nm of 0.8) and co-cultivated during three days at
25 C in the dark on co-culture medium containing MS salts and
vitamins (Murashige and Skoog, 1962), 3% (w/v) sorbitol, 22 M
TDZ, 2.6 M NAA and 0.8% (w/v) plant agar; pH (5.65.8).
After the three day co-culture the explants were transferred
onto a regeneration medium where 150 mg/l ticarcillin, 250 mg/l
cefotaxime and 50 mg/l kanamycin were added.
Explants were kept in the dark for two weeks at 25 C before
they were moved to an environment with a photoperiod of 16/8 h.
Every two weeks the explants were subcultured and when shoots
appeared on the callus they were transferred onto elongation
medium containing: MS salts and vitamins, 3% (w/v) sucrose, 1%
myo-inositol, 3.1 M BAP, 0.5 M NAA, 2.8 m GA3, 150 mg/l ticarcillin, 250 mg/l cefotaxime, 50 mg/l kanamycin and 0.8% (w/v) plant
agar; pH (5.65.8). The resulting transgenic lines were transferred
to fresh medium every 45 weeks.
The procedure to regenerate marker-free shoots through recombination was carried out by cutting the top four youngest leaves
from three-week old in vitro transgenic mother lines in strips. The
leaf strips were embedded overnight in a liquid MS solution containing 10 mM dexamethasone (DEX) (SigmaAldrich, St. Louis,
Mo., USA) (MS salts and vitamins, 3% (w/v) sucrose, 10 M dexamethasone, pH 5.65.8). After overnight induction the explants
were cultured on a regeneration medium without kanamycin
(described above) supplemented with 1 M dexamethasone to
continue the recombination activity and 150 mg/l 5-uorocytosine
(5-FC) (SigmaAldrich, St. Louis, Mo., USA) to select against the
cells still harboring the fusion marker gene. The following regeneration process was identical as described above for the regeneration
of transgenic plants after Agrobacterium-mediated transformation.
Two different strategies were applied to regenerate putative cisgenic shoots, a normal negative selection strategy where the 5-FC
was added to the regeneration medium after an overnight culture in DEX-medium and a delayed negative selection strategy
where the 5-FC was added to the regeneration medium after two
weeks culture on regeneration medium containing only DEX and an
overnight DEX-culture. An additional control test was performed
by regenerating the transgenic explants immediately on a DEXmedium with 5-FC and omitting the overnight DEX-culture.
Cisgenic shoots and in vitro control shoots were micropropagated on elongation medium and six weeks old shoots were
grafted on M9 rootstocks. Grafting was carried out through a V incision in the in vitro shoot and a vertical incision in a shoot of the M9
rootstock as described by (Joshi, 2010). A latex tape (Sealtex, Maryland, USA) was used to hold the in vitro shoot in the incision of
the rootstock. Subsequently the graftings were covered by transparent plastic bags to maintain a high humidity and from the 3rd
day on cuttings were made every three days in the bags to gradually reduce the humidity level until no condensation was visible
on the bags. After about two weeks the bags were removed and the

