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Transgenic strawberry: State of the art for improved traits

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Strawberry (Fragaria x ananassa Duch.), a member of the Rosaceae family, is one of the most important fruit crops cultivated worldwide. Strawberry is unique within the Rosaceae because it is a rapidly growing herbaceous perennial with a small genome, short reproductive cycle, and facile vegetative and generative propagation for genetic transformation. For these reasons, strawberry has been recognized as excellent germplasm for genetic and molecular studies for the Rosaceae family. Although traditional breeding methods have achieved steady improvement in agronomic traits, the lack of useful economic characters still remains a major challenge. Genetic transformation has opened a new era for greater creativity in strawberry breeding and germplasm by offering an effective method for creating new varieties that selectively targets a specific interested gene or a few heterologous traits. Enormous advances have been made in strawberry genetic transformation since the first transgenic strawberry plant was obtained in 1990. This paper reviews recent progress in genetic transformation of strawberry on increasing resistance to viruses, fungi, insects, herbicides, stress, and achieving better quality. Problems and prospects for future applications of genetic transformation in strawberry are also discussed.
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Research review paper
Transgenic strawberry: State of the art for improved traits
Yonghua Qin
a,b,
, Jaime A. Teixeira da Silva
c
, Lingxiao Zhang
d
, Shanglong Zhang
a,
a
Department of Horticulture, Zhejiang University, Hangzhou 310029, China
b
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
c
Department of Horticultural Science, Faculty of Agriculture, Kagawa University, Ikenobe, 761-0795, Kagawa, Japan
d
Delta Research and Extension Center, Mississippi State University, Stoneville, MS38776, USA
Received 4 November 2007; received in revised form 18 December 2007; accepted 18 December 2007
Available online 31 December 2007
Abstract
Strawberry (Fragaria×ananassa Duch.), a member of the Rosaceae family, is one of the most important fruit crops cultivated worldwide. Strawberry
is unique within the Rosaceae because it is a rapidly growing herbaceous perennial with a small genome, short reproductive cycle, and facile vegetative
and generative propagation for genetic transformation. For these reasons, strawberry has been recognized as excellent germplasm for genetic and
molecular studies for the Rosaceae family. Although traditional breeding methods have achieved steady improvement in agronomic traits, the lack of
useful economic characters still remains a major challenge. Genetic transformation has opened a new era for greater creativity in strawberry breeding and
germplasm by offering an effective method for creating new varieties that selectively targets a specific interested gene or a few heterologous traits.
Enormous advances have been made in strawberry genetic transformation since the first transgenic strawberry plant was obtained in 1990. This paper
reviews recent progress in genetic transformation of strawberry on increasing resistance to viruses, fungi, insects, herbicides, stress, and achieving better
quality. Problems and prospects for future applications of genetic transformation in strawberry are also discussed.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Strawberry; Genetic transformation; Germplasm improvement
Contents
1. Introduction ............................................................. 220
2. Establishment of a genetic transformation system of strawberry ................................... 220
3. Transgenic strawberry for insect resistance .............................................. 222
4. Transgenic strawberry for phytopathogenic fungi, bacteria and virus resistance ........................... 223
5. Transgenic strawberry for stress resistance .............................................. 224
6. Transgenic strawberry for herbicide resistance ............................................ 225
7. Transgenic strawberry for fruit quality improvement ......................................... 226
8. Problems and prospects ....................................................... 227
8.1. Efficiency of transformation.................................................. 228
8.2. Gene targeting and isolation.................................................. 228
8.3. Unpredictability and variability ................................................ 228
8.4. GMO safety issues ...................................................... 228
Acknowledgement ............................................................. 229
References ................................................................. 229
A
vailable online at www.sciencedirect.com
Biotechnology Advances 26 (2008) 219 232
www.elsevier.com/locate/biotechadv
Corresponding authors. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. Tel./fax: +86 571 8697 1009.
E-mail addresses: qyh6k@163.com (Y. Qin), shlzhang@zju.edu.cn (S. Zhang).
0734-9750/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.12.004
Author's personal copy
1. Introduction
Strawberry (Fragaria×ananassa Duch.), genus Fragaria of
the Rosaceae family, is one of the most important economical fruit
crops that adapts to various environmental growing conditions
throughout the world (Hancock, 1999). Strawberries are not only
attractive for their physicalappearance and physiological features,
such as bright colors, delicious taste, fine texture and fresh aroma,
but also for their high economical and nutritional values, such as
essential minerals, organic (amino) acids, vitamins and antiox-
idant properties.
Strawberries can be grown in a wide range of climates and,
for this reason, commercial production for the fresh fruit market
and processing industry has increased dramatically in recent
years, and its worldwide production among the berries ranks
second after fresh grape. Strawberries have experienced one of
the highest increases in rates of consumption in all fruit crops,
and the demand has continued to grow rapidly during the past
decade (FAOSTAT, 2007). The recent harvested area of
strawberry in the world was approximately 250,000 ha. Annual
production of strawberry was 3.3, 3.5 and 3.7 million tons in
2003, 2004 and 2005, respectively. The United States is the
largest producer of strawberries, accounting for over a quarter of
total production in the world and the second largest total
harvested area after Poland (FAOSTAT, 2007). The future of
strawberry production and sales is very promising. Strawberry
production, sales price, trading prospect, and consumption are
expected to increase continuously over the next decade.
Although years of efforts from traditional breeding have
resulted in steady improvement in various aspects of strawberry
production, many areas, such as yield, fruit size and shape,
berry quality, complex ploidy of strawberries , viz. diploidy,
tetra-, hexa-, octo- and even decaploid, as well as interspecific
hybrids with intermediate ploidy levels, still need to be explored
(Galletta and Maas, 1990; Potter et al., 2000; Faedi et al., 2002;
Marta et al., 2004; Folta and Davis, 2006). The process of
developing polyploid strawberries to select desirable agrono-
mical traits through traditional breeding programs is very
inefficient, labor-intensive, and time-consuming (Galletta and
Maas, 1990; Faedi et al., 2002; Marta et al., 2004). Therefore,
genetic engineering and biotechnology applications are increas-
ingly being used to improve strawberry qualities and expand the
breeding base and germplasm utilization. Genetic engineering
offers a realistic possibility to create varieties that selectively
target a gene or a few heterologous traits for introduction into
the strawberry genome, by facilitating the process of obtaining
desirable agronomical traits that exhibit increased strawberry
resistance to pests, herbicides, diseases, environmental stresses
as well as desired enhancement of fruit qualities. Great advances
have been made in strawberry genetic transformation since the
first transgenic strawberry plant was introduced in 1990 (Nehra
et al., 1990a,b). This review paper provides an overview of the
advances on strawberry transgenic research, consolidating the
large body of information regarding genetic modification of this
important genus. Furthermore, an assessment of the perfor-
mance of transgenic strawberry and its potential in strawberry
genetic transformation are proposed.
2. Establishment of a genetic transformation system
of strawberry
The key to successfully develop a genetic modified
strawberry cultivar is to establish an efficient regeneration and
transformation system, which provides the protocol for the
selection and recovery of genetically modified plants following
gene transfer. The application of strawberry culture in vitro in
genetic transformation and biotechnology has recently been
reviewed in depth by Debnath and Teixeira da Silva (2007) and
thus the focus of this review will be purely on transgenic
strategies for cultivar improvement. Various characteristics of
some well-studied examples of strawberry (Fragaria ×ana-
nassa and Fragaria vesca L.) genetic transformation using
reporter genes are summarized in Table 1. Reporter genes, such
as β-glucuronidase (GUS or uidA), neomycin phosphotransfer-
ase ( nptII), and hygromycin phosphotransferase (hpt) were first
used to screen the optimum conditions for the establishment of
an efficient genetic transformation system of strawberry.
The most effective and widely used method for strawberry
gene engin eering is Agrobacterium tumefaciens-mediated
transformation using strawberry leaf disk, petiole, aminae,
stalk and calli as explants and a bacterial plasmid as a vector
for the genes of interest (Table 1). Moreover, there are reports in
which biolistics (Wang et al., 2004), protoplast electropo-
ration (Nyman and Wallin, 1992) and a method combining
Agrobacterium infection and biolistic bombardment (Cordero
de Mesa et al., 2000) have been also used to obtain transgenic
strawberry.
