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Received: 27 February, 2010. Accepted: 21 August, 2010.
Original Research Paper
Genes, Genomes and Genomics ©2011 Global Science Books
Towards the Production of Genetically Modified
Strawberries which are Acceptable to Consumers
Jan G. Schaart
1*
•
Trygve D. Kjellsen
2
•
Lisbeth Mehli
3
•
Reidun Heggem
4
•
Tor-Henning Iversen
5
•
Henk J. Schouten
1
•
Frans A. Krens
1
1
Wageningen UR Plant Breeding, Wageningen University and Research Centre. P.O. Box 16, 6700 AA Wageningen, the Netherlands
2
Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
3
Faculty of Technology, Sør-Trøndelag University College, 7004 Trondheim, Norway
4
Centre for Rural Research, 7491 Trondheim, Norway
5
Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Corresponding author: * jan.schaart@wur.nl
ABSTRACT
This manuscript discusses different aspects that are relevant to genetically modified strawberry plants with improved characteristics and
‘acceptable’ to consumers and growers of strawberry. It starts with a consumer acceptance survey, held in Norway, Denmark and the UK,
studying public perception of genetic modification in general and specifically of genetically modified strawberries with altered properties.
This study revealed that genetically modified plants are better accepted by consumers if only genes from the species itself are used for the
genetic modification. Subsequently, the results of a functional analysis of the strawberry polygalacturonase inhibiting protein gene
(FaPGIP) are described. This indicates that this gene is a possible candidate to induce resistance to Botrytis cinerea when upregulated in
strawberry fruits. For this analysis, the FaPGIP gene was overexpressed in transgenic strawberry plants using the cauliflower mosaic
virus 35S (CaMV35S) promoter. This showed that FaPGIP overexpression led to resistance to Botrytis in transgenic leaves. For the
generation of intragenic (i.e. genetically modification using native genetic elements only) strawberry plants, a transformation vector was
constructed in which FaPGIP was combined with a strawberry fruit-specific promoter and terminator that were isolated from a strawberry
expansin gene (FaExp2). This vector also included elements that allow the elimination of (foreign) selectable marker genes after
genetically modified plant lines have been established. Using this vector, genetically modified strawberry plants were produced that
contained only genes from the species itself, and therefore these plants were called intragenic, rather than transgenic. Unfortunately,
further evaluations of the intragenic strawberry plants could not demonstrate any enhanced level of resistance to Botrytis in fruits.
_____________________________________________________________________________________________________________
Keywords: genetic modification, intragenic, consumer acceptance, polygalacturonase inhibiting protein gene (FaPGIP), soft rot
resistance, Botrytis cinerea
Abbreviations: FaPGIP, strawberry polygalacturonase inhibiting protein gene; FaExp2, strawberry expansin gene; CaMV35S, cauli-
flower mosaic virus 35S
INTRODUCTION
Breeding for improvement of strawberry is difficult. Many
traits, such as disease resistances, firmness and vulnerability
of the fruit, productivity and of course its taste, have to be
considered in the selection of a successful strawberry culti-
var. In addition, genetic variation in Fragaria. x ananassa
is very limited, while genetic variation is a prerequisite for
progress in conventional breeding. Furthermore, breeding is
hampered because strawberry is an octoploid, hybrid spe-
cies, originating from a rather recent cross between two
wild octoploid Fragaria species, F. virginiana and F. chilo-
ensis (Darrow 1966). The complicated genetic constitution
of the strawberry genome has kept most researchers from
investing in the development of methods that could improve
breeding of strawberry. Only a few years ago, the first re-
sults towards the production of a genetic map for strawberry
have been published (Haymes et al. 2000; Lerceteau-Kohler
et al. 2003), opening up possibilities for molecular marker-
assisted breeding.