graftings were grown in a greenhouse (day/night temperature of

20 /15 , photoperiod of 16/8 h, 70% rH).
2.3. Presence of cisgenes and copy number determination
The presence/absence of the genes HcrVf2, trfA, nptIII and the
fusion marker gene nptII/codA in the in vitro and greenhouse plants
was veried by PCR using Qiagen Multiplex PCR kit (Qiagen, Hilden,
Germany). The primers used are listed in Table 1. The primers
used for detection of HcrVf2 were RT1for and RT2rev (Vinatzer
et al., 2001) and give a specic band at 856 bp and an additional
non-specic band at 1050 bp, this additional band is also visible
in cv. Gala. Primers for the marker gene, nptII/codA, were used
to demonstrate the successful excision of the cassette containing the positive/negative fusion marker gene, whereas primers for
trfA, nptIII and the primer pair pmf bb3/pmf bb4 were used to verify backbone integration or absence in the lines. The presence of
Agrobacterium was tested to avoid false positive results (using the
primers picA1/picA2 listed in Table 1) The genomic plant DNA was
isolated with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)
following the manufacturers protocol. PCR reactions consisted of
1X QIAGEN Multiplex PCR Master Mix, 0.2 M of each primer in a
total volume of 15 l. PCR reactions were performed applying the
manufacturers protocol conditions (95 C for 15 min, followed by
35 cycles at 94 C for 30 s, 60 C for 90 s, 72 C for 90 s, and a nal
extension at 72 C for 10 min). The amplicons were separated and
identied on agarose gel (1%).
In order to assess the copy number, the transgenic mother
lines (T7.1, T11.1, T12.1) with their respective cisgenic sublines
(C7.1.49, C11.1.53, C12.1.49) were analyzed by Southern blot
as described by Szankowski et al. (2009). Genomic DNA was
digested by XhoI and the blotted membrane was hybridized using
digoxygenin-labeled PCR probes on nptII (amplied using the
primers 167nptIIfor/367nptIIrev listed in Table 1). The copy number of the T-DNA insert in the cisgenic sublines is based on the copy
number in the transgenic lines.
2.4. Qualitative expression of cisgenes
RNA was isolated using PureLinkTM Plant RNA Reagent (Invitrogen, Paisley, Scotland) using 100 mg of fresh young leaves from
in vitro and greenhouse-grown trans- and cisgenic lines and control cvs. Gala and Florina. The RNA was subsequently treated with
Ambion TURBO DNA-freeTM DNase (Applied Biosystems, Foster
City, CA) to eliminate genomic DNA contamination. Primers EF1for
and EF1rev (Table 1) that amplify a fragment of the elongation
factor gene EF1 alpha (EF1-, GenBank accession no. DQ341381)
were used to check DNA contamination in the RNA samples. The
PCR reaction was performed as described above using 1 l of RNA.
The amplied fragments were analyzed through agarose gel electrophoresis.
10 l RNA was converted into cDNA using the M-MLV Reverse
Transcriptase, RNase H Minus Kit with oligo(dT)18 primers
(Promega, Madison, WI, USA). All steps were performed according to manufacturers instructions. The expression of the reference
gene EF1-, target gene HcrVf2 and fusion marker gene codA/nptII
were tested with the primers EF1for and EF1rev, RT1for and RT2rev,
codAfor and codArev, using 2 l cDNA and the conditions as mentioned above.
3. Results
3.1. Generation of transgenic lines
Six independent transformation experiments were performed
and resulted in the regeneration of a total of ten transgenic (T) lines

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

307

Fig. 2. Backbone integration. Polymerase chain reaction analysis using primers specic for nptIII to detect backbone integration. pMF1 (plasmid with HcrVf2) was used as a
positive 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 regenerate
transgenic shoots. The overall transformation efciency was 0.6%
(Table 2). A control transformation experiment that was performed
with an empty pMF1 vector had a transformation efciency of 1%
(data not shown).
In all transgenic lines the presence of HcrVf2 and codA/nptII was
proven by PCR and RT-PCR (data not shown). Eight out of ten transgenic lines (T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3, T11.2) showed
nptIII insertion, conrming backbone integration (Fig. 2).
3.2. Generation of cisgenic lines
All ten transgenic lines were subjected to recombination and
subsequent regeneration through an induction step of the R recombinase by culturing the transgenic explants on a dexamethasone
amended medium. For each transgenic line 200 explants were used
and a maximum of 36 putative cisgenic sublines from which the
transgenic motherlines contain backbone were kept for analyses,
whereas all the putative cisgenic sublines were kept from the two
transgenic motherlines that are free from backbone integration
(T11.1, T12.1) (Table 3). The delayed negative selection medium
regenerated more shoots than the normal negative selection strategy with an increase of 8% for putative cisgenic sublines from
T7.1 and 26% for T7.2 regenerated cisgenic sublines. The control
test where the overnight culture in liquid MS medium with DEX
was omitted resulted in efciencies comparable to the strategy
with overnight DEX-culture (data not shown). The regenerants of
transgenic line 7.1 suffered great losses due to fungal contamination; therefore no quantitative data are reported for these sublines.
Except in putative cisgenic sublines of T8.1, T11.2 and T12.1, no
codA/nptII marker gene was found, conrming that the recombination was efcient. All the putative cisgenic sublines of T8.2 and
T8.3 still contained backbone along with the HcrVf2 gene, therefore
these sublines are not cisgenic (Table 3).
Three cisgenic lines, C7.1.49, C11.1.53 and C12.1.49 were chosen
for more extensive research, based on the fact that their transgenic
mother lines T11.1 and T12.1 are backbone-free and the trans-