The first transformation of strawberry was attempted in 1990
(Nehra et al., 1990a,b
). Nehra et al. (1990a,b) inoculated
strawberry leaf disks and calli with A. tumefaciens strain MP90
which contains nptII and gus genes to yield a transformation
frequency of 6.5% for leaf disks and 3% for calli where all of the
transgenic calli and shoots expressed varying levels of nptII and
GUS enzyme activity. Integration of both marker genes into the
strawberry genome was further confirmed by Southern blot
analysis. Subsequently, James et al. (1990a,b) infected strawberry
leaf disks and stalks with A. tumefaciens carrying a disarmed
binary vector plasmid pBIN6 containing the nos (nopaline
synthase) and nptII genes and plasmid pSS1 containing the
nptII and ipt (isopentenyl-transferase) genes. The nos and nptII
genes in the transgenic plants were expressed using pBIN6 as the
vector. However, the phenotype of transgenic plants with ipt
genes was dwarfed, caspitose and difficult to induce roots.
Similarly, Zhang and Wu (1998) developed a transformation
system for the commercial strawberry cultivar Tudla and
obtained transformed plants. After the leaf pieces were co-
cultivated for 3 d with Agrobacterium strain EHA105 containing
the binary vector pMOG410, then incubated on regeneration
medium containing 40 mg/L Kanamycin (Kan) for four weeks,
the transformed buds appeared obviously on the explants. The
regeneration percentage of transformed buds was 4.5% and a few
transformants expressed strong GUS activity following histo-
chemical analysis. Selection in this study was surprisingly high
since strawberry tissues are extremely sensitive to Kan, and
concentrations even at as low as 10 mg/L impair shoot
220 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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Table 1
Well-studied examples of strawberry genetic transformation using reporter genes
Species Cultivar+ others Explants Transgene Promoter/vector TrM TrE (%) Verification Reference
PCR Southern Others
Fragaria×ananassa Red Coat Callus, leaf GUS, nptII 35S, pBI121 AM 36.5 No Yes Yes Nehra et al. (1990a,b)
Rapella Leaf, leaf stalk NOS, nptII, ipt 35S, pBIN6 and pSS1 AM 0.95 No Yes Yes James et al. (1990a,b)
77101 Protoplasts GUS, nptII 35S, pRT88HPT Electroporation 15×10
4
No Yes Yes Nyman and Wallin (1992)
Selekta Leaf GUS, PAT 35S, pBI121 and pBIN19 AM 10 No Yes Yes du Plessis et al. (1997)
Chandler Leaf GUS, nptII 35S, pBI121 AM 4.22 Yes No Yes Barceló et al. (1998)
Totem Leaf GUS, nptII 35S, pAG1501 AM No Yes Yes Mathews et al. (1998)
Teodora, Egla Stipule GUS, nptII 35S pBin121 pVG3850 AM Yes No Yes Monticelli et al. (2002)
Marmolada Onebor Leaf, stipule,
petioles
nptII 35S pKIWI105 p35SGUS AM Yes Yes Yes Martinelli et al. (1997)
Chandler Calli GUS, nptII 35S, pGUSINT AM-biolistic 20 No Yes Yes Cordero de Mesa et al.
(2000, 2004)
BHN FL9003130 or
BHN 92664-501
Leaf GUS, nptII eFMV/eBigMac/
nos pCGN
AM 6.57 No Yes Yes Janette et al. (2001)
Kinuama, Senga Sengana,
Tudla, Toyonoka
Leaf GUS, nptII 35S, pMOG410 AM 3.76.8 Yes No Yes Zhang and Wu (1998);
Zhang et al. (2001)
Pajaro Leaf GUS, nptII 35S, pBI121 AM 6.6 Yes No Yes Ricardo et al. (2003)
Hecker, La Sans Rivale Leaf GUS, nptII AtSUC2, pBISPG AM 10.4 for Hecker;
7.4 for La Sans Rivale
Yes Yes Yes Zhao et al. (2004)
Induka, Elista Leaf laminae GUS, nptII 35S, pBIN19 AM 8.3 for Elista;
4.2 for Induka
Yes No Yes Gruchala et al. (2004)
Allstar Leaf GUS, nptII 35S pBLGC AM 1.1 No No Yes Zhang and Wang (2005)
LF9 Petiole nptII 35S, pCAMBIA,
pHK1001
AM
No No Yes Folta et al. (2006)
Chandler Leaf nptII, trfA 35S pBINPLUS,
pGUSINT, pBIN19
AM 65.7 Yes Yes No Abdal-Aziz et al. (2006)
Fragaria vesca L. Germinated plants
from seeds
Leaf GUS, nptII 35S, pBI121 AM 6.9 Yes No Yes El-Mansouri et al. (1996)
Alpine accession FRA197 Leaf GUS, nptII 35S, pBI121 AM Yes Yes Yes Haymes and Davis (1998)
Germinated plants
from seeds
Leaf, petiole GUS, nptII 35S, pCIRCE AM 15 Yes Yes Yes Alsheikh et al. (2002)
Alpine accession
FRA197 and 198
Leaf, petiole GUS, nptII AtSUC2, pBISPG AM 64.4 for FRA197;
67.9 for FRA198
Yes Yes Yes Zhao et al. (2004)
Germinated plants
from seeds
Leaf, petiole nptII, GUS, GFP 35S, pCAMBIA1304
pBBR1MCS-5.virGN54D
AM 11100 Yes No Yes Phillip (2005)
Alpine accession PI Leaf GFP, hpt 35S, pCAMBIA1304 AM 100 Yes Yes Yes Oosumi et al. (2006)
: not mentioned in the paper.
TrM: transformation method; TrE: transformation efficiency; AM: Agrobacterium-mediated transformation; GUS: β-glucuronidase; nptII: neomycin phosphotransferase; ipt: isopentenyl-transferase gene; CaMV 35S:
cauliflower mosaic virus 35S promoter; PA T: phoshinothricin acetyl transferase; eFMV: enhanced Figwort Mosaic Virus promoter; eBigMac: enhanced Big Mac promoter; GFP: green fluorescent protein; hpt:
hygromycin phosphotransferase; AtSUC2: Arabidopsis sucrose-H
+
symporter gene.
221Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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regeneration (Barceló et al., 1998; Alsheikh et al., 2002; Gruchala
et al., 2004; Qin and Zhang, 2007).
The key to Agrobacterium-mediated transformation in
strawberry depends not only on the establishment of an efficient
regeneration system (Debnath and Teixeira da Silva, 2007) but
also on the selection and recovery of transformed cells following
organogenesis. Mathews et al. (1998) reported an efficient
protocol to obtain pure transgenic plants. Two experiments were
conducted with and without repeated (iterative) cultures to
induce transgenic shoots on selection medium. The concentra-
tions of Kan in the non-iterative method were kept constant,
while in the iterative protocol, the Kan levels increased gradually
during subculture. The results showed that there were non-
chimeric plants using the iterative protocol. Therefore, they
believed that the iterative culture was an effective technique to
obtain pure transgenic lines.
Genotype is an important factor that significantly influences
transformation efficiency. Strawberry cultivars with different
genetic backgrounds may respond quite differently to a given
regeneration and transformation protocol (Table 1). Wawrzyńc-
zak et al. (2000) explored the transformation efficiency of five
strawberry cultivars Elsanta, Kaster, K-1349, K-1476 and
Senga Sengana by Agrobacterium-mediated transformation of
leaf explants. Transformation efficiency in different genotypes
varied, with the highest rate for Senga Sengana (5.71%) and
the lowest rate for K-1349 (1.67%). The incorporation of the
transgene was confi rmed by PCR and its expression by GUS
assay. Cordero de Mesa et al. (2000) used Agrobacterium-
coated gold particles and a gene gun to bombard Chandler
calli. After twenty-five weeks in culture, they reported a 20%
transformation rate. Gruchala et al. (2004) tested the regenera-
tion capability and transformation efficiency of two strawberry
cultivars in attempts to establish a transformation system for
strawberry and found that it was highly genotype-dependent.
On MS medium with 0.4 mg/L IBA and 1.8 mg/L BA, the
number of regenerated shoots Induka (3.5 shoots/explant) was
about twice more than Elista (1.8 shoots/explant). However,
after plant transformation using A. tumefaciens LBA4404 strain
containing plasmid pBIN19 with nptII and gus genes, the
number of transgenic Elista was about 2-fold higher (8.3
shoots/100 explants) than Induka (4.2 shoots/100 explants).
Besides genotype, plant transformation efficiency also
depends on many factors such as antibiotic types and concentra-
tions, inoculation and co-culture period and the presence or
absence of acetosyringone (AS). Zhang and Wang (2005) initiated
a detailed study to investigate factors that influenced the
transformation efficiency of Allstar strawberry; and discovered
that the optimal conditions for Agrobacterium-mediated transfor-
mation of Allstar leaf disks were: inoculation for 1015 min, co-
culture for 3 d plus 50 µM AS, and then transferred to a selection
medium containing 25 mg/L Kan plus 450 mg/L Carbenicillin
(Carb). All of these factors contribute to the further improvement
of transformation efficiency. A transformation frequency of 1.1%
was achieved based on Kan resistance assays.
