ArticlePDF Available
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
Trygve D. Kjellsen
Lisbeth Mehli
Reidun Heggem
Tor-Henning Iversen
Henk J. Schouten
Frans A. Krens
Wageningen UR Plant Breeding, Wageningen University and Research Centre. P.O. Box 16, 6700 AA Wageningen, the Netherlands
Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Faculty of Technology, Sør-Trøndelag University College, 7004 Trondheim, Norway
Centre for Rural Research, 7491 Trondheim, Norway
Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Corresponding author: *
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
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).
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 consumers 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
The above mentioned sociological studies suggested rela-
Will lead to
No effect Make things Do not know
Consumers’ attitudes towards genetic modification in general
sk for environm ent
Last longer
20% cheaper
Less pes
Organic principl
Would not buy
Would buy
Would you buy genetically modified strawberries rather than
conventional strawberries if the modified strawberries were....
Partly agree Partly
disag ree
disagre e
Do not know
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.
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).
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
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.
Increased lesion size (mm)
after B. cinerea
compared to water inoculation
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
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.
Genes, Genomes and Genomics 5 (Special Issue 1), 102-107 ©2011 Global Science Books
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.
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
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.
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
17147 bps
Recombinase R-LBD
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).
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
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
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-
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with the pMF1- 1.6pFaExp2-FaPGIP-tFaExp2..
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... Fruit-specific antisense suppression of the endob-1,4-glucanase (EGase) catalyzing the disassembly of hemicellulose molecules in the cell wall increased strawberry fruits' firmness [12]. Overexpression of the endogenous polygalacturonase-inhibiting protein gene FaPGIP driven by the promoter of the fruitspecific gene FaExp2 was achieved to regulate strawberry's resistance to the fruit rot fungus Botrytis cinerea [13]. Overexpression and CRISPR/Cas9-directed mutagenesis of RAP, a glutathione S transferase (GST) (a carrier transferring anthocyanins from the cytosol into the vacuole) reversed the coloration of strawberry fruits [14]. ...
... Intragenic black carrot has been proposed [40]. Intragenic strawberry was also developed, although the resistance trait had not been improved as expected [13]. ...
... So far, use of the novel intragenesis tool has scarcely been reported in strawberry except to increase fruit resistance to Botrytis cinerea [13]. In addition, although the Agrobacterium-mediated transformation of strawberry is easy to perform, it is challenging to use DNA blotting in determining the transgenic copy number in the octoploid genome. ...
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Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.
... (NBAB, 2020: 40) As we have indicated, this is not supported by a critical reading of the study design and the analysis. It is also claimed in the conclusion that this study, unlike previous studies, shows that the opinions are more nuanced than 'for' or 'against' (NBAB, 2020: 40 nuanced assessments of biotechnology innovations has been repeatedly demonstrated in surveys from the last twenty years (Gaskell et al., 2003, Schaart et al., 2011. ...
Conference Paper
Full-text available The status that charitable food aid resources have attained and the loss of responsibility by the Welfare State for the right to food undermines the latter doubly. In a first step, retrenchment and austerity reduce its operational space, which weakens it in its functions in the face of food insecurity. In a second step, the effect of charitable food aid resources undermines the idea about its redistributive function, which has been a basis for its legitimacy. The main hypothesis of this work is that this double undermining of the welfare state is constantly reinforced. It defends, through a logical-critical reflection based on specialized bibliography, that charitable food aid resources are a threat to the fundamental ethos of the welfare state, to its ability to respond to collective problems. The retrenchment and the austerity have strengthened the charitable food resources. Organizationally, the fact of not reaching the objective for which they arose, that is to say, ending food insecurity, paradoxically, reinforces them. Therefore, situations such as those triggered by Covid reinforce them even more. Closing the circle of dynamics, the effects of the work of these resources, where collective solidarity is channelled away from the traditional public redistributive mechanisms, represents a new motive of erosion. The main conclusion is that this phenomenon generates feelings of guilt or shame, and stigmatizes the recipients of food aid. It expels these people in vulnerable situations from the normalized consumption circuit. On the other hand, the institutionalization of food aid resources distracts from the origin of poverty, hides the true reasons for food need. Despite all this, it is important to bear in mind that it is the only resource available to many food-insecure people.
