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Biotechnology Advances 26 (2008) 219 – 232

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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

⁎ Corresponding authors. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. Tel./fax: +86 571 8697 1009.
E-mail addresses: [email protected] (Y. Qin), [email protected] (S. Zhang).

0734-9750/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.12.004
220 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

1. Introduction 2. Establishment of a genetic transformation system

of strawberry
Strawberry (Fragaria × ananassa Duch.), genus Fragaria of
the Rosaceae family, is one of the most important economical fruit The key to successfully develop a genetic modified
crops that adapts to various environmental growing conditions strawberry cultivar is to establish an efficient regeneration and
throughout the world (Hancock, 1999). Strawberries are not only transformation system, which provides the protocol for the
attractive for their physical appearance and physiological features, selection and recovery of genetically modified plants following
such as bright colors, delicious taste, fine texture and fresh aroma, gene transfer. The application of strawberry culture in vitro in
but also for their high economical and nutritional values, such as genetic transformation and biotechnology has recently been
essential minerals, organic (amino) acids, vitamins and antiox- reviewed in depth by Debnath and Teixeira da Silva (2007) and
idant properties. thus the focus of this review will be purely on transgenic
Strawberries can be grown in a wide range of climates and, strategies for cultivar improvement. Various characteristics of
for this reason, commercial production for the fresh fruit market some well-studied examples of strawberry (Fragaria × ana-
and processing industry has increased dramatically in recent nassa and Fragaria vesca L.) genetic transformation using
years, and its worldwide production among the berries ranks reporter genes are summarized in Table 1. Reporter genes, such
second after fresh grape. Strawberries have experienced one of as β-glucuronidase (GUS or uidA), neomycin phosphotransfer-
the highest increases in rates of consumption in all fruit crops, ase (nptII), and hygromycin phosphotransferase (hpt) were first
and the demand has continued to grow rapidly during the past used to screen the optimum conditions for the establishment of
decade (FAOSTAT, 2007). The recent harvested area of an efficient genetic transformation system of strawberry.
strawberry in the world was approximately 250,000 ha. Annual The most effective and widely used method for strawberry
production of strawberry was 3.3, 3.5 and 3.7 million tons in gene engineering is Agrobacterium tumefaciens-mediated
2003, 2004 and 2005, respectively. The United States is the transformation using strawberry leaf disk, petiole, aminae,
largest producer of strawberries, accounting for over a quarter of stalk and calli as explants and a bacterial plasmid as a vector
total production in the world and the second largest total for the genes of interest (Table 1). Moreover, there are reports in
harvested area after Poland (FAOSTAT, 2007). The future of which biolistics (Wang et al., 2004), protoplast electropo-
strawberry production and sales is very promising. Strawberry ration (Nyman and Wallin, 1992) and a method combining
production, sales price, trading prospect, and consumption are Agrobacterium infection and biolistic bombardment (Cordero
expected to increase continuously over the next decade. de Mesa et al., 2000) have been also used to obtain transgenic
Although years of efforts from traditional breeding have strawberry.
resulted in steady improvement in various aspects of strawberry The first transformation of strawberry was attempted in 1990
production, many areas, such as yield, fruit size and shape, (Nehra et al., 1990a,b). Nehra et al. (1990a,b) inoculated
berry quality, complex ploidy of strawberries, viz. diploidy, strawberry leaf disks and calli with A. tumefaciens strain MP90
tetra-, hexa-, octo- and even decaploid, as well as interspecific which contains nptII and gus genes to yield a transformation
hybrids with intermediate ploidy levels, still need to be explored frequency of 6.5% for leaf disks and 3% for calli where all of the
(Galletta and Maas, 1990; Potter et al., 2000; Faedi et al., 2002; transgenic calli and shoots expressed varying levels of nptII and
Marta et al., 2004; Folta and Davis, 2006). The process of GUS enzyme activity. Integration of both marker genes into the
developing polyploid strawberries to select desirable agrono- strawberry genome was further confirmed by Southern blot
mical traits through traditional breeding programs is very analysis. Subsequently, James et al. (1990a,b) infected strawberry
inefficient, labor-intensive, and time-consuming (Galletta and leaf disks and stalks with A. tumefaciens carrying a disarmed
Maas, 1990; Faedi et al., 2002; Marta et al., 2004). Therefore, binary vector plasmid pBIN6 containing the nos (nopaline
genetic engineering and biotechnology applications are increas- synthase) and nptII genes and plasmid pSS1 containing the
ingly being used to improve strawberry qualities and expand the nptII and ipt (isopentenyl-transferase) genes. The nos and nptII
breeding base and germplasm utilization. Genetic engineering genes in the transgenic plants were expressed using pBIN6 as the
offers a realistic possibility to create varieties that selectively vector. However, the phenotype of transgenic plants with ipt
target a gene or a few heterologous traits for introduction into genes was dwarfed, caspitose and difficult to induce roots.
the strawberry genome, by facilitating the process of obtaining Similarly, Zhang and Wu (1998) developed a transformation
desirable agronomical traits that exhibit increased strawberry system for the commercial strawberry cultivar ‘Tudla’ and
resistance to pests, herbicides, diseases, environmental stresses obtained transformed plants. After the leaf pieces were co-
as well as desired enhancement of fruit qualities. Great advances cultivated for 3 d with Agrobacterium strain EHA105 containing
have been made in strawberry genetic transformation since the the binary vector pMOG410, then incubated on regeneration
first transgenic strawberry plant was introduced in 1990 (Nehra medium containing 40 mg/L Kanamycin (Kan) for four weeks,
et al., 1990a,b). This review paper provides an overview of the the transformed buds appeared obviously on the explants. The
advances on strawberry transgenic research, consolidating the regeneration percentage of transformed buds was 4.5% and a few
large body of information regarding genetic modification of this transformants expressed strong GUS activity following histo-
important genus. Furthermore, an assessment of the perfor- chemical analysis. Selection in this study was surprisingly high
mance of transgenic strawberry and its potential in strawberry since strawberry tissues are extremely sensitive to Kan, and
genetic transformation are proposed. concentrations even at as low as 10 mg/L impair shoot
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 3–6.5 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 1–5 × 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, nptII 35S pKIWI105 p35SGUS AM – Yes Yes Yes Martinelli et al. (1997)

Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

petioles
Chandler Calli GUS, nptII 35S, pGUSINT AM-biolistic 20 No Yes Yes Cordero de Mesa et al.
(2000, 2004)
BHN FL90031–30 or Leaf GUS, nptII eFMV/eBigMac/ AM 6.57 No Yes Yes Janette et al. (2001)
BHN 92664-501 nos pCGN
Kinuama, Senga Sengana, Leaf GUS, nptII 35S, pMOG410 AM 3.7–6.8 Yes No Yes Zhang and Wu (1998);
Tudla, Toyonoka 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; Yes Yes Yes Zhao et al. (2004)
7.4 for La Sans Rivale
Induka, Elista Leaf laminae GUS, nptII 35S, pBIN19 AM 8.3 for Elista; Yes No Yes Gruchala et al. (2004)
4.2 for Induka
Allstar Leaf GUS, nptII 35S pBLGC AM 1.1 No No Yes Zhang and Wang (2005)
LF9 Petiole nptII 35S, pCAMBIA, AM – No No Yes Folta et al. (2006)
pHK1001
Chandler Leaf nptII, trfA 35S pBINPLUS, AM 65.7 Yes Yes No Abdal-Aziz et al. (2006)
pGUSINT, pBIN19
Fragaria vesca L. Germinated plants Leaf GUS, nptII 35S, pBI121 AM 6.9 Yes No Yes El-Mansouri et al. (1996)
from seeds
Alpine accession FRA197 Leaf GUS, nptII 35S, pBI121 AM – Yes Yes Yes Haymes and Davis (1998)
Germinated plants Leaf, petiole GUS, nptII 35S, pCIRCE AM 15 Yes Yes Yes Alsheikh et al. (2002)
from seeds
Alpine accession Leaf, petiole GUS, nptII AtSUC2, pBISPG AM 64.4 for FRA197; Yes Yes Yes Zhao et al. (2004)
FRA197 and 198 67.9 for FRA198
Germinated plants Leaf, petiole nptII, GUS, GFP 35S, pCAMBIA1304 AM 11–100 Yes No Yes Phillip (2005)
from seeds pBBR1MCS-5.virGN54D
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; PAT: 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.