The cultivated strawberry, Fragaria ×ananassa Duch., is an
octoploid (2n =8x =56) species and the high ploidy level makes
genetic and molecular studies difficult. F. vesca L., a diploid
(2n =2x= 14) strawberry, is an attractive model for studying
ripening in non-climacteric fruit, breeding, genetic a nd
molecular research in Rosaceae because of its small genome
size, small plant size and a short life cycle for transformation
(Hancock and Luby, 1993; El-Mansouri et al., 1996; Sargent
et al., 2004; Zhao et al., 2004; Phillip, 2005; Oosumi et al.,
2006). El-Mansouri et al. (1996) infected leaf disks of F. vesca
using A. tumefaciens LBA4404 carrying the plasmid pBI121
containing gus and nptII genes. Transformation efficiency was
57% ac cording to the GUS assay. Haymes and Davis (1998)
were the first to show the suitability of Alpine F. vesca for
transgene research in strawberry by demonstrating the transmis-
sion of the gus and nptII genes to the R1 progeny. However, no
transformation efficiency was reported by Haymes and Davis
(1998). Alsheikh et al. (2002) and Zhao et al. (2004) studied
F. vesca, they achieved 15% and 64.467.9% transformation
efficiency, respectively. In recent years, several scientists de-
scribed a new transformation procedure that uses leaf explants
from newly unfolded trifoliate leaves obtained from stock plants
67 weeks after seed germination, following co-cultivation with
Agrobacterium strain GV3101, and then selection on MS medium
containing 4 mg/L hygromycin. Using this protocol, 100%
transformation efficiency was obtained for 6 of 14 F. vesca ac-
cessions tested (Oosumi et al., 2006). Phillip (2005) developed an
efficient high-throughput Agrobacterium-mediated transforma-
tion protocol of F. vesca following the procedure of Oosumi et al.
(2006) with a minor modification. Transformation efficiencies
ranging from 11 to 100% were obtained for two F. vesca
accessions. Multiplex PCR, for amplification of the nptII and
GFP genes, was performed on a random sample of GFP plants
to verify integration of the T-DNA (Phillip, 2005).
Over the past decade, many laboratories have developed
transformation systems for different strawberry cultivars
(Table 1). These techniques provide an important foundation for
conferring commercial strawberry cultivars resistance to insects,
viruses, fungi, herbicides, stress and improving fruit quality
through biotechnology.
3. Transgenic strawberry for insect resistance
Tarnished plant bugs (Lygus lineolaris) and strawberry bud
weevils (Anthonomus signatus Say) are two common insects in
strawberries. These two insects can cause major damage to
strawberry flowers and buds, reducing yields significantly.
However, over-application of insecticides can be a serious
problem to the environment and negatively affect human health
as well as accelerating insecticide resistance. Environmentally-
friendly synthetic bioinsecticides are highly specific, and are
variably efficacious due to the influences of various biotic and
abiotic factors. Therefore, scientists have been looking for new
strategies to develop and improve insect-resistant strawberries.
Novel biotechnological tools have facilitated the introdu ction of
genes into strawberr y to comb at insects. One potential
possibility is for the strawberry plant itself to produce specific
proteins with insecticidal activity since a plant harboring a
protease inhibitor is part of the natural defense system against
insect predation. Plants transformed with foreign plant protease
222 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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inhibitor genes can enhance the resistance to insects. The
cowpea (Vigna unguiculata) protease trypsin inhibitor (CpTi)
gene, an insecticidal gene, has been successfully introduced into
strawberry. Transgenic strawberry lines constitutively expres-
sing the CpTi gene could protect against the feeding of vine
weevil (Otiorhynchus sulcatus) under greenhouse and field
conditions (James et al., 1992; Graham et al., 1995, 1997, 2001,
2002). Compared with control lines, transgenic strawberry lines
significantly improved plant growth and development. The
CpTi gene significantly affected vine weevil by reducing the
survival of weevil larvae and the number of pupae following an
insect bioassay. Moreover, there was no significant effect of the
CpTi transgenic lines on the numbers of Carabid and other non-
target arthropods.
4. Transgenic strawberry for phytopathogenic fungi,
bacteria and virus resistance
Strawberry is susceptible to various phytopathogenic fungi,
bacteria and viruses, which are associated with strawberry
diseases, causing fruit deformation, leaf yellowing, root and
crown disease, plant growth stunting, and substantial yield loss to
strawberry production as well as plant death (Simpson, 1991;
Mass, 1998).These diseases are difficult to control through
traditional breeding methods due to lack of both curative methods
and resistant varieties (Simpson, 1991). Using chemical insecti-
cides or fungicides to control strawberry diseases are not very
practical because strawberry fruit is a directly consumed product.
Development of biotechnology has provided a new opportunity to
enhance disease resistance for strawberry breeding. Finstad and
Martin (1995) obtained transformed strawberry plants using a
coat protein (cp) gene from Strawberry mild yellow edge
potexvirus (SMYELV-CP), which conferred resistance to the
virus. The results of PCR, Southern blot, and ELISA showed that
the cp gene was successfully incorporated and stably expressed.
Subsequently, Martinelli et al. (1996) obtained transgenic
strawberry using A. tumefaciens LBA4404 carrying the plasmid
pKyLX71 containing osmotin and nptII genes. Compared to
control plants, transgenic strawberry lines had higher levels of
fungicidal activity and significantly increased resistance to wilt
and gray mold diseases.
Gray mold caused by Botrytis cinerea and anthracnose
diseases produced by Colletotrichum fungi are the two most
destructive strawberry diseases that bring the majority of yield
losses to strawberry farmers (Sutton et al., 1988; Sutton, 1990;
Horowitz et al., 2002; Legard et al., 2003; Cesar et al., 2006).
Plants respond to biotic stress by inducing a set of genes encoding
diverse proteins, many of which are believed to play a self-
defensive role against pathogens. Among these proteins, the most
significant one is chitinase which is an endo-type enzyme that
hydrolyzes chitin, a structural component of the cell walls of
fungi, shells of crustaceans, and the integument and peritrophic
membranes of insects. Chitinase is a component of normal pre-
existing defense mechanisms in many plants that can be used as a
possible biocontrol agent instead of chemical fungicides
(Schlumbaum et al., 1986; Samac et al., 1990; Collinge et al.,
1993; Karasuda et al., 2003). Researchers have reported that
enhanced chitinase levels in transgenic strawberry plants can
indeed reduce the damage caused by powdery mildew fungi
(Asao et al., 1997, 2003). Chalavi et al. (2003) isolated a chitinase
gene (pcht28)fromLycopersicon chilense and transferred it into
Joliette strawberry using Agr obacterium-mediated transforma-
tion. Introduction of the pcht28 gene was verified by Southern
blot analysis and its expression by Northern blot. In growth
chamber studies, the transgenic strawberry plants that expressed
pcht28 had significantly higher resistance to Verticillium dahliae.
Ricardo et al. (2006)
obtained transgenic strawberry lines by
expressing three defense genes: ch5B (encoding a chitinase
protein from kidney bean (Phaseolus vulgaris)), gln2 (encoding a
glucanase protein from tobacco (Nicotiana tabacum)), and ap24
(encoding a thaumatin-like protein from tobacco). They evaluated
the effects of these genes on the protection against gray mold and
anthracnose diseases produced by local strains of B. cinerea and
C. acutatum, respectively. Sixteen transgenic lines expressing
one or a combination of two defense genes were obtained. The
results showed that the expression of the ch5B gene in transgenic
strawberry increased the resistance to gray mold while it had no
significant effect on anthracnose disease resistance. The resis-
tance was correlated with the presence of the foreign ch5B protein
and an increase of chitinolytic activity in leaves. These results
demonstrate that chitinases play a key role in defense against
fungal disease in strawberry. Recent findings by Mercado et al.
(2007) had indicated that expression of a β-1,3-glucanase gene
isolated from the antagonist soil fungus Trichoderma harzianum
in strawberry enhanced anthracnose resistance.
Glucose oxidase (GO) is closely related to resistance of plant
fungal diseases. It catalyzes the oxidation of β-
D-glucose to D-
glucono-1,5-lactone and hydrogen peroxide (H
2
O
2
) while the
production of H
2
O
2
is toxic to phytopathogenic fungi, which is
usually responsible for most major diseases in many crops (Kim
et al., 1988). A study was initiated to isolate the GO gene from
Aspergillus niger and introduce it into strawberry by Agrobac-
terium-mediated transformation. Compared to control plants, a
higher concentration of H
2
O
2
was detected in GO transgenic
strawberry lines, which resulted in increased resistance to gray
mold (Jin et al., 2005).