Table 2
Data of the transformation experiments with their respective amount of explants
used for each experiment, the amount of regenerated transgenic shoots and the
transformation efciency.
Transformation

# Explants

# Transgenic shoots

Transformation
efciency (%)

7
8
9
10
11
12

250
380
250
250
250
255

4
3
0
0
2
1

1.6
0.8
0
0
0.8
0.4

Total

1635

10

0.6

genic mother line T7.1 was chosen as a transgenic line carrying

HcrVf2 and backbone DNA. Expected fragments of the two genes
(HcrVf2 and codA) were detected in the plasmid (pMF1) and in
the DNA isolated from the transgenic mother lines. PCR analysis
of DNA, isolated from shoots obtained after the recombinase activating DEX treatment (C7.1.49, C11.1.53, C12.1.49), indicated the
successful removal of the fusion marker gene codA/nptII, which is
located between the recombination sites. HcrVf2 was still present,
conrming the cisgenic character of the regenerated plants. In the
untransformed in vitro cv. Gala, neither the transgenes nor the specic band for the cisgene HcrVf2 were detected (Fig. 3). RT-PCR was
performed for the HcrVf2 gene and fusion marker gene codA/nptII,
showing that the HcrVf2 gene is expressed in trans- and cisgenic
lines and the expression of the marker gene can only be detected in
the transgenic lines, no expression of the marker gene is observed
in the cisgenic lines (Fig. 4). Backbone presence was investigated
through the genes trfA and nptIII. The presence of one of these genes
indicates backbone integration. PCR analysis of transgenic line T7.1
shows us bands for trfA and nptIII, but trfA and nptIII are absent
in its cisgenic line C7.1.49. The lack of amplied products in the
other trans- and cisgenic lines (T11.1, C11.1.53, T12.1 and C12.1.49)
indicates the absence of trfA and nptIII (Fig. 5).
The loss of the backbone from T7.1 to C7.1.49 has been investigated more in detail by testing other putative cisgenic sublines
of T7.1 (C7.1.17a, C7.1.63 and C7.1.3 g) with the primer pair,
pmf bb3/pmf bb4, located on the backbone close to the RB. Results
show that during the recombination step toward a putative cisgenic line, two different recombination genotypes occurred. In the
rst genotype (C7.1.49 and C7.1.3 g) the backbone has been cut out
along with the marker gene and in the second genotype (C7.1.63
and C7.1.17a) the backbone was still present but the marker gene
was absent (Fig. 6).
Southern blot analysis with nptII probe of the transgenic lines
(T7.1, T11.1 and T12.1), from which the three selected cisgenic lines
originated, showed single integration events of the T-DNA in all
three lines (Fig. 7).
The chosen lines were micro-grafted on M9 rootstocks grown
in the greenhouse for future phenotypic screening.

4. Discussion
Schouten et al. (2006) dened the concept of cisgenic plants
whereby all introduced genes and control sequences are derived
from a crossable donor plant and sparked a long, still ongoing debate involving numerous articles or letters (Schubert and
Williams, 2006; Van Bueren et al., 2007; Houdebine, 2007; Jacobsen
and Schouten, 2008; Jacobsen and Nataraja, 2008; Akhond and
Machray, 2009). The cisgenic approach found most interest in dealing with plant diseases of crops which are vegetative propagated,
directly consumed and have quality aspects related to a particular
cultivar. Cisgenes are being discussed in articles with its potential
use in potato against potato late blight (Jacobsen, 2007; Kuhl et al.,

308

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

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 was
amplied 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 included
cv. 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 controls
and 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 negative
controls. pMF1 (plasmid with HcrVf2) was used as positive control for the PCR reaction.