Another example of modern breeding technologies is
genetic modification. In strawberry, the first genetic modi-
fication protocols were developed in the early 90ties (James
et al. 1990; Nehra et al. 1990a, 1990b) and this approach
has gained increasing interest over the last decade (Debnath
and Teixeira da Silva 2007). In principle, genetic modifica-
tion allows a relatively quick improvement of existing im-
portant strawberry cultivars, for example, by the introduc-
tion of disease resistance genes. However, the availability
of suitable genes and specific regulatory sequences that will
result in desired improvements has been the rate-limiting
step until recently. Identification and isolation of such genes
and sequences still requires specific investments, but comes
more and more within our reach with the ever increasing
power of DNA sequencing techniques. Furthermore, the
public attitude toward genetically modified crops in general
is, at least in Europe, still sceptic, hampering the introduc-
tion of genetically modified strawberries in the immediate
future. In addition to this, strict regulations, like the EU
Directive 2001/18/EC, require very expensive testing to
warrant environmental and food safety, and thereby limit
the use of this modern technology by small and medium-
sized enterprises. Nevertheless, for many important crops
transformation methods have been developed, many
genetically improved lines have been produced and several
transgenic crops have been commercialized and are grown
on a world-wide scale (ISAAA 2008).
CONSUMER ACCEPTANCE OF INTRAGENIC
CROPS
In the multidisciplinary EU-project entitled ‘Sustainable
production of transgenic strawberry plants. Ethical conse-
quences and potential effect on producers, environment and
®
Genes, Genomes and Genomics 5 (Special Issue 1), 102-107 ©2011 Global Science Books
consumers’ (QLK5-CT-1999-01479) one of the aims was to
produce genetically modified strawberry plants with en-
hanced levels of resistance towards B. cinerea. This would
be attained by enhancing the expression level of the PGIP
(polygalacturonase inhibiting protein) gene which was
known to give resistance towards Botrytis in transgenic
tomato plants in which a PGIP gene from pear was intro-
duced (Powell et al. 2000). To enhance consumer and pro-
ducer acceptance of genetically modified strawberry plants,
it was considered desirable that only genes and regulatory
elements from strawberry itself were used for the improve-
ment and that the ultimate genetically modified strawberry
plants were completely free of any foreign regulatory and
coding DNA sequences. Nielsen (2003) introduced the term
intragenesis for this condition. In case solely species-own
DNA is used for the genetic modification of a plant, he pro-
posed to call such plants intragenic rather than transgenic.
Rommens (2004) and Rommens et al. (2004, 2007) elabo-
rated on this topic in several articles in which they reviewed
crop improvement using the plants own DNA only. In the
EU-project mentioned above, also the attitude of consumers
toward genetic modification in general, and particularly
towards genetically modified strawberries, was monitored
(study performed in 2002-2003). In this survey it was
shown that the attitude of consumers in Norway, Denmark
and the UK towards genetic modification in general was
rather negative (Fig. 1A), but in more specific cases, regar-
ding genetically modified strawberry plants that had under-
gone different hypothetical modifications, consumer accep-
tance increased when traits beneficial to consumers could
be introduced (Fig. 1B). Furthermore, it was shown that
modifications involving the use of strawberry-own DNA
exclusively (Fig. 1C). This latter finding was confirmed by
a consumer’s survey in the USA, which showed that the
majority of the respondents would eat vegetables with an
extra gene from the same species or from another vegetable
species, while this was only a minority in case viral genes
had been used (Lusk and Sullivan 2002; Lusk and Rozan
2006).
GENETIC MODIFICATION USING SPECIES-OWN
DNA SEQUENCES
The above mentioned sociological studies suggested rela-
0%
10%
20%
30%
40%
50%
worse
Will lead to
improvements
No effect Make things Do not know
A
Consumers’ attitudes towards genetic modification in general
0%
20%
40%
60%
80%
100%
R
i
sk for environm ent
Last longer
B
i
gger
and
r
edder
20% cheaper
Tast
e
bet
t
er
Less pes
t
i
c
ides
Organic principl
es
H
ealthier
Would not buy
Would buy
B
Would you buy genetically modified strawberries rather than
conventional strawberries if the modified strawberries were....