Thaumatin-like proteins (TLP), which belong to the PR-5
(pathogenesis-related) group of proteins, have different degrees
of specificities to antifungal activity. TLP has been successfully
used to enhance plant resistance to fungal pathogens ( Chen
et al., 1999; Datta et al., 1999; Schestibratov and Dolgov, 2005).
Schestibratov and Dolgov (2005) reported that a thaumatin II
cDNA driven by the CaMV 35S promoter was introduced into
strawberry via Agrobacterium-mediated transformation. The
tested transgenic lines expressing the TLP showed a signifi-
cantly higher level of resistance to gray mold after infection
with a conidial suspension.
Antimicrobial peptide (AMP) is a class of micromolecule
polypeptides that plants synthesize to defend themselves against
the invasion of environmental microbes; most of them are
cationic peptides with good thermal stability (Theis and Stahl,
2004; Brogden, 2005). AMP is an inducible insect peptide with
a broad range of activities in resisting bacteria and fungi in
plants (Broekaert et al., 1995; Mariana and Wagner, 2005). A
223Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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number of studies have demo nstrated the e xpression of
heterologous AMP in plants with different degrees of enhanced
resistance to pathogens (Huang et al., 1997; Sharma et al., 2000;
Arenas et al., 2006). Qin et al. (2005a,b,c), Qin and Zhang,
2007) extensively investigated the factors that influenced the
regeneratio n and t ransf ormation efficiency of Toyonoka
strawberry. By establishing an efficient regeneration system,
antimicrobial peptide-D gene (APD), driven by a 35S prom oter
to Toyonoka via Agrobacterium-mediated transformation, can
be successfully transferred. Kanamycin-resistant (Kan
R
) buds
and shoots were obtained from the explants (Fig. 1A, B). PCR
analysis and Southern hybridization showed that the APD gene
was integrated into the strawberry genome (Figs. 2 and 3).
Among the six transformants tested, one contained two copies,
two contained five copies, and the others contained a single
copy. No hybridization signal was detected in DNA from the
non-transformed control plants (Fig. 3). Transgenic strawberry
was acclimatized in the greenhouse for further analysis
(Fig. 1C).
5. Transgenic strawberry for stress resistance
Abiotic stresses, such as salinity and low temperatures, are
part of the limiting factors in strawberry growth and develop-
ment. Abiotic stresses can cause a series of morphologic al,
physiological, biochemical and molecular changes that
adversely affect plant growth and productivity (Awang et al.,
1993; Keutgen and Keutgen, 2003 ). Conventional breeding for
salt tolerance has been attempted for a long time; however, it has
resulted in little progress (Kaya et al., 2002a,b; Dziadczyk et al.,
2003; 2005). The direct introduction of a number of genes by
genetic engineering offers a convenient and effective way to
improve stress tolerance and achieve prodigious progre ss.
Researchers have identified genes that play a key role in stress
tolerance by identifying proteins regulated in response to stress
(Teixeira da Silva, 2006b ), which include betaine aldehyde
dehydrogenase (BADH)(Weretilnyk and Hanson, 1987, 1990),
late embryogenesis abundant proteins (LEAs)(Wise, 2003),
cold-induced transcription factor (CBF 1)(Gilmour et al., 1998)
and antifreeze protein (AFP)(Georges et al., 1990). Transgenic
strawberry plants with the above genes exhibited constitutive
activation of stress responsive genes and enhanced salt (Liu
et al., 1997; Wang et al., 2004) and freezing tolerance (Firsov
and Dolgov, 1999; Owens et al., 2002, 2003; Khammuang et al.,
2005).
Liu et al. (1997) obtained transgenic strawberry plants using
the BADH gene driven by the 35S promoter. The expression of
this gene was confirmed by Northern blot and a BADH enzyme-
assay. The transgenic plants grew normally i n medium
containing 0.40.7% (w/v) NaCl while all untransformed
plants died on medium with 0.4% NaCl after 20 d. The relative
electronic conductivity and membrane permeability demon-
strated that the damage of the membrane structure of transgenic
plants was lower than the controls'. This may be one of the
Fig. 1. The Kan
R
buds and plantlets from strawberry leaf disks infected with Agrobacterium EH105 harboring APD gene. A: Kan
R
buds; B: Kan
R
plantlets;
C: acclimatized Kan
R
plantlets.
Fig. 2. PCR analysis of 122 bp fragment corresponding to APD. Lane 1: marker
(DL2000); lane 2: positive control; lanes 39: transgenic strawberry plants; lane
10: non-transformed plants.
Fig. 3. Southern blot analysis of DNA from control and transgenic strawberry
plants digested with Hind III and probed with a 122 bp fragment of APD. Lanes
16: transgenic strawberry plants; lane 7: non-transformed plant.
224 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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reasons for the higher salt tolerance of transgenic plants. LEAs
have been suggested to increase salt tolerance in plants by
binding water and maintaining the structure of other proteins
and membranes (Close, 1996). A group of scientists transferred
the LEA
3
gene into strawberry by particle bombardment, which
confirmed the integration of the gene into strawberry genomes
by Southern blots. Results of NaCl salt stress experiments
indicated that the LEA
3
gene significantly increased the
resistance of strawberry to salt tolerance (Wang et al., 2004).
Many rosaceous fruit crops suffer yield reductions due to
early season freezes during or just prior to bloom (Ki and
Warmund, 1992). Re-engineering plants for greater freezing
tolerance through plant transformation is a potential way to
reduce the damage caused by freezing. Great progress has been
made in strawberry in the past few years in terms of improving
freezing tolerance through genetic transformation. By develop-
ing a protocol for Agrobacterium transformation of strawberry
leaf explants, transgenic plants have been obtained containing
the AFP gene of winter flounder (Firsov and Dolgov, 1999;
Khammuang et al., 2005). The integration of nptII and AFP
gene was confirmed by PCR analysis (Firsov and Dolgov,
1999). However, no further study of agronomic traits was
reported.
To determine the function of CBF1 in enhancing strawberry
freezing tolerance, Owens et al. (2002, 2003) obtained
transgenic strawberry plants via Agrobacterium-mediated
transformation harboring the CBF1 gene driven by the 35S
promoter. Two transformants expressed the gene at low levels in
both leaves and receptacles with pistils. No difference in
freezing tolerance was detected between receptacles with
attached pistils of the transformants and wild type (wt) plants.
However, the freezing tolerance values of temperature at which
50% electroly te leakage (a rapid index of plant vitality for biotic
or abiotic stress) occurred in detached leaf disks from the two
transgenic lines was 8.2 °C and 10.3 °C respectively, which
were significantly greater than that of the wt Honeoye
( 6.4 °C).
WCOR410 belongs to a different subtype of the D-11 protein
family, the so-called acidic dehydrins, playing a role in
preventing the destabilization of the plasma membrane during
water stress and low temperature, and could be a determining
factor for increased cell resistance to freezing. Expression of the
acidic dehydrin gene Wcor410 cloned from wheat was found to
be associated with the development of freezing tolerance
(Danyluk et al., 1994, 1998). Transgenic strawberry plants
expressing the Wcor410 gene had a 5 °C improvement of
freezing tolerance than wt or transformed leaves not expressing
the Wcor410 protein (Houde et al., 2004). The results suggest
that Wcor410 is involved in the cryoprotection of the plasma
membrane against freezing stress to improve freezing tolerance
in leaves of transgenic strawberry.
6. Transgenic strawberry for herbicide resistance
Various weeds constantly compete with strawberry plants for
available water, nutrients and light energy; weed pressure is
further aggravated by disease, nematodes and insects, and thus
affects strawberry growth and causing yield reductio n.
Historically, very few herbicides can be used for weed control
in strawberry production due to its perennial nature. Recently,
genetic engineering has been applied to develop herbicide-
resistant varieties in strawberry through the introduction of
glufosinate and glyphosate-resistant genes. Scientists have
obtained transgenic strawberry pla nts via Agrobacterium-
mediated transformation containing the gus or pat genes drive n
by the 35S promoter in binary vectors pBI121 and pBIN19,
respectively (du Plessis et al., 1997). A 10% transformation
frequency was obtained on a shoot-inducing medium containing
50 mg/L Kan and most Kan
R
shoots that originated from
explants transformed with pBI121 expressed the gus gene.