221
222 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

regeneration (Barceló et al., 1998; Alsheikh et al., 2002; Gruchala (2n = 2x = 14) strawberry, is an attractive model for studying
et al., 2004; Qin and Zhang, 2007). ripening in non-climacteric fruit, breeding, genetic and
The key to Agrobacterium-mediated transformation in molecular research in Rosaceae because of its small genome
strawberry depends not only on the establishment of an efficient size, small plant size and a short life cycle for transformation
regeneration system (Debnath and Teixeira da Silva, 2007) but (Hancock and Luby, 1993; El-Mansouri et al., 1996; Sargent
also on the selection and recovery of transformed cells following et al., 2004; Zhao et al., 2004; Phillip, 2005; Oosumi et al.,
organogenesis. Mathews et al. (1998) reported an efficient 2006). El-Mansouri et al. (1996) infected leaf disks of F. vesca
protocol to obtain pure transgenic plants. Two experiments were using A. tumefaciens LBA4404 carrying the plasmid pBI121
conducted with and without repeated (iterative) cultures to containing gus and nptII genes. Transformation efficiency was
induce transgenic shoots on selection medium. The concentra- 5–7% according to the GUS assay. Haymes and Davis (1998)
tions of Kan in the non-iterative method were kept constant, were the first to show the suitability of ‘Alpine’ F. vesca for
while in the iterative protocol, the Kan levels increased gradually transgene research in strawberry by demonstrating the transmis-
during subculture. The results showed that there were non- sion of the gus and nptII genes to the R1 progeny. However, no
chimeric plants using the iterative protocol. Therefore, they transformation efficiency was reported by Haymes and Davis
believed that the iterative culture was an effective technique to (1998). Alsheikh et al. (2002) and Zhao et al. (2004) studied
obtain pure transgenic lines. F. vesca, they achieved 15% and 64.4–67.9% transformation
Genotype is an important factor that significantly influences efficiency, respectively. In recent years, several scientists de-
transformation efficiency. Strawberry cultivars with different scribed a new transformation procedure that uses leaf explants
genetic backgrounds may respond quite differently to a given from newly unfolded trifoliate leaves obtained from stock plants
regeneration and transformation protocol (Table 1). Wawrzyńc- 6–7 weeks after seed germination, following co-cultivation with
zak et al. (2000) explored the transformation efficiency of five Agrobacterium strain GV3101, and then selection on MS medium
strawberry cultivars ‘Elsanta’, ‘Kaster’, ‘K-1349’, ‘K-1476’ and containing 4 mg/L hygromycin. Using this protocol, 100%
‘Senga Sengana’ by Agrobacterium-mediated transformation of transformation efficiency was obtained for 6 of 14 F. vesca ac-
leaf explants. Transformation efficiency in different genotypes cessions tested (Oosumi et al., 2006). Phillip (2005) developed an
varied, with the highest rate for ‘Senga Sengana’ (5.71%) and efficient high-throughput Agrobacterium-mediated transforma-
the lowest rate for K-1349 (1.67%). The incorporation of the tion protocol of F. vesca following the procedure of Oosumi et al.
transgene was confirmed by PCR and its expression by GUS (2006) with a minor modification. Transformation efficiencies
assay. Cordero de Mesa et al. (2000) used Agrobacterium- ranging from 11 to 100% were obtained for two F. vesca