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

309

Fig. 5. Backbone integration. Polymerase chain reaction analysis using primers specic for trfA (a) and nptIII (b) to detect backbone integration. pMF1 (plasmid with HcrVf2)
was used as positive control. Cvs. Gala and Florina, two untransformed plants and water were used as negative control.
Table 3
Frequencies of the possible genotypes after recombination for each transgenic mother line; assessed by PCR on HcrVf2, codA and trfA.
Transgenic
mother lines

# Shoots
tested

% HcrVf2: +
codA: +
trfA: +

% HcrVf2: +
codA: +
trfA:

% HcrVf2: +
codA:
trfA: +

% HcrVf2: +
codA:
trfA:
cisgenic

% Agro bacterium
contamination

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

N/A
36
36
18
18
36
9
126
36
108

N/A
0
0
0
44
0
0
0
6
0

N/A
0
0
0
0
0
0
0
0
24

N/A
3
22
72
0
100
100
0
88
0

N/A
94
78
22
50
0
0
81
6
74

N/A
3
0
6
6
0
0
19
6
2

2007; Haverkort et al., 2008; Park et al., 2009), in apple against

apple scab (Gessler et al., 2009; Joshi et al., 2009; Joshi, 2010) and
in downy mildew of melon (Benjamin et al., 2009).
So far, no cisgenic crops have been reported in scientic literature that satisfy the denition of Schouten et al. (2006). Lately,
experiments have been performed to evaluate the use of additional
native genes (cisgenes) in poplar to enhance the growth rate (Han
et al., 2010; Strauss et al., 2009), however without developing a
cisgenic poplar line.
Therefore, this is the rst report of the development of a cisgenic
crop tting the denition of Schouten et al. (2006). Our cisgenic
Gala lines were shown to contain the apple scab resistance gene
HcrVf2, which induces resistance to apple scab (Barbieri et al., 2003;
Belfanti et al., 2004; Joshi, 2010; Szankowski et al., 2009), and lacking any foreign genes.
The above results proof the functionality of the method. However, the high degree of backbone integration (80%) observed in this
method could pose a considerable problem.
From the analysis of the transgenic mother lines (T7.1, T11.1,
T12.1) we deduce that there is only one copy of the T-DNA integrated into the plant genome.
The DEX treatment which activates the recombinase gene is not
impairing apple explant regeneration. The subsequent elimination

of the foreign genes, i.e. the fusion marker gene and the R recombinase gene, occurred as expected, with exception of the lines T8.1,
T11.2 and T12.1. The negative selection with 5-FC showed a better recovery of putative cisgenic shoots when a delayed negative
selection strategy was used, but overall no negative effects were
observed on the growth of the shoots.

Fig. 6. Analysis of two different genotypes after recombination of transgenic line

T7.1 using the primers pmf bb3/pmf bb4. pMF1 (plasmid with HcrVf2) was used as
positive control. Untransformed cv. Gala and water were used as negative control.

310

T. Vanblaere et al. / Journal of Biotechnology 154 (2011) 304311

Fig. 7. Copy number analyses by Southern blot hybridisation. Genomic DNA of trans- and cisgenic lines were digested by XhoI, run on agarose gel, transferred to a membrane
and the blotted membrane was hybridized using digoxygenin-labeled PCR probe on nptII. pMF1 (plasmid with HcrVf2) was used as positive control and cv. Gala as negative
control for the nptII gene.