Table 2
Comprehensively well-studied examples of genetic transformation in strawberry fruit quality improvement
Species Cultivar Explants Transgene Promoter TrM Verification Reference
PCR Southern Others
Fragaria ananassa Symphony,
Senga Sengana
Stem tissues Invertase 35S AM Yes No No Bachelie et al. (1997)
Calypso Leaf Cel1 antisense 35S AM Yes Yes Yes Woolley et al. (2001)
Israeli cultivars Leaf, stipule rolB TPRP-F1 AM Yes Yes No Kafkas et al. (2002)
Chandler Leaf PL antisense (njjs25) 35S AM Yes Yes Yes Jiménez-Bermúdez et al. (2002)
AN93.231.53 Leaf DefH9-iaaM NOS AM No Yes Yes Mezzetti et al. (2004a,b)
Kaster Leaf iaglu 35S AM Yes Yes Yes Wawrzyńczak et al. (2005)
Calypso Leaf Cel1, Cel2 and Cel1/Cel2 FBP7 AM No No Yes Palomer et al. (2006)
M14 Leaf annfaf 35S AM Yes Yes No Na et al. (2006)
Anther Leaf AGPase antisense APX AM Yes Yes Yes Park et al. (2006)
Pájaro Leaf ch5b, gln2, ap24 35S AM No Yes Yes Ricardo et al. (2006)
Calypso Leaf FaOMT sense and antisense 35S AM No No Yes Lunkenbein et al. (2006b)
Elsanta Leaf CHS antisense 35S AM No Yes Yes Lunkenbein et al. (2006a)
Chandler Leaf PL antisense 35S AM No No Yes Sesmero et al. (2007)
Fragaria vesca L. Alpina W. Original Leaf DefH9-iaaM DefH9 AM No Yes Yes Mezzetti et al. (2004a,b)
TrM: transformation method; NOS: nopali ne synthase promo ter; APX: fruit-dominant ascorbat e peroxidase promoter; rolB: rhizogeges; DefH9: ovule-specific
promoter; FBP7: flor al binding protein 7 promoter; CaMV 35S: cauliflower mosaic virus pro mote r; TPRP-F1: ovary specific promoter; PL: pectate lyase; iaglu:
IAA-glucose synthase gene; AGPase: ADP-glucose pyrophosphorylase; annfaf: annexin of Fragaria× ananassa fruit; FaOMT: Fragaria× ananassa O-
methyltransferase; CHS: chalcone synthase; iaaM
: acid-tr yptophan monooxygenase.
225Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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Field trials revealed that most transgenic lines which contained
the pat gene were resistant to the herbicide glufosinate-
ammonium. Subsequently, Zhang et al. (2001) obtained
transgenic plants using the gus and bar genes via A.
tumefaciens-mediated transformation by developing an efficient
and stable regeneration system and genetic transformation
system for the commercial strawberry cult ivar Tudla. A high
level of GUS activity was detected in five transgenic strawberry
lines from 10 independent shoots putatively transformed with
the gus gene. The transgenic strawberry plants transformed with
the bar gene were able to differentiate on medium containing
10 mg/L glufosinate and showed complete resistance to 450 mg/
L glufosinate after three applications of foliage spray in the
field. The transgenic plants flowered and set frui ts normally
while the untransformed plant died after 10 d. Meanwhile,
Morgan et al. (2002) obtained strawber ry varieties tolerant to
glyphosate following integration of the CP4.EPSP (5-enolpyr-
uvylshikimate 3-phosphate) synthase gene through Agrobac-
terium-mediated transformation. Among the 73 independent
transformants that were sprayed with commercial levels of
Roundup Ultra
®
in the nursery, a range of tolerance to the
herbicide was shown in those transformants ranging from
complete tolerance to no resistance. Introduction of the CP4.
EPSP gene was confirmed by Southern blots and its expression
was verified by Northern analysis. Preliminary assessment of
fruit characteristics and yield data suggested that some
glyphosate-resistant lines produced good quality fruit typical
of Camarosa (Morgan et al., 2002).
7. Transgenic strawberry for fruit quality improvement
Strawberry is a delicate fruit with a short shelf-life, mainly
due to a rapid loss of firmness in texture. To prevent fruit
softening in order to prolong shelf-life and to improve fruit
quality of strawberry, extensive studies have been carried out
(Table 2). Antisense technology is a useful tool to prevent
strawberry fruit from softening by suppressing particular genes
involved in fruit softening without altering fruit quality
(Mathews et al., 1995; Woolley et al., 2001; Jiménez-Bermúdez
et al., 2002; Palomer et al., 2006; Sesmero et al., 2007).
Ethylene, a gaseous hormone, is produced in all higher plants
and can stimulate fruit ripening. The 1-aminocyclopropane-1-
carboxylic acid (ACC) synthase is the limiting factor in ethylene
production and it can be manipulated through biotechnology to
delay fruit ripening. The control of ethylene production has
been studied extensively. S-adenosylmethionine (SAM) is the
metabolic precur sor of ACC synthase, the proximal precursor to
ethylene. In recent years, biotechnology has been used to delay
the ripening of strawberry using the SAMase gene by down-
regulating ACC synthase. Integration of the gene was
confirmed by Southern blot. However, no further report was
found regarding transgenic fruit ripening and shelf-life
(Mathews et al., 1995).
Pectate lyase (PL) is an extracellular enzyme involved in cell
wall disassembly and maceration during fruit ripening (Jimé-
nez-Bermúdez et al., 2002; Marín-Rodríguez et al., 2002). To
effecti vely control or delay fruit softening in strawberry,
Jiménez-Bermúdez et al. (2002) transferred the antisense
orientation of the PL sequence driven by the 35S promoter
into strawberries. In most transformed Apel lines, total yield
was significantly reduced. However, suppression of the PL
mRNA gene in strawberry by an antisense transformation
significantly increased the firmness of ripe fruits and extended
the post-harvest shelf-life without significantly affecting other
fruit characteristics such as color, size, shape and weight during
fruit ripening. In all six Apel lines tested, expression of the PL
gene in ripened fruit was 30% lower than that of the control, and
three of them were completely suppressed. Compared to control
fruit, transformed ripened Apel fruit had a lower degree of in
vitro swelling and a lower amount of ionically bound pectins.
The post-harvest soft ening of Apel fruit was also diminished.
Recently, using an antisense sequence of a strawberry PL gene,
Sesmero et al. (2007) evaluated the effect of this transgenic
modification on the texture of frozen and thawed fruits and the
jam after strawberry processing. The mRNA transcript level of
PL was significantly reduced in two independent lines (90% in
Apel 14 and 99% in Apel 23). At harvest, the ripened fruits'
firmness from the two lines was significantly higher than that of
the controls. Transgenic fruits resisted the cooking process
better than the conventional ones in terms of having a higher
amount of fruit berries in these jams and these processed fruits
were firmer than the control. The degree of firmness was posi-
tively correlated with the degree of PL silencing. In contrast,
jams of these transgenic lines were similar in firmness but
slightly less viscous than the control. These results indicated
that the PL gene has a great potential to be used for preventing
fruit softening in strawberry through biotechnology. Suppres-
sion or silencing of the PL gene in strawberry fruit can improve
quality traits of strawberry jam, such as texture and content of
whole berries.
Plant endo-β-(1,4)-glucanases (EGases) are hydrolytic
enzymes that are active against β-(1,4)-glucan links. Early
research indicated that EGase activity was involved in cell wall
weakening, which ranged from cell wall expansion to fruit
ripening and disassembly (Trainotti et al., 1999a,b). Fruit
softening during ripening is associated with the overlapping
presence of two divergent EGases, Cel1andCel2. Woolley et al.
(2001) studied the role of the Cel1 protein in fruit softening using
antisense technology and obtained transgenic plants with reduced
cel1 mRNA levels. However, they found that the constitutive
antisense down-regulation of Cel1 transcripts in strawberry plants
did not significantly alter EGase activity and fruit firmness. To
further explore the role of Cel1andCel2 EGases in strawberry
fruit softenin g, Palomer et al. (2006) obtained transgenic
strawberry plants using antisense constructs for individual
silencing of Cel1andCel2 and a chimeric antisense Cel1/Cel2
under the control of a fruit-specific promoter (FBP7). The results
showed that constant down-regulation of Cel1 expression
throughout ripening was accompanied by reduced Cel1protein
accumulation. However, no difference in fruit firmness was found
between transgenic lines and control plants with a reduction of
Cel1 protein level and EGase activity. Moreover, there was no
significant reduction of Cel2 protein accumulation in any of the
Cel2 transgenic or Cel1/Cel2 double-transgenic lines. Therefore,
226 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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they suggested that Cel1 alone is not the major determinant of
strawberry fruit softening during ripening while Cel2mightbe
responsible for fruit development prior to ripening, thus
accounting for the lack of Cel2 protein down-regulation observed
(Palomer et al., 2006). These studies paved the way for
understanding the biochemical mechanisms of strawberry fruit
softening.