0%
10%
20%
30%
40%
50%
Completely
agree
Partly agree Partly
disag ree
Completely
disagre e
Do not know
C
Statement: It is more acceptable that one moves genes inside a
species rather than moving them between different species
Fig. 1 Sociological inquiry among 720 consumers in Norway, Denmark and the UK.
103
Intragenic strawberries. Schaart et al.
tively high levels of public acceptance of genetically modi-
fied crop plants that have only genes from the species itself
or from a cross-compatible species. In such genetically
modified crop plants the introduction of native DNA se-
quences is referred to as intragenesis or cisgenesis. In cis-
genesis the newly introduced DNA is a natural genome
fragment, containing a gene of interest together with its
own introns, 5- and 3-untranslated regions and regulatory
elements (promoter and terminator) (Schouten et al. 2006).
Like cisgenesis, intragenesis also uses donor gene sequen-
ces from the species itself or from a natural crossable donor
species, but in intragenesis new genes can be created by
combining functional genetic elements such as promoters,
coding parts (with or without introns) and terminators of
different natural genes, and insert this new chimeric gene
into existing varieties (Rommens 2004; Rommens et al.
2004; Rommens 2007; Schouten and Jacobsen 2008).
ISOLATION AND CHARACTERISATION OF
STRAWBERRY PGIP
For the ultimate production of intragenic or cisgenic crops
the availability of specific genes and regulatory sequences
within a species is a prerequisite. Up to date, for a number
of plant species the complete genome sequence is available
or will become available soon, which facilitates identifica-
tion and isolation of the required gene and promoter se-
quences. However, for most crop species up till now, only
limited information on genes and regulatory sequences is
available and approaches like amplification using degene-
rated primers for the isolation of new genes and genome
walking for the isolation of desired promoter and terminator
sequences have to be employed (Agius et al. 2005). After
isolation of species-specific gene and regulatory sequences,
accurate functional characterisation of the sequences needs
to be performed, in order to be able to anticipate the effects
of the envisaged modification.
For the aimed introduction of B. cinerea resistance in
strawberry, we focussed on the FaPGIP gene sequences
from strawberry. Plant-pathogenic fungi, like Botrytis, pro-
duce cell wall degrading enzymes with which they attack
the plant. Studies have shown that PGIP from a variety of
origins is able to inhibit B. cinerea polygalacturonase (a cell
wall degrading enzyme) activity in vitro (Sharrock and
Labavitch 1994; Yao et al. 1995). It was also shown that
introduction of a PGIP from pear into transgenic tomato
plants resulted in an enhanced level of resistance towards B.
cinerea (Powell et al. 2000). Richter et al. (2006) and Janni
et al. (2008) also showed that overexpression from PGIP of
raspberry or bean in transgenic pea and wheat, respectively,
increased resistance to infections by fungal pathogens.
Finally, the important role of PGIP in conferring resistance
to Botrytis was demonstrated by antisense expression of
PGIP in Arabidopsis, which reduced accumulation of PGIP
and subsequently resulted in an enhanced susceptibility to
Botrytis (Ferrari et al. 2006). This information suggested
that for strawberry, overexpression of the PGIP gene would
be a suitable option to achieve an enhanced Botrytis resis-
tance level.
We isolated and characterised a PGIP gene from straw-
berry (Mehli et al. 2004; Schaart et al. 2005) and showed
th
at in the natural situation this FaPGIP
was expressed at
relatively low level in leaves and immature fruit tissue, but
that it was upregulated during strawberry fruit ripening.
Inoculation of fruits with B. cinerea spores led to a rapid
upregulation of FaPGIP expression to a level that, depen-
ding on the strawberry cultivar tested, was 4-40 times
higher than found for the control red fruits. This upregula-
tion was however transient and FaPGIP was downregulated
again two days after inoculation. These observations
prompted us to aim at modifying FaPGIP gene expression
in such a way that sufficient FaPGIP activity would be pre-
sent in B. cinerea susceptible tissues and stay present.