... It is also claimed in the conclusion that this study, unlike previous studies, shows that the opinions are more nuanced than 'for' or 'against' (NBAB, 2020: 40). However, this is not new as tendency towards nuanced assessments of biotechnology innovations has been repeatedly demonstrated in surveys from the last twenty years (Gaskell et al., 2003, Schaart et al., 2011. ...
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The UN’s Sustainable Development Goals saw the global community agree to end hunger and malnutrition in all its forms by 2030. However, the number of chronically undernourished people is increasing continuously. Ongoing climate change and the action needed to adapt to it are very likely to aggravate this situation by limiting agricultural land and water resources and changing environmental conditions for food production. Climate change and the actions it requires raise questions of justice, especially regarding food security. These key concerns of ethics and justice for food security due to climate change challenges are the focus of this book, which brings together work by scholars from a wide range of disciplines and a multitude of perspectives. These experts discuss the challenges to food security posed by mitigation, geoengineering, and adaptation measures that tackle the impacts of climate change. Others address the consequences of a changing climate for agriculture and food production and how the Covid-19 pandemic has affected food security and animal welfare.
... It is also claimed in the conclusion that this study, unlike previous studies, shows that the opinions are more nuanced than 'for' or 'against' (NBAB, 2020: 40). However, this is not new as tendency towards nuanced assessments of biotechnology innovations has been repeatedly demonstrated in surveys from the last twenty years (Gaskell et al., 2003, Schaart et al., 2011. ...
... For the management of Fusarium wilt, chemical control, biological control, crop rotation and use of resistant cultivars are the most popular methods (Koike and Gordon, 2015). However, it has been difficult to develop resistant cultivars for strawberry, because many traits, such as fruit productivity and quality of fruits, have to be considered in the selection of successful resistant strawberry cultivars (Schaart et al., 2011). Moreover, octoploidy (2n = 8× = 56) and heterozygosity impose difficulties when this species is bred according to conventional strategies, and so manipulation on a gene level through transgenic technology is generally also carried out (Husaini, 2010). ...
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This book provides a broad, well-structured review of strawberries and their cultivation under current environmental conditions. Methods of strawberry cultivation have undergone many improvements, and this book covers aspects from plant propagation, architecture, genetic resources, breeding, abiotic stresses and climate change to evolving diseases and their control. The first chapter gives a general introduction to strawberry with some important statistics. It is followed by Section 1 on Genetics, Breeding and Omics, which contains five chapters. Chapters 2 and 3 discuss modern aspects related to available genetic resources and breeding for yield, quality and disease traits. The scope of metabolomics is discussed with special reference to white strawberry ( Fragaria chiloensis subsp. chiloensis f. chiloensis ) in Chapter 4. Chapter 5 is an interesting study on the transcriptomic profile of some key genes in relation to systemic acquired resistance, while chapter 6 discusses different aspects of genetic transformation in strawberry. Section 2 on Cultivation Systems and Propagation contains three chapters. These chapters discuss at length the plant growth, flowering, root growth and architecture, replant problems and plant propagation techniques. Section 3 on Disease and Stress Management contains six chapters dealing with biotic and abiotic stresses of strawberry. The first four chapters discuss traditional and emerging fungal diseases, their diagnosis and modern biointensive management strategies. Chapters 14 and 15 deal with the emerging challenges posed due to climate change and its impact on the changing magnitude and dimensions of abiotic stresses on strawberry. The book is aimed at those involved in strawberry research and development, and also to those who are interested in cultivation of strawberry for commercial gain.
... Additionally, it is difficult to develop resistant cultivars because several traits, such as fruit productivity and quality of fruits, have to be considered during the development of successful resistant cultivars (Schaart et al., 2011). Lemon balm (Melissa officinalis L.), which belongs to the family Lamiaceae, is an important medicinal herb that has been widely used in traditional medicine (Meftahizade et al., 2010). ...