coated gold particles and a gene gun to bombard ‘Chandler’ accessions. Multiplex PCR, for amplification of the nptII and
calli. After twenty-five weeks in culture, they reported a 20% GFP genes, was performed on a random sample of GFP plants
transformation rate. Gruchala et al. (2004) tested the regenera- to verify integration of the T-DNA (Phillip, 2005).
tion capability and transformation efficiency of two strawberry Over the past decade, many laboratories have developed
cultivars in attempts to establish a transformation system for transformation systems for different strawberry cultivars
strawberry and found that it was highly genotype-dependent. (Table 1). These techniques provide an important foundation for
On MS medium with 0.4 mg/L IBA and 1.8 mg/L BA, the conferring commercial strawberry cultivars resistance to insects,
number of regenerated shoots ‘Induka’ (3.5 shoots/explant) was viruses, fungi, herbicides, stress and improving fruit quality
about twice more than ‘Elista’ (1.8 shoots/explant). However, through biotechnology.
after plant transformation using A. tumefaciens LBA4404 strain
containing plasmid pBIN19 with nptII and gus genes, the 3. Transgenic strawberry for insect resistance
number of transgenic ‘Elista’ was about 2-fold higher (8.3
shoots/100 explants) than ‘Induka’ (4.2 shoots/100 explants). Tarnished plant bugs (Lygus lineolaris) and strawberry bud
Besides genotype, plant transformation efficiency also weevils (Anthonomus signatus Say) are two common insects in
depends on many factors such as antibiotic types and concentra- strawberries. These two insects can cause major damage to
tions, inoculation and co-culture period and the presence or strawberry flowers and buds, reducing yields significantly.
absence of acetosyringone (AS). Zhang and Wang (2005) initiated However, over-application of insecticides can be a serious
a detailed study to investigate factors that influenced the problem to the environment and negatively affect human health
transformation efficiency of ‘Allstar’ strawberry; and discovered as well as accelerating insecticide resistance. Environmentally-
that the optimal conditions for Agrobacterium-mediated transfor- friendly synthetic bioinsecticides are highly specific, and are
mation of ‘Allstar’ leaf disks were: inoculation for 10–15 min, co- variably efficacious due to the influences of various biotic and
culture for 3 d plus 50 µM AS, and then transferred to a selection abiotic factors. Therefore, scientists have been looking for new
medium containing 25 mg/L Kan plus 450 mg/L Carbenicillin strategies to develop and improve insect-resistant strawberries.
(Carb). All of these factors contribute to the further improvement Novel biotechnological tools have facilitated the introduction of
of transformation efficiency. A transformation frequency of 1.1% genes into strawberry to combat insects. One potential
was achieved based on Kan resistance assays. possibility is for the strawberry plant itself to produce specific
The cultivated strawberry, Fragaria × ananassa Duch., is an proteins with insecticidal activity since a plant harboring a
octoploid (2n = 8x = 56) species and the high ploidy level makes protease inhibitor is part of the natural defense system against
genetic and molecular studies difficult. F. vesca L., a diploid insect predation. Plants transformed with foreign plant protease
 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232 223