We mention the appearance of two genotypes after recombination of line 7.1. A possible hypothesis could be the fact that a
read-through has happened during T-DNA integration that carries
on over the whole backbone of the vector and continues over the
right border (Wenck et al., 1997; Twyman et al., 2002), inserting
another copy of the recombination site. Therefore three recombination sites can lead to two possible recombination events, the whole
backbone with the cassette containing the marker gene could be cut
out during the recombination step or only the cassette containing
the marker gene could be cut out, leaving the backbone present.
In this research we established the marker-free system with the
pMF1 vector in cv. Gala which has led to the development of three
cisgenic lines (C7.1.49, C11.1.53, C12.1.49), containing only the
gene of interest, HcrVf2, through a transformation method based
on recombination. The motherlines of these cisgenic lines contain
only one T-DNA insert and the gene is expressed in all three lines.
The appearance of the cisgenic plants, grafted on M9 rootstocks,
in the greenhouse is not distinguishable from untransformed cv.
Gala. Further analyses will be performed on these cisgenic lines in
order to conduct a full biosafety assessment and an assessment of
the effectiveness of the introduced resistance. Cisgenesis is a very
promising strategy to bring us a step closer to transforming plants
without adding new traits to the gene pool and making genetically
engineered plants more consumer friendly.
Acknowledgements
The authors wish to acknowledge the nancial support by the
Swiss National Science Foundation NRP59 and COST Action 864.
Contributions of Caterina Matasci are gratefully acknowledged.
References
Akhond, M.A.Y., Machray, G.C., 2009. Biotech crops: technologies, achievements and
prospects. Euphytica 166, 4759.
Barbieri, M., Belfanti, E., Tartarini, S., Vinatzer, B.A., Sansavini, S., SilfverbergDilworth, E., Gianfranceschi, L., Hermann, D., Patocchi, A., Gessler, C., 2003.
Progress of map-based cloning of the Vf-resistance gene and functional verication: preliminary results from expression studies in transformed apple.
Hortscience 38, 329331.
Belfanti, E., Silfverberg-Dilworth, E., Tartarini, S., Patocchi, A., Barbieri, M., Zhu, J.,
Vinatzer, B.A., Gianfranceschi, L., Gessler, C., Sansavini, S., 2004. The HcrVf2 gene
from a wild apple confers scab resistance to a transgenic cultivated variety.
Proceedings of the National Academy of Sciences of the United States of America
101, 886890.
Benjamin, I., Kenigsbuch, D., Galperin, M., Abrameto, J.A., Cohen, Y., 2009. Cisgenic
melons over expressing glyoxylate-aminotransferase are resistant to downy
mildew. European Journal of Plant Pathology 125, 355365.
Gardiner, S.E., Bus, V.G.M., Rusholme, R.L., Chagn, D., Rikkerink, E.H.A, 2007. Apple.
In: Kole, C. (Ed.), Genome Mapping and Molecular Breeding in Plant, Volume 4.
Fruits and Nuts. Springer-Verlag, Berlin, Heidelberg.
Gaskell, G., Allum, N., Bauer, M., Durant, J., Allansdottir, A., Bonfadelli, H., Boy, D.,
de Cheveigne, S., Fjaestad, B., Gutteling, J.M., Hampel, J., Jelsoe, E., Jesuino, J.C.,
Kohring, M., Kronberger, N., Midden, C., Nielsen, T.H., Przestalski, A., Rusanen, T.,