For functional analysis of FaPGIP in strawberry, we
produced transgenic strawberry plants in which FaPGIP
was overexpressed using the constitutive CaMV35S promo-
ter. Because this promoter provides strong expression in
strawberry leaf tissue (Schaart et al. 2011), its use allows
early screening of B. cinerea resistance in transgenic straw-
berry leaf tissue. Inoculation of detached leaves of straw-
berry plants with B. cinerea showed that for a certain num-
ber of these transgenic plants, inoculation did not result in a
significantly different reaction as compared to control
(water) inoculations on the same leaf (Fig. 2), indicative for
enhanced resistance. For non-transgenic control plants as
well as for some of the transgenic plants, inoculation with B.
cinerea resulted in a clear destruction of leaf tissue giving
significantly larger lesions than the control (water) inocu-
lations. These results indicated that overexpression of
FaPGIP was able to confer resistance to B. cinerea in trans-
genic strawberry plants, at least in leaf tissue. The cor-
relation between the level of resistance to B. cinerea and ex-
pression pattern and levels of FaPGIP was not investigated
in these plants.
Because our ultimate aim was to achieve intragenic
rather than transgenic strawberry lines, we did not induce
flowering and fruiting of the transgenic plants in which the
CaMV35S promoter was used to drive FaPGIP expression.
A
-0.5
0
0.5
1
1.5
2
NT
1-04
1-07
1-08
1-21
1-23
1-24
1-27
1-28
1-29
2-02
2-04
2-08
2-22
2-23
2-27
*
*****
Increased lesion size (mm)
after B. cinerea
inoculation,
compared to water inoculation
B
NT
1-27
1-21
1-28
water
spores
Fig. 2 B. cinerea colonisation test on detached leaves of non-transgenic
control (NT) and genetically modified strawberry plants transformed
with a construct containing FaPGIP under the regulation of the
CaMV 35S promoter. Detached leaves were wounded with a needle,
giving an approximately 1 mm diameter lesion. Two μl (10
5
spores/ml) of
germinating B. cinerea spores (line BCNL) were pipetted on each wound.
The left half of the leaf was inoculated with spores, while the right half
was inoculated with water. For each transgenic line 3 leaves were inocu-
lated at six positions per inoculum (spores vs. water) per leaf. Leaves were
incubated in separate containers for 7 days, after which the diameter of
each lesion was measured. (A) Example of B. cinerea-inoculated leaves, 7
days after inoculation. NT = non-transgenic control; 1-21, 1-27 and 1-28
are leaves from three different transgenic lines. (B) Average differences in
lesion size (mm) of non-transgenic control (NT) and several transgenic
lines. Difference is calculated with respect to the average of all water con-
trol lesion diameters (1.95 mm; SD= 0.54). Statistical analysis was done
with two-way ANOVA. Transgenic lines marked with an asterisk differ
significantly from the non-transgenic control at P-values < 0.05.
104
Genes, Genomes and Genomics 5 (Special Issue 1), 102-107 ©2011 Global Science Books
SELECTION OF SUITABLE STRAWBERRY GENE
PROMOTER
In strawberry, primary B. cinerea infections take place
through the flower after which the fungus remains latent in
immature fruits. Once the strawberry fruit ripens, B. cinerea
causes fruit rot which subsequently can lead to secondary
infections of the so far unaffected other ripe and unripe
fruits. In order to restrain B. cinerea in an effective way,
FaPGIP upregulated expression should be extended at least
into the ripe fruit stage, but preferentially also in flowers
and immature fruits. In order to achieve an effective
FaPGIP expression pattern, specific promoter sequences
had to be identified. Initially, for a transgenic approach we
focussed on the heterologous CaMV35S and the petunia
fbp7-promoter sequences that were already available, and
we tested these promoter sequences for their expression
pattern in transgenic strawberry plants (Schaart et al. 2002).