Strawberry is a non-climacteric fruit and its ripening
mechanism is unclear. Annexin plays an important role in
fruit development and ripening (Wilkinson et al., 1995). In
order to eluci date the function of the annfaf (annexin of Fra-
garia ×ananassa fruit) gene in ripening processes of strawberry,
Na et al. (2006) isolated the gene from strawberry fruit and
transferred it into the strawberry genome using an antisense
fusion annfaf gene. Southern blot confirmed that the gene was
integrated into the strawberry genome as a single copy. Results
from this study provided the foundation for selecting deficient
transgenic plants, which could be beneficial for further studies
on the mechanism of maturation of strawberry fruits and for
breeding of traits related to freshness in strawberry (Wang et al.,
2001; Na et al., 2006).
Advanced knowledge on strawberry fruit productivity
through genetic engineering is available. Auxin (IAA) produced
by fert ilized ovules is essential for strawberry fruit development
and quality (Manning, 1994; 1998). To explore this role,
scientists have introduced the parthenocarpic chimeric gene
(DefH9-iaaM) into two strawberry species (F. ananassa cv.
AN93.231.53 and F. vesca cv. Alpina W. Original)byA.
tumefaciens-mediated transformation (Mezzetti et al., 2002;
2004a,b; Mezzetti and Costantini, 2006). Transgenic strawberry
lines showed significant increases in fruit size, number and fruit
yield. Compared with the conventional cultivars, fruit yields
were increased approximately 184% and 139% in transgenic
cultivated strawberry AN93.231.53 and Alpina W. Original,
respectively. The total IAA contents of DefH9-iaaM transgenic
young flower buds had increased about 1.5 times compared
with the untransformed flower buds. Moreover, the increase in
fruit production did not reduce fruit total sugar content, an
important parameter related to fruit quality. These results
indicated that IAA plays an important role in plant fecundity in
Rosaceae species. Meanwhile, Wawrzyńczak et al. (2005)
obtained transgenic strawberry plants with the maize IAA-
glucose synthase gene (iaglu) via the Agrobacterium-mediated
method. Genomic integration and expression of transgenes was
verified by PCR, Southern blot a nd RT-PCR analysis.
Compared to wt plants, there was a significant increase of
ester-conjugated IAA levels in the tissue of all transformants
harboring the iaglu gene while free IAA levels were
significantly decreased in two transgenic lines tested. The
level of amide-conjugated hormone wa s not affected by
transformation with iaglu . Compared to wt plants, all transgenic
plants had a dwarfish genotype (i.e., shorter leaf petioles,
smaller leaf laminas, less crown diameter and shorter runners)
even though they produced more roots in vitro.
Carbohydrate content and balance play an important role in
determining the flavor and processing quality of the fruit.
Invertases are known to exist in multiple forms which are
responsible for catalyzing the breakdown of sucrose in many
fruit species (Sturm, 1999). To study the role of invertase in
strawberry ripening, Bachelie et al. (1997) cloned two invertase
genes from potat o, encoding cell wall and vacuolar forms
respectively, and integrated them into two strawberry cultivars
Symphony and Senga Sengana via A. tumefaciens. The
presence of the inver tase genes in strawber ry was confirmed by
PCR analysis. However, no further study was found regarding
transgenic fruit characteristics such as sugar balance, flavor and
processing quality. Recently, Park et al. (2006) generated
transgenic plants that incorporated an antisense cDNA of ADP-
glucose pyrophosphorylase (AGPase) small subunit (FagpS)
driven by the strawberry fruit-dominant ascorbate peroxidase
(APX) p romoter, to evaluate the effects on carbohydrate
contents during fruit development. The results showed that
the levels of AGPase mRNA were drastically reduced in the red
stage of fruits in all the transgenic plants. The suppression of the
AGPase small subunit in transgenic plants resulted in a 1637%
increment of total soluble sugar content and a 2747% decrease
of the starch content in mature fruit without significantly
affecting other fruit characteristics such as color, weight and
hardness. Results from previous studies suggested that, through
biotechnological alternation, the AGPase gene might be used
for improving soluble sugar content and decreasing starch
content in strawberry fruits.
Vitamin C is an essential component of human nutrition.
Agius et al. (2003) cloned a GalUR promoter from strawberry
that encodes an NADPH-dependent
D-galacturonate reductase,
an enzyme involved in the biosynthesis of vitamin C in
strawberry fruit. The result of expression analysis showed that
GalUR was correlated with changes in ascorbic acid content
in strawberry fruit during ripening. Over-expression of the gene
in Arabidopsis thaliana enhanced vitamin C content 23 fold.
The study suggested that the gene might be useful for increasing
vitamin C levels of strawberry fruit though genetic transforma-
tion (Agius et al., 2003).
Recently a great stride has been taken in identifying and
characterizing, through biochemical and molecular means, the
major enzymes and genes involved in flavonoid and proantho-
cyanidin biosynthesis during fruit development (Almeida et al.,
2007) and these findings would allow the potential cloning of
strawberry-derived genes into strawberry, i.e. intra-generic
genetic transformation, perhaps facilitating the improvement
of color and flavor traits.
8. Problems and prospects
Genetic engineering (GE), also termed transgenic biotechnol-
ogy, refers to the transfer of individual genes between unrelated
species (animal or plant), through the use of recombinant DNA
techniques. Through plant genetic engineering, a novel gene can
be introduced from one plant species to another plant species to
improve the later plant, a genetically modified organism (GMO).
The application of GE in strawberry is an effective breeding
method to make strawberry plants more resistant to biotic and
abiotic stresses, improve qualitative and quantitative fruit quality,
increase yields, and better stress resistance, while also being
227Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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environmentally friendly. In past decades, great progress has been
made in strawberry breeding and germplasm improvement
through plant GE. Although, such approaches have demonstrated
a great promise and future, it is still at a nascent and experimental
phase, requiring further development and testing in the field
before any commercial application. These studies have also
proposed many tough challenges for plant biologists and breeders
in creating new and better strawberry varieties. The major
challenges ahead include:
8.1. Efficiency of transformation
Fundamental research is still needed to improve transforma-
tion frequency in delivering genes, as well as in developing a
highly-efficient and stable regeneration system, although great
strides have already been achieved. Progress in Agrobacterium-
mediated genetic transformation of strawberry had been
hampered greatly by their generally poor response to tissue
culture, although several novel methods and genotype-inde-
pendent protocols have now started to emerge (Debnath and
Teixeira da Silva, 2007), which will be a massive boost for the
GE protocols. Though a few strawberry transformation systems
have been developed for several strawberry cultivars by many
research groups since 1990s, most of these methods were
inefficient and were mostly not repeated (Finstad and Martin,
1995; Barceló et al., 1998; Hokanson and Maas, 2001; Folta and
Dhingra, 2006). The most important aspect of transformation
efficiency is the choice of species and cultivars. Genotype is an
important factor that significantly influences the efficiency of
transformation (Tables 1 and 2). High variability of efficiency
was observed among strawberry cultivars with different genetic
backgrounds, even within the same cultivar (Bachelie et al.,
1997; Barceló et al., 1998; Cordero de Mesa et al., 2000, 2004;
Jiménez-Bermúdez et al., 2002; Abdal-Aziz et al., 2006;
Lunkenbein et al., 2006a,b; Sesmero et al., 2007). The door is
beginning to open for identifying strawberry germplasm from
different genetic backgrounds that can be easily transformed
(Oosumi et al., 2006; Folta et al., 2006). On a new and exciting
front Hanhineva and Kärenlampi (2007) proposed the use of
temporary immersion bioreactor systems to mass-propagate
hygromycin-resistant strawberries.
8.2. Gene targeting and isolation
Identification and isolation of interesting genes for transferring
into targeted strawberry varieties are one of the major goals for a
strawberry biotechnology program. It is difficult to isolate an
interesting gene for a strawberry plant since strawberries are
polyploid, and also most desirable traits are controlled by multiple
genes (Faedi et al., 2002; Folta and Davis, 2006). Isolation and
transformation of genes correlated with fruit softening, yield and
quality are still at the exploratory stage (Harpster et al., 1998;
Llop-Tous et al., 1999; Trainotti et al., 1999a,b; Palomer et al.,
2004; Manning, 1994, 1998). Genes identified and transferred in
most of the studies discussed so far are still from other crops, such
as barley (Wang et al., 2004), cowpea (James et al., 1992; Graham
et al., 1995, 1997, 2001, 2002), wheat (Houde et al., 2004), potato
(Bachelie et al., 1997), rice (Asao et al., 1997, 2003), tomato
(Chalavi et al., 2003), katemfe (Schestibratov and Dolgov, 2005),
maize (Wawrzyńczak et al., 2005), kidney bean (Ricardo et al.,
2006), and tobacco (Ricardo et al., 2006). The challenge for
scientists is to expand the efforts to identify and understand
additional gene regulation and expression in strawberry growth
and development.