In this study, the in vivo potential of lemon balm water extract on Fusarium wilt control in strawberry and the antifungal properties of secondary metabolites in the extract were investigated. Runner plants of strawberry (Fragaria × ananassa Duch., ‘Sachinoka’) were treated with water extracts (20%, w/v) of lemon balm (Melissa officinalis L.) and inoculated with Fusarium oxysporum f. sp. fragariae (Fof). Four weeks after Fof inoculation, lower disease incidence and indices in both shoots and roots were observed in lemon balm-treated plants. These effects could be attributed to reduced Fusarium populations due to the fungistasis and fungicidal effects induced by the extract in the rhizospheric soil. Consequently, dry weights of shoots and roots in the plants treated with lemon balm extracts were higher than those of the control. Based on the results of ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analyses, rosmarinic acid was the metabolite with the highest concentration and was also the most stable metabolite in the water extract. In addition, the antifungal effect of rosmarinic acid against Fof was confirmed by in vitro tests. Therefore, water extracts of lemon balm could suppress Fusarium wilt in strawberry plants and rosmarinic acid was one of the key metabolites with antifungal properties present in the water extract.
The concept of cisgenesis was born in 1999 in a working group of two Christian political parties in the Netherlands. In this working group it was concluded that when ‘species-own genes’ are used for genetic modification, gene flow from cisgenic crops to wild relatives in vegetations does not lead to genetic contamination of these wild relatives, as the cisgenes have been taken from these wild relatives. Further, the integrity of the species would be less affected in case of cisgenes compared to the use of foreign genes. The word ‘cisgenese’ has been published in a Dutch book from this working group. In a European Research project that started in 2000, cisgenic strawberries were developed for fruit rot resistance. Social scientists in this project showed that cisgenic strawberries are more acceptable for growers and consumers compared to transgenic strawberries. After 2004 the definition of cisgenesis became gradually stricter. In 2009 and 2012 motions for exempting cisgenic crops from the GMO regulation were accepted by the majorities of the Senate and Parliament in the Netherlands. However, this could not be implemented in this country, as the Dutch GMO regulation is based on the European GMO regulation. This chapter describes the birth of cisgenesis, its evolving definition, and discussions exempting cisgenic crops from GMO regulations.KeywordsCisgenesisDefinitionsGMO regulationEthics
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Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.
Improving fruit quality and resistance to pathogens in strawberry by friendly biotechnological approaches represents a major issue to reduce the use of chemicals and pesticides and an important goal for breeding. We have designed new RNAi intragenic silencing constructions by combining specific strawberry promoters and pathogen defence-related candidate genes to silence endogenous target genes in a fruit ripening related manner thus avoiding any unwanted effect on plant growth and development. For this, a ∼3000-bp DNA fragment upstream FvDOF2 and FvFAAT2 coding regions was fused to RNAi designed cassettes for FaWRKY1 and FaNPR3.1 genes. Four different binary plasmid constructions were generated using pMinMYB as a recipient plasmid frame carrying the MdMYB10 gene from a red-fleshed apple as natural selectable marker. The fruit-specific transcription factor FvDOF2 regulates the production of volatile eugenol in ripe fruit receptacles whereas gene FvAAT2 encodes a fruit-related acyltransferase involved in strawberry aroma biogenesis. Also, FaWRKY1 transcription factor negatively regulates resistance to Colletotrichum acutatum in fruit upon infection and FaNPR3.1 is the strawberry orthologue of the Arabidopsis AtNPR3/AtNPR4 salicylic acid (SA) receptors acting as transcriptional co-repressors in plant immunity. Using these plasmid constructs, stable red transformant intragenic strawberry lines silencing target genes are being produced and will be tested for increase resistance to different pathogens. © 2021 International Society for Horticultural Science. All rights reserved.
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‘New Breeding Techniques (NBTs)’ have been one of hot issues, since their future will be affected profoundly by national as well as international regulatory landscapes. In this review, we compare characteristics of NBTs with conventional and genetic modification, and analyze genetically modified organism (GMO) regulatory systems in the context of possible regulation of NBTs. NBTs are very heterogeneous in terms of principles, methodologies, and final products. As Living Modified Organisms (LMO) is defined in the Cartagena Protocol on Biosafety (CPB) as an organism containing novel combination of genetic materials obtained by the use of modern biotechnology, CPB as well as other national legislations locate itself somewhere in the middle between product-based and process-based regulations. It is also noted that jurisdictions with regulatory systems more oriented to product-based one tend to be more productive and decide or may decide to exempt site-directed nucleases-1 from GMO regulation. In this context, Korean legislations are reviewed to clarify the commons and differences in GMO definitions. Act on Transboundary Movement of LMO Act, Food Sanitation Act and Agricultural and Fishery Products Quality Control Act are three major acts to regulate GMOs. It is noted that there are differences in the definition of LMO or GM food/products especially between the LMO Act and the Food Sanitation Act. Such differences may cause conflicts between Acts when policy-decision regarding the regulation of NBTs is made. Therefore, it is necessary to reorganize legislations before policies regarding the regulation of any techniques from biotechnology are made.