Both promoter sequences seemed to be able to direct ex-
pression of the -glucuronidase reporter gene in flowers as
well in different developmental fruit stages, and are, there-
fore, suitable to induce the intended upregulation of
FaPGIP. However, to follow the intragenic approach, suita-
ble promoter sequences have to be isolated from strawberry
itself. For this purpose, a strawberry expansin gene, FaExp2,
that showed fruit ripening-specific expression (Civello et al.
1999; Aharoni et al. 2002; Salentijn et al. 2003) was selec-
ted and its promoter was isolated and characterized using
transgenic plants in which the promoter was fused to a gus
reporter gene (Schaart et al. 2011). It was shown that the
FaExp2 promoter fragments regulated gus expression in a
fruit-specific way, which was in agreement with the des-
cribed FaExp2 expression pattern. Interestingly, plants with
the 1.6 Kb FaExp2-promoter fragment showed a much
higher gus expression than a shorter 0.7 Kb FaExp2-pro-
moter fragment. In order to achieve high levels of FaPGIP
expression for inhibition of B. cinerea in the ultimate intra-
genic strawberry plants, the 1.6pFaExp2-fragment was con-
sidered to be most suitable and was subsequently chosen for
further experimentation.
USE OF SELECTABLE MARKER-REMOVAL
SYSTEM
For the efficient production of genetically modified plants
the use of selectable marker genes is a prerequisite. In many
transformation protocols either herbicide or antibiotic resis-
tance genes have been shown to act as very effective selec-
table markers for genetically modified tissue and they have
found wide application. However, public debate concerning
health and environmental risks has focused particularly on
such resistance genes, which make them undesirable in the
final products. The public concerns have resulted in the
development of selection methods which make use of alter-
native, less objectionable selectable marker genes. Such
genes are mostly genes of bacterial origin, like the phospho-
mannose-isomerase gene which enables transgenic plants to
proliferate on mannose, which cannot be metabolised by
many plant species (Joersbo et al. 1998).
Next to the use of alternative selectable marker genes,
systems have been developed which allow the elimination
of selectable marker genes after they have been used. Such
a marker removal system is especially valuable for vege-
tatively propagated crops, like strawberry, and for crops
with long reproductive cycles. In view of the higher level of
acceptance of genetically modified plants which are devoid
of foreign gene sequences, the use of elimination systems is
pref
erable to the use of alternative selectable marker genes.
We therefore developed and tested a recombinase based
system for elimination of undesired DNA sequences in
strawberry (Schaart et al. 2005, 2010). We demonstrated
that this method could be applied effectively using our stan-
dard strawberry transformation protocol and that by marker
removal, marker-free plants could effectively be produced.
PRODUCTION OF INTRAGENIC STRAWBERRY
PLANTS
In the end, the combined use of all aspects described above,
the strawberry PGIP gene to confer resistance to Botrytis,
the strawberry fruit-specific promoter from the FaExp2
gene to direct gene expression to high levels in strawberry
fruits and a marker-removal system for elimination of for-
eign DNA sequences from the predestined intragenic plants,
enables the production of genetically modified plants which
contain only gene and promoter sequences from strawberry
itself. To demonstrate the possibility of producing such
intragenic plants, we constructed a transformation vector in
which FaPGIP was combined with regulatory sequences of
FaExp2. For this, next to the 1.6 kb promoter also a 500 bp
sequence fragment which is flanking the 3-end of FaExp2
was isolated and was used as terminator sequence (tExp2).
The 1.6pFaExp2-FaPGIP-tFaExp2 chimeric gene was then
introduced in the binary vector pMF1 for production of
marker-free genetically modified plants (Schaart et al.
2011) (Fig. 3). In this binary vector an inducible recom-
binase gene and the bifunctional selectable marker gene are
flanked by recombination sites. Chemical induction of re-
combinase activity enables recombination mediated remo-
val of undesired gene sequences at the desired point in time.
For a detailed description of the pMF1 vector and of the
marker removal protocol, see Schaart et al. (2004, 2010).