8.3. Unpredictability and variability
Unpredictable and variable expres sion of a foreign gene is a
major problem that hinders the success of biotechnology
programs in strawberry research. With the development of
several novelties in plant GE (Teixeira da Silva, 2006a), many
economically valuable heterogeneous genes have been trans-
ferred into strawberry plants (Hokanson and Maas, 2001; Folta
and Dhingra, 2006). However, the subsequent results have
shown that almo st all transgene expression remains largely
unpredictable and a great variation of expression level among
transgenic lines exists. A number of strategies are available to
target the expression to specific tissues and/or to provide a high
and stable level of gene expression using a specific promoter
(Agius et al., 2005; Zhao et al., 2004) or by an induced promoter
(Li et al., 2005), o r an expression vector harboring matrix
attachment regions (Allen et al., 1996; Abranches et al., 2005),
or a small subunit leader and transit peptide (Wong et al., 1992;
Janette et al., 2001) or even site-specific recombination systems
(Lyznik et al., 2003; Djukanovic et al., 2006; D'Halluin et al.,
2008). Within the next few years, undoubtedly, the most
beneficial conditions for transformation will be categorized to
allow for maximization and stabilization of gene expression
levels in strawberry.
8.4. GMO safety issues
Risk assessment for human health and environment related
to transgenic plants is another serious obstacle for public
acceptance and commercialization (Van den Eede et al., 2004;
Kulikov, 2005 ). Recently, regulatory authorities and consumers
are concerned about the environmental safety of GMO and are
demanding for comm ercial transgenic plants to be free of
unnecessary genes, such as marker genes (antibiotic resistance)
or vector backbone sequenc es (Ebinuma et al., 1997; Endo
et al., 2002; Schaart et al., 2004; Abdal-Aziz et al., 2006;
Ballester et al., 2006; Ludmila et al., 2006; Mihály et al., 2006).
Schaart et al. (2004), for example, used an inducible site-
specific recombinase to obtain marker-free transgenic straw-
berry plants using a bifunctional selectable marker gene for the
initial positive selection of transgenic tissue. When shoots had
regenerated on Kan, leaf explants from Kan
R
shoots were treated
with dexamethasone, which induces the recombinase gene, and
subsequently subjected to a second round of regeneration in the
presence of 5-fluorocytosine (5-FC). The codA gene converted
the non-toxic 5-FC to cytotoxic fluorouracil (Stougaard, 1993),
leading to the death of cells that still contain the marker genes.
After negative selection, they successfully obtained 30 putative
marker-free transgenic plants. In another practical success story,
228 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
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Hoffmann et al. (2006) optimized RNAi silencing by agroinfil-
trating developing fruits attached to plants with a construct
containing a sense and its corresponding antisense sequence of a
chalcone synthase gene (CHS) separated by an intron. Silencing
of the CHS gene could be detected by the absence of red
coloration in ripened fruits following agroinfiltration. Lunkenbein
et al. (2006a,b) obtained transgenic plants containing an antisense
CHS gene to assess the impact of down-regulation of this gene on
pigment accumulation in ripened fruit, while other transgenic
plants harboring a down-regulated O-methyltransferase gene
almost completely depleted the levels of DMMF (2,5-dimethyl-4-
methoxy-3(2H)-furanone, the product of methylation of the most
important aroma (4-hydroxy-2,5-dimethyl-3(2H)-furanone) dur-
ing strawberry fruit ripening. These studies would most likely
represent the state of the art in strawberry genetic engineering at
present.
Legislations and regulations towards genetically modified
plants (GMPs) vary among countries, particularly in the EU,
which are not allowing deliberate release of GMPs carrying
antibiotic resistance genes into the environment or permitting
their commerci alization. Therefore, developing procedures to
avoid the use of antibiotic selection or to allow elimination of
marker genes from a GMP (GM-gene-deletor) will be a high
research priority in the coming years (Luo et al., 2007).
The applications of biotechnology that permits particular
genes to be expressed in a specific manner can accelerate the
process of breeding new and better strawberry varieties.
Ongoing efforts to improve strawberry germplasm using
molecular approaches will be greatly speeded up with the
development of biotechnology and the increased understanding
of basic biological sciences. To obtain bro ad-spectrum
resistance plus good fruit quality, the introduction of multiple
genes simultaneously will be extensively applied to strawberry
breeding. Although such products are still a long way from
commercialization, the next decade p romises to be an exciting
one for strawberry biotechnology.
Acknowledgement
The authors sincerely thank Dr. Ye Grace Chen at Harvard
University for her critical review and comments on this
manuscript, and Dr. Lanlan Zhang at Zhejiang University for
reference collection.
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232 Y. Qin et al. / Biotechnology Advances 26 (2008) 219232
... Although strawberry leaf tissue has been widely studied to develop efficient transformation and regeneration methods through somatic tissue or callus formation (Nehra et al., 1990;El Mansouri et al., 1996;Haymes and Davis, 1997;Balokhina et al., 2000;Jimenez-Bermudez et al., 2002;Agius et al., 2003;Oosumi et al., 2006;Qin et al., 2008;Zhang et al., 2014;Song et al., 2019), the regeneration of transgenic strawberry plants takes a long time from transformation to identification (Barcelo et al., 1998;Folta and Dhingra, 2006;Haddadi et al., 2015). For example, it takes at least 15 months from transformation to gaining transgenic fruits for gene function analysis in ripening (Hoffmann et al., 2006;Miyawaki et al., 2012). ...
... First establishment of rapid stable transgenic system by the Agrobacterium-mediated transformation of early germination seeds in strawberry Strawberry is not only considered as a model plant for the Rosaceae family (Folta and Davis, 2006) but also regarded as an ideal model plant for studying non-climacteric fruit ripening . Thus, in the past years, strawberry transient and stable genetic transformations have been extensively studied (Nehra et al., 1990;El Mansouri et al., 1996;Haymes and Davis, 1997;Balokhina et al., 2000;Jimenez-Bermudez et al., 2002;Agius et al., 2003;Oosumi et al., 2006;Qin et al., 2008;Zhang et al., 2014;Haddadi et al., 2015;Song et al., 2019). However, some limits exist in the present transformation system, and it is necessary to develop a rapid and stable transformation system in strawberry. ...
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A strawberry RIPK1, a leu-rich repeat receptor-like protein kinase, is previously demonstrated to be involved in fruit ripening as a positive regulator; however, its role in vegetable growth remains unknown. Here, based on our first establishment of Agrobacterium-mediated transformation of germinating seeds in diploid strawberry by FvCHLH/FvABAR, a reporter gene that functioned in chlorophyll biosynthesis, we got FvRIPK1-RNAi mutants. Downregulation of FvRIPK1 inhibited plant morphogenesis, showing curled leaves; also, this silencing significantly reduced FvABAR and FvABI1 transcripts and promoted FvABI4, FvSnRK2.2, and FvSnRK2.6 transcripts. Interestingly, the downregulation of the FvCHLH/ABAR expression could not affect FvRIPK1 transcripts but remarkably reduced FvABI1 transcripts and promoted FvABI4, FvSnRK2.2, and FvSnRK2.6 transcripts in the contrast of the non-transgenic plants to the FvCHLH/FvABAR-RNAi plants, in which chlorophyll contents were not affected but had abscisic acid (ABA) response in stomata movement and drought stress. The distinct expression level of FvABI1 and FvABI4, together with the similar expression level of FvSnRK2.2 and FvSnRK2.6 in the FvRIPK1- and FvABAR/CHLH-RNAi plants, suggested that FvRIPK1 regulated plant morphogenesis probably by ABA signaling. In addition, FvRIPK1 interacted with FvSnRK2.6 and phosphorylated each other, thus forming the FvRIPK1–FvSnRK2.6 complex. In conclusion, our results provide new insights into the molecular mechanism of FvRIPK1 in plant growth.
... In another study, genetic modification of strawberry was done by transferring antisense orientation of PL (pectate lyase) sequence into strawberries thereby suppressing PL gene. The genetic transformation resulted in production of firmer fruit having enhanced postharvest life without causing other changes in fruit quality attributes such as colour, shape, size or weight (Jiménez-Bermúdez et al., 2002;Qin, Silva, Zhang, & Zhang, 2008;Sesmero, Quesada, & Mercado, 2007). However, the PL gene suppression reduced yield of the transgenic strawberry plants. ...
... The cytosolic DHAR gene led to an increase in ascorbic acid content in tubers and leaves while, chloroplastic DHAR enhanced ascorbic acid content only in leaves. The overexpression of gene encoding for NADPHdependent D galacturonate reductase (GalUR) that is involved in ascorbic acid biosynthesis in strawberry, enhanced vitamin C level in strawberry fruit about 2 to 3-fold (Agius et al., 2003;Qin et al., 2008). In plants, ascorbic acid synthesis mainly takes place by L-galactose pathway. ...