inhibitor genes can enhance the resistance to insects. The enhanced chitinase levels in transgenic strawberry plants can
cowpea (Vigna unguiculata) protease trypsin inhibitor (CpTi) indeed reduce the damage caused by powdery mildew fungi
gene, an insecticidal gene, has been successfully introduced into (Asao et al., 1997, 2003). Chalavi et al. (2003) isolated a chitinase
strawberry. Transgenic strawberry lines constitutively expres- gene (pcht28) from Lycopersicon chilense and transferred it into
sing the CpTi gene could protect against the feeding of vine ‘Joliette’ strawberry using Agrobacterium-mediated transforma-
weevil (Otiorhynchus sulcatus) under greenhouse and field tion. Introduction of the pcht28 gene was verified by Southern
conditions (James et al., 1992; Graham et al., 1995, 1997, 2001, blot analysis and its expression by Northern blot. In growth
2002). Compared with control lines, transgenic strawberry lines chamber studies, the transgenic strawberry plants that expressed
significantly improved plant growth and development. The pcht28 had significantly higher resistance to Verticillium dahliae.
CpTi gene significantly affected vine weevil by reducing the Ricardo et al. (2006) obtained transgenic strawberry lines by
survival of weevil larvae and the number of pupae following an expressing three defense genes: ch5B (encoding a chitinase
insect bioassay. Moreover, there was no significant effect of the protein from kidney bean (Phaseolus vulgaris)), gln2 (encoding a
CpTi transgenic lines on the numbers of Carabid and other non- glucanase protein from tobacco (Nicotiana tabacum)), and ap24
target arthropods. (encoding a thaumatin-like protein from tobacco). They evaluated
the effects of these genes on the protection against gray mold and
4. Transgenic strawberry for phytopathogenic fungi, anthracnose diseases produced by local strains of B. cinerea and
bacteria and virus resistance C. acutatum, respectively. Sixteen transgenic lines expressing
one or a combination of two defense genes were obtained. The
Strawberry is susceptible to various phytopathogenic fungi, results showed that the expression of the ch5B gene in transgenic
bacteria and viruses, which are associated with strawberry strawberry increased the resistance to gray mold while it had no
diseases, causing fruit deformation, leaf yellowing, root and significant effect on anthracnose disease resistance. The resis-
crown disease, plant growth stunting, and substantial yield loss to tance was correlated with the presence of the foreign ch5B protein
strawberry production as well as plant death (Simpson, 1991; and an increase of chitinolytic activity in leaves. These results
Mass, 1998).These diseases are difficult to control through demonstrate that chitinases play a key role in defense against
traditional breeding methods due to lack of both curative methods fungal disease in strawberry. Recent findings by Mercado et al.
and resistant varieties (Simpson, 1991). Using chemical insecti- (2007) had indicated that expression of a β-1,3-glucanase gene
cides or fungicides to control strawberry diseases are not very isolated from the antagonist soil fungus Trichoderma harzianum
practical because strawberry fruit is a directly consumed product. in strawberry enhanced anthracnose resistance.
Development of biotechnology has provided a new opportunity to Glucose oxidase (GO) is closely related to resistance of plant
enhance disease resistance for strawberry breeding. Finstad and fungal diseases. It catalyzes the oxidation of β-D-glucose to D-
Martin (1995) obtained transformed strawberry plants using a glucono-1,5-lactone and hydrogen peroxide (H2O2) while the
coat protein (cp) gene from Strawberry mild yellow edge production of H2O2 is toxic to phytopathogenic fungi, which is
potexvirus (SMYELV-CP), which conferred resistance to the usually responsible for most major diseases in many crops (Kim
virus. The results of PCR, Southern blot, and ELISA showed that et al., 1988). A study was initiated to isolate the GO gene from
the cp gene was successfully incorporated and stably expressed. Aspergillus niger and introduce it into strawberry by Agrobac-
Subsequently, Martinelli et al. (1996) obtained transgenic terium-mediated transformation. Compared to control plants, a
strawberry using A. tumefaciens LBA4404 carrying the plasmid higher concentration of H2O2 was detected in GO transgenic
pKyLX71 containing osmotin and nptII genes. Compared to strawberry lines, which resulted in increased resistance to gray
control plants, transgenic strawberry lines had higher levels of mold (Jin et al., 2005).
fungicidal activity and significantly increased resistance to wilt Thaumatin-like proteins (TLP), which belong to the PR-5
and gray mold diseases. (pathogenesis-related) group of proteins, have different degrees
Gray mold caused by Botrytis cinerea and anthracnose of specificities to antifungal activity. TLP has been successfully
diseases produced by Colletotrichum fungi are the two most used to enhance plant resistance to fungal pathogens (Chen
destructive strawberry diseases that bring the majority of yield et al., 1999; Datta et al., 1999; Schestibratov and Dolgov, 2005).
losses to strawberry farmers (Sutton et al., 1988; Sutton, 1990; Schestibratov and Dolgov (2005) reported that a thaumatin II
Horowitz et al., 2002; Legard et al., 2003; Cesar et al., 2006). cDNA driven by the CaMV 35S promoter was introduced into
Plants respond to biotic stress by inducing a set of genes encoding strawberry via Agrobacterium-mediated transformation. The
diverse proteins, many of which are believed to play a self- tested transgenic lines expressing the TLP showed a signifi-
defensive role against pathogens. Among these proteins, the most cantly higher level of resistance to gray mold after infection
significant one is chitinase which is an endo-type enzyme that with a conidial suspension.
hydrolyzes chitin, a structural component of the cell walls of Antimicrobial peptide (AMP) is a class of micromolecule
fungi, shells of crustaceans, and the integument and peritrophic polypeptides that plants synthesize to defend themselves against
membranes of insects. Chitinase is a component of normal pre- the invasion of environmental microbes; most of them are
existing defense mechanisms in many plants that can be used as a cationic peptides with good thermal stability (Theis and Stahl,
possible biocontrol agent instead of chemical fungicides 2004; Brogden, 2005). AMP is an inducible insect peptide with
(Schlumbaum et al., 1986; Samac et al., 1990; Collinge et al., a broad range of activities in resisting bacteria and fungi in
1993; Karasuda et al., 2003). Researchers have reported that plants (Broekaert et al., 1995; Mariana and Wagner, 2005). A
224 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