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In order to achieve specific expression of transgenes in strawberry fruits, the availability of tissue- (receptacle) specific promoter sequences is desired. For this reason, 5′-upstream sequences of the strawberry expansin gene FaExp2, which is expressed in a fruit-specific manner, have been isolated. To characterise the promoter activity of the isolated sequences, fragments of 0.7 kb (0.7pFaExp2)and 1.6 kb (1.6pFaExp2) have been fused to the β-glucuronidase reporter gene (gus). In transgenic strawberry plants transformed with either 0.7pFaExp2-gus or1.6pFaExp2-gus, a fruit-specific expression pattern was observed for both promoter constructs. However, quantitative RT-PCR revealed that gus expression levels driven by the 1.6pFaExp2 promoter fragment were much more higher. In addition to the expression in fruits, both promoter fragments also seemed to direct gene expression in the achenes and to some extent in epidermal and subepidermal tissues of petioles and stems of flowers and fruits. It is concluded that both promoter sequences are suitable for directing transgene expression in strawberry fruits in a specific way
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The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the -glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.
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Genetic transformation of plants often results in multiple copies of the introduced DNA at a single locus. To ensure that only a single copy of a foreign gene resides in the plant genome, we used a strategy based on site-specific recombination. The transformation vector consists of a transgene flanked by recombination sites in an inverted orientation. Regardless of the number of copies integrated between the outermost transgenes, recombination between the outermost sites resolves the integrated molecules into a single copy. An example of this strategy has been demonstrated with wheat transformation, where four of four multiple-copy loci were resolved successfully into single-copy transgenes.
Introduction Selectable Markers and Public Concern Marker-Free Transformation Technology Transformation without Selectable Marker Specific Issues Associated with Transformation without Selectable Marker Generation of Amylose-Free Potato Lines by Transformation without Selectable Marker Marker Elimination Conclusion Acknowledgment References
Firmness is an important selection criterium in the breeding of fruit, including strawberry (Fragaria × ananassa Duch.). Clear differences in fruit-firmness are observed between cultivars. In order to identify candidate genes which might be associated with such textural differences, gene expression levels were compared for a soft and a firm cultivar (cv. Gorella and cv. Holiday, respectively). DNA-microarrays representing 1701 strawberry cDNAs were used for simultaneous hybridization of two RNA populations derived from red ripe fruit of both cultivars. In total 61 clones (3.6% of the total cDNAs on the arrays) displayed differential expression, including 10 clones (8 different ones) which showed homology to cell wall related genes in the public databases. The results from the microarray experiments were further confirmed by RNA gel blots, which were also used to examine gene expression in a third cultivar, Elsanta, showing an intermediate texture phenotype (offspring of a cross between Gorella and Holiday). Interestingly, two genes encoding proteins catalysing successive reactions in lignin metabolism (cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase) showed the highest difference in expression level.
The influence of polygalacturonase inhibitor protein (PGIP) from mature green Bartlett pears on the action of a mixture of cell wall hydrolases from Botrytis cinerea on isolated pear cell walls, and on a soluble extract of these walls, was studied. The aim of this work was to test the hypothesis of Cervone et al. that PGIP may modify cell wall degradation to result in the production of an increased proportion of pectic oligomers in the particular size class most effective for elicitation of defence responses. Products of the partially inhibited and uninhibited enzyme system from B. cinerea culture supernatant were compared as elicitors of ethylene production by pear fruit cells in suspension culture (which would indicate the presence of oligogalacturonides in the size class known to elicit plant defence responses), and on the basis of direct analytical data relating to oligomer length. Pear PGIP caused partial inhibition of the crude mixture of Botrytis enzymes, and increased the ratio of dimeric to monomeric uronide products. However, no accumulation of larger oligomeric breakdown intermediates was detected, and no impact on ethylene elicitor activity of the digestion products was observed. Differential inhibition of the B. cinerea polygalacturonase isozymes by pear PGIP was demonstrated following their separation by isoelectric focusing. The fact that some isozymes remained relatively uninhibited may have accounted for the absence of any accumulation of oligomeric breakdown intermediates.
Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA.
Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.