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Genetic engineering techniques offer opportunities for deliberate modulation and enhance the bio-functionality and bio-availability of horticultural crops, particularly fruit and vegetable which can aid in realising the goals of food and nutritional security for the burgeoning world population. The need of the hour calls upon us to use modern-day technologies such as genetic engineering in horticultural field as well for eradication of globally widespread hunger and malnutrition while the population is escalating and climate change is imminent. However, rigorous testing and trials to elucidate any unfounded fear and notion are warranted before the release of genetically modified (GM) crops for ensuring environmental and food safety. In this review, there is an attempt by the authors to compile and discuss the modulation in postharvest biology, edible quality, functional properties and storage life of genetically modified fruit and vegetable along with challenges in their use and consumer acceptance. The review also provides an outline of GM fruit and vegetable research prioritisation along with road map for future work.
... However, while the Southern blot provided evidence for transformation or the potential for low copy numbers of T-DNA inserts, it did not determine the copy number of DNA insertions in strawberry of the other two cases [10,66]. In previously engineered strawberries, the copy numbers of T-DNA inserts ranged from 1 to 5, markedly varying with the strawberry variety and the Agrobacterium strain used [61,[67][68][69][70][71]. Apparently, Agrobacterium strains have the distinct capacity to deliver T-DNA into different hosts, even plants of the same species. ...
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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.
... Strawberries can be cultivated in a wide range of agro-climatic conditions (Martinelli 1992). Hence, commercial production for the fresh fruit market and processing industry has increased dramatically in last two decades (Qin et al. 2008;FAOSTAT 2017). ...
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The objective of this study was to develop a protocol for large-scale propagation of strawberry cv. ‘Sweet Charlie’ and ‘Winter Dawn’ using the micropropagation technique. Shoot cultures were obtained from shoot tips on Murashige and Skoog (MS) medium with 4% table sugar, 0.75% agar, 5 mg L−1 6-benzyladenine and 0.01 mg L−1 kinetin. These shoots were multiplied and maintained on MS medium with 1 mg L–1 6-benzyladenine and 0.1 mg L–1 kinetin. Rooting of in vitro raised shoots was successfully conducted by pulse treatment with 500 mg L–1 indole-3-butyric acid for 30 s and subsequent culturing in MS medium with 1 mg L–1 indole-3-butyric acid, 0.1 g L–1 activated charcoal and 6% table sugar. Plants were successfully acclimatized and survived in field conditions.
... Thidiazuron (TDZ) has also been used as the cytokinin in both diploid and octoploid Fragaria (Zhao et al. 2004;Landi and Mezzetti 2006). The most widely used genetic transformation method for strawberry is Agrobacterium tumefaciencsmediated transformation system (Qin et al. 2008). Controlling the bacteria growth after Agrobacterium-mediated transformation is essential yet challenging as antibiotic can be phytotoxic especially at high concentration . ...
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Fragaria vesca L., a diploid relative of the cultivated octoploid strawberry Fragaria x ananassa, consists of seasonal Fragaria vesca f. vesca (F. vesca) and everbearing Fragaria vesca f. semperflorens (F. v. semperflorens). Study on F. vesca L. showed that F. v. semperflorens had higher in vitro regeneration capabilities than F. vesca although they belong to the same genus and species in the hierarchical rank. F. vesca explants and F. v. semperflorens could not regenerate from either leaf or petiole segments on media without plant growth regulators. Plantlet regeneration from leaf explants was highest on media supplemented with 1 mg/litre 6-benzylaminopurine (BAP) in comparison with those cultured on media supplemented with 2, 3 and 4 mg/litre BAP. Plantlet regeneration from F. v. semperflorens petiole explants was best achieved at 0.1 mg/litre indole-3-butyric acid compared to 0, 0.2 and 0.4 mg/litre.
... Thidiazuron (TDZ) has also been used as the cytokinin in both diploid and octoploid Fragaria (Zhao et al. 2004;Landi and Mezzetti 2006). The most widely used genetic transformation method for strawberry is Agrobacterium tumefaciencsmediated transformation system (Qin et al. 2008). Controlling the bacteria growth after Agrobacterium-mediated transformation is essential yet challenging as antibiotic can be phytotoxic especially at high concentration . ...
... Thidiazuron (TDZ) has also been used as the cytokinin in both diploid and octoploid Fragaria (Zhao et al. 2004;Landi and Mezzetti 2006). The most widely used genetic transformation method for strawberry is Agrobacterium tumefaciencsmediated transformation system (Qin et al. 2008). Controlling the bacteria growth after Agrobacterium-mediated transformation is essential yet challenging as antibiotic can be phytotoxic especially at high concentration . ...
... Thidiazuron (TDZ) has also been used as the cytokinin in both diploid and octoploid Fragaria (Zhao et al. 2004;Landi and Mezzetti 2006). The most widely used genetic transformation method for strawberry is Agrobacterium tumefaciencsmediated transformation system (Qin et al. 2008). Controlling the bacteria growth after Agrobacterium-mediated transformation is essential yet challenging as antibiotic can be phytotoxic especially at high concentration . ...
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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.
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Abstract In order to evaluate the role of paclobutrazol (PBZ) and prohexadione-calcium (ProCa) in reduction of vegetative growth and improvement of reproductive growth of strawberry (cultivar Paros), a 4-replicate completely randomized design was carried out in greenhouse. ProCa in three concentrations of 0, 100 and 150 mg L-1 and PBZ in four concentrations of 0, 60, 90 and 120 mg L-1 were sprayed on the strawberry plants in two stages (four weeks after planting and three weeks after the first stage). The results showed that the application of PBZ, ProCa and also their interaction had a significant effect on plant height, plant fresh weight, petiole length, specific leaf weight, root weight ratio, crown number, fruit number, fruit yield and harvest index. Application of 120 mg L-1 of PBZ without the use of ProCa resulted in the lowest plant height. The interaction of 100 mg L-1 of ProCa and 60 mg L-1 PBZ resulted in the highest number of flowers and crowns. The use of these retardants did not have a significant effect on fruit weight. The effect of ProCa and its interaction with PBZ was significant on the number of flowers and the interaction of PBZ and ProCa was significant on fruit set. Treatment with 100 mg L-1 ProCa without PBZ was the most effective treatment on increasing fruit set. In general, the results showed that the use of retardants reduced the vegetative growth and increased reproductive growth of the Paros strawberry genotype. The concentration of 100 mg L-1 ProCa and 60 mg L-1 PBZ was the most effective treatment for increase in reproductive growth of the examined strawberry cultivar.
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A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T0, T1, and T2 transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants.
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Orthologs of CBF1, a cold-induced transcription factor important in the cold acclimation response in Arabidopsis thaliana were cloned from strawberry (Fragaria x ananassa Duchesne) and sour cherry (Prunus cerasus L.) with degenerate PCR primers. The putative orthologs [Fragatia x ananassa CBF1 (FaCBF1) and Prunus cerasus CBF1 (PcCBF1)] have 48% amino acid identity to CBF1 and mRNA levels were up-regulated in leaves of both crops following exposure to 4 degreesC from 15 minutes to 24 hours. However, mRNA of FaCBF1 and PcCBF1 was not detected in pistils of strawberry and sour cherry following 4 degreesC exposure. Agrobacterium-mediated transformation of a CaMV35S-CBF1 construct was conducted on Fragaria x ananassa 'Honeoye' crown discs. Two transgenic lines were regenerated that expressed the transgene at low levels in both leaves and receptacles. Receptacles of the transgenic lines showed no significant change in freezing tolerance when compared to wild type plants, although the temperature at which 50% electrolyte leakage occurred in detached leaf discs from the two transgenic lines was -8.2 degreesC and -10.3 degreesC, respectively. These freezing tolerance values were significantly greater than the value for the wild-type 'Honeoye' leaf discs of -6.4 degreesC.
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A "defensin-like" antibacterial peptide from Mytilus edulis chilensis, was sub-cloned into a binary vector for expression in plant tissues. The resulting new clone was electroporated into A. tumefaciens to transform tobacco plants. The presence of the construct in transgenic tobacco lines was demonstrated through RT-PCR, Northern and Western blots. Transformed positive plants were selected and grown for challenging. Tobacco leaves were infiltrated with Pseudomonas syringae pv . syringae and visual lesions determined at different times post-exposure. Of seven plants exposed, four gave variable protection up to seven days post-infection while one of them appears to be fully protected. These results suggest that defensin-like antimicrobial peptides from molluscs are a good source to provide resistance of tobacco plants to Pseudomonas syringae pv . syringae .