Fig. 1. The KanR buds and plantlets from strawberry leaf disks infected with Agrobacterium EH105 harboring APD gene. A: KanR buds; B: KanR plantlets;
C: acclimatized KanR plantlets.

number of studies have demonstrated the expression of salt tolerance has been attempted for a long time; however, it has
heterologous AMP in plants with different degrees of enhanced resulted in little progress (Kaya et al., 2002a,b; Dziadczyk et al.,
resistance to pathogens (Huang et al., 1997; Sharma et al., 2000; 2003; 2005). The direct introduction of a number of genes by
Arenas et al., 2006). Qin et al. (2005a,b,c), Qin and Zhang, genetic engineering offers a convenient and effective way to
2007) extensively investigated the factors that influenced the improve stress tolerance and achieve prodigious progress.
regeneration and transformation efficiency of ‘Toyonoka’ Researchers have identified genes that play a key role in stress
strawberry. By establishing an efficient regeneration system, tolerance by identifying proteins regulated in response to stress
antimicrobial peptide-D gene (APD), driven by a 35S promoter (Teixeira da Silva, 2006b), which include betaine aldehyde
to ‘Toyonoka’ via Agrobacterium-mediated transformation, can dehydrogenase (BADH) (Weretilnyk and Hanson, 1987, 1990),
be successfully transferred. Kanamycin-resistant (KanR) buds late embryogenesis abundant proteins (LEAs) (Wise, 2003),
and shoots were obtained from the explants (Fig. 1A, B). PCR cold-induced transcription factor (CBF1) (Gilmour et al., 1998)
analysis and Southern hybridization showed that the APD gene and antifreeze protein (AFP) (Georges et al., 1990). Transgenic
was integrated into the strawberry genome (Figs. 2 and 3). strawberry plants with the above genes exhibited constitutive
Among the six transformants tested, one contained two copies, activation of stress responsive genes and enhanced salt (Liu
two contained five copies, and the others contained a single et al., 1997; Wang et al., 2004) and freezing tolerance (Firsov
copy. No hybridization signal was detected in DNA from the and Dolgov, 1999; Owens et al., 2002, 2003; Khammuang et al.,
non-transformed control plants (Fig. 3). Transgenic strawberry 2005).
was acclimatized in the greenhouse for further analysis Liu et al. (1997) obtained transgenic strawberry plants using
(Fig. 1C). the BADH gene driven by the 35S promoter. The expression of
this gene was confirmed by Northern blot and a BADH enzyme-
5. Transgenic strawberry for stress resistance assay. The transgenic plants grew normally in medium
containing 0.4–0.7% (w/v) NaCl while all untransformed
Abiotic stresses, such as salinity and low temperatures, are plants died on medium with 0.4% NaCl after 20 d. The relative
part of the limiting factors in strawberry growth and develop- electronic conductivity and membrane permeability demon-
ment. Abiotic stresses can cause a series of morphological, strated that the damage of the membrane structure of transgenic
physiological, biochemical and molecular changes that plants was lower than the controls'. This may be one of the
adversely affect plant growth and productivity (Awang et al.,
1993; Keutgen and Keutgen, 2003). Conventional breeding for