Sakellaris, G., Torgersen, H., Twardowski, T., Wagner, W., Olofsson, A., Oehman,
S., 2000. Biotechnology and the European public. Nature Biotechnology 18,
935938.
Gessler, C., Patocchi, A., 2007. Recombinant DNA technology in apple. Advances in
Biochemical Engineering/Biotechnology 107, 113132.
Gessler, C., Vanblaere, T., Szankowski, I., Broggini, G., 2009. Cisgenic approach to
disease resistance in Apple. Phytopathology 99, S42.
Han, K.M., Dharmawardhana, P., Arias, R.S., Ma, C., Busov, V., Strauss, S.H., 2010.
Gibberellin-associated cisgenes modify growth, stature and wood properties in
Populus. Plant Biotechnology Journal 9, 162178.
Haverkort, A.J., Boonekamp, P.M., Hutten, R., Jacobsen, E., Lotz, L.A.P., Kessel, G.J.T.,
Visser, R.G.F., van der Vossen, E.A.G., 2008. Societal costs of late blight in
potato and prospects of durable resistance through cisgenic modication. Potato
Research 51, 4757 (Special Issue: Late Blight and Genetic Modication).
Hood, E.E., Gelvin, S.B., Melchers, L.S., Hoekema, A., 1993. New agrobacterium helper
plasmids for gene-transfer to plants. Transgenic Research 2, 208218.
Houdebine, L.M., 2007. Transgnique ou cisgnique: quel niveau de risque? Cahiers
Agricultures 16, 6364.
Jacobsen, E., 2007. Cisgenesis: next step in advanced traditional potato breeding.
Potato Production and Innovative Technologies, 348352.
Jacobsen, E., Nataraja, K.N., 2008. Cisgenics facilitating the second green revolution
in India by improved traditional plant breeding. Current Science 94, 13651366.
Jacobsen, E., Schouten, H.J., 2008. Cisgenesis, a new tool for traditional plant breeding, should be exempted from the regulation on genetically modied organisms
in a step by step approach. Potato Research 51, 7588 (Special Issue: Late Blight
and Genetic Modication.).
Joshi, S.G., 2010. Towards durable resistance to apple scab using cisgenes. Wageningen, The Netherlands: Wageningen University, PhD Thesis.
Joshi, S.G., Soriano, J.M., Schaart, J.G., Broggini, G.A.L., Szankowski, I., Jacobsen, E.,
Krens, F.A., Schouten, H.J., 2009. Approaches for development of cisgenic apples.
Transgenic Plant Journal 3, 4046.
Kuhl, J.C., Zarka, K., Coombs, J., Kirk, W.W., Douches, D.S., 2007. Late blight resistance
of RB Transgenic potato lines. Journal of the American Society for Horticultural
Science 132, 783789.
Laurens, F., 1998. Review of the current apple breeding programmes in the world:
objectives for scion cultivar improvement. Acta Horticulture 484, 163170.
Lespinasse, Y., 1989. Breeding pome fruits with stable resistance to diseases. 3.
Genes, resistance mechanisms, present work and prospects. IOBC (WPRS) Bull.
2, 100115.
MacHardy, W.E., 1996. Apple Scab Biology, Epidemiology and management. APS
Press, St. Paul, Minnesota.
MacHardy, W.E., Gadoury, D.M., Gessler, C., 2001. Parasitic and biological tness of
Venturia inaequalis: relationship to disease management strategies. Plant Disease 85, 10361051.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays
with tobacco tissue cultures. Physiologia Plantarum 15, 473497.
Park, T.H., Vleeshouwers, V.G.A.A., Jacobsen, E., Van Der Vossen, E., Visser, R.G.F.,
2009. Molecular breeding for resistance to Phytophthora infestans (Mont.) de
Bary in potato (Solanum tuberosum L.): a perspective of cisgenesis. Plant Breeding
128, 109117.
Rommens, C.M., Haring, M.A., Swords, K., Davies, H.V., Belknap, W.R., 2007. The intragenic approach as a new extension to traditional plant breeding. Trends in Plant
Science 12, 397403.
Schaart, J.G., Krens, F.A., Pelgrom, K.T.B., Mendes, O., Rouwendal, G.J.A., 2004. Effective production of marker-free transgenic strawberry plants using inducible
site-specic recombination and a bifunctional selectable marker gene. Plant
Biotechnology Journal 2, 233240.
Schouten, H.J., Krens, F.A., Jacobsen, E., 2006. Do cisgenic plants warrant less stringent oversight? Nature Biotechnology 24, 753.
Schubert, D., Williams, D., 2006. Cisgenic as a product designation. Nature Biotechnology 24, 13271329.
Strauss, S.H., Busov, V., Ma, C., Van Wormer, K., 2009. Cisgenic and intragenic
approaches to genetic modication of growth and form in poplar. In Vitro Cellular & Developmental Biology-Animal 45, S24S25.