Using this vector for transformation of strawberry and for
successive removal of the selectable marker and recom-
binase gene from the transgenic plants that were obtained,
resulted in 14 putative intragenic strawberry plants. PCR
analysis showed that in 11 out of 14 of these plants the new
1.6pFaExp2-FaPGIP-tFaExp2 gene combination was pre-
sent and that the selectable marker gene was successfully
removed (data not shown) and that these plants could be
labelled as intragenic. The presence of binary vector DNA
(which is of foreign origin) was not checked in these puta-
tive intragenic plants. In similar experiments using a pMF1-
based vector in strawberry transformation demonstrated
however, that in a considerable number of transformed
plants (up to 50%) pMF1 vector backbone sequences were
pMF1-
pFaExp2-FaPGIP-tFaExp2
17147 bps
RK2
ColE1
nptIII
trfA
RS
pCaMV35S
CodA-
NptII
tNos
pCaMV35S
Recombinase R-LBD
tNos
RS
tFaExp2
FaPGIP
p1.6FaExp2
RB
LB
Fig. 3 pMF1 binary vector with the intragene 1.6pFaExp2-FaPGIP-
tFaExp2 for obtaining marker-free GM plants that overexpress
FaPGIP in a fruit-specific way. White boxed sequences are located on
the binary vector backbone. The black and grey boxed sequences are
located on the T-DNA, which is flanked by RB and LB (right and left T-
DNA border sequences, respectively) and which is transferred to the
plants cell and incorporated into the plant genome. The grey boxed se-
quences are flanked by RS, Recombination sites, and these sequences will
be removed after induction of recombinase activity (see Schaart et al.
2011 for detailed explanation).
105
Intragenic strawberries. Schaart et al.
co-integrated with the gene of interest. This result indicates
that the number of true intragenic (marker- and vector back-
bone-free) plants obtained described here is likely to be
lower. Although the aim of the EU project was just to
demonstrate the possibility to produce intragenic strawberry
plants, we obviously were interested in the performance of
the newly introduced FaPGIP gene under the regulation of
the FaExp2 promoter and terminator. For this the intragenic
strawberry plants were transferred to the greenhouse (Fig.
4) for production of fruits for further characterisation. For
evaluation of the level of Botrytis resistance in ripening
fruits, Botrytis spores were injected (50 μl of conidial sus-
pension of 10
5
spores.ml
-1
in fruits at different developmen-
tal stages and fruit rot incidence was monitored one week
after injection of the fruits. Unfortunately, this assay could
not demonstrate any increase in Botrytis resistance in the
intragenic fruits as compared to control fruits. Because we
have not quantified FaPGIP transcript or FaPGIP protein
levels in the intragenic fruits, we cannot conclude whether
the lack of improved resistance was due to poor FaPGIP
expression in the fruits tested or that PGIP alone was in-
sufficient to stop Botrytis colonisation in the intragenic
strawberry fruits or that the number of spores that were
injected was too high to discriminate between resistant and
susceptible.
CONCLUSION
In this short communication different steps have been des-
cribed to come to genetically modified plants in which only
gene sequences from the species itself have been introduced.
To demonstrate the successful production of intragenic
strawberry plants, an intragene was constructed by com-
bining the regulatory properties of the strawberry FaExp2
gene with the functional gene properties of the strawberry
FaPGIP gene. This new gene combination was successfully
introduced into strawberry plants after which the undesired
selectable marker genes, that were essential for the produc-
tion of the genetically modified strawberry plants, were
removed. This resulted ultimately in the production of intra-
genic strawberry plants.
Because the intragenic strawberry plant did not show
the expected phenotype, i.e. enhanced resistance to Botrytis,
other intragenes should be constructed and tested to ulti-
mately reach the goal of producing Botrytis resistant intra-
genic strawberry lines. Cultivating such intragenic straw-
berries will result in reduction of fungicide applications,
which will be favourable to producers, consumers and envi-
ronment, and because of its intragenic nature, it is en-
visaged that such a particular intragenic strawberry will find
good acceptance by producers and consumers of straw-
berries. In the end, the use of intragenic strawberry plants
may lead to a new way of sustainable crop production prac-
tices.
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