Fig. 2. PCR analysis of 122 bp fragment corresponding to APD. Lane 1: marker Fig. 3. Southern blot analysis of DNA from control and transgenic strawberry
(DL2000); lane 2: positive control; lanes 3–9: transgenic strawberry plants; lane plants digested with Hind III and probed with a 122 bp fragment of APD. Lanes
10: non-transformed plants. 1–6: transgenic strawberry plants; lane 7: non-transformed plant.
 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232 225

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, Stem tissues Invertase 35S AM Yes No No Bachelie et al. (1997)
Senga Sengana
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: nopaline synthase promoter; APX: fruit-dominant ascorbate peroxidase promoter; rolB: rhizogeges; DefH9: ovule-specific
promoter; FBP7: floral binding protein 7 promoter; CaMV 35S: cauliflower mosaic virus promoter; 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-tryptophan monooxygenase.

reasons for the higher salt tolerance of transgenic plants. LEAs were significantly greater than that of the wt ‘Honeoye’
have been suggested to increase salt tolerance in plants by (− 6.4 °C).
binding water and maintaining the structure of other proteins WCOR410 belongs to a different subtype of the D-11 protein
and membranes (Close, 1996). A group of scientists transferred family, the so-called acidic dehydrins, playing a role in
the LEA3 gene into strawberry by particle bombardment, which preventing the destabilization of the plasma membrane during
confirmed the integration of the gene into strawberry genomes water stress and low temperature, and could be a determining
by Southern blots. Results of NaCl salt stress experiments factor for increased cell resistance to freezing. Expression of the
indicated that the LEA3 gene significantly increased the acidic dehydrin gene Wcor410 cloned from wheat was found to
resistance of strawberry to salt tolerance (Wang et al., 2004). be associated with the development of freezing tolerance
Many rosaceous fruit crops suffer yield reductions due to (Danyluk et al., 1994, 1998). Transgenic strawberry plants
early season freezes during or just prior to bloom (Ki and expressing the Wcor410 gene had a 5 °C improvement of
Warmund, 1992). Re-engineering plants for greater freezing freezing tolerance than wt or transformed leaves not expressing
tolerance through plant transformation is a potential way to the Wcor410 protein (Houde et al., 2004). The results suggest
reduce the damage caused by freezing. Great progress has been that Wcor410 is involved in the cryoprotection of the plasma
made in strawberry in the past few years in terms of improving membrane against freezing stress to improve freezing tolerance
freezing tolerance through genetic transformation. By develop- in leaves of transgenic strawberry.
ing a protocol for Agrobacterium transformation of strawberry
leaf explants, transgenic plants have been obtained containing 6. Transgenic strawberry for herbicide resistance
the AFP gene of winter flounder (Firsov and Dolgov, 1999;
Khammuang et al., 2005). The integration of nptII and AFP Various weeds constantly compete with strawberry plants for
gene was confirmed by PCR analysis (Firsov and Dolgov, available water, nutrients and light energy; weed pressure is
1999). However, no further study of agronomic traits was further aggravated by disease, nematodes and insects, and thus
reported. affects strawberry growth and causing yield reduction.
To determine the function of CBF1 in enhancing strawberry Historically, very few herbicides can be used for weed control
freezing tolerance, Owens et al. (2002, 2003) obtained in strawberry production due to its perennial nature. Recently,
transgenic strawberry plants via Agrobacterium-mediated genetic engineering has been applied to develop herbicide-
transformation harboring the CBF1 gene driven by the 35S resistant varieties in strawberry through the introduction of
promoter. Two transformants expressed the gene at low levels in glufosinate and glyphosate-resistant genes. Scientists have
both leaves and receptacles with pistils. No difference in obtained transgenic strawberry plants via Agrobacterium-
freezing tolerance was detected between receptacles with mediated transformation containing the gus or pat genes driven
attached pistils of the transformants and wild type (wt) plants. by the 35S promoter in binary vectors pBI121 and pBIN19,
However, the freezing tolerance values of temperature at which respectively (du Plessis et al., 1997). A 10% transformation
50% electrolyte leakage (a rapid index of plant vitality for biotic frequency was obtained on a shoot-inducing medium containing
or abiotic stress) occurred in detached leaf disks from the two 50 mg/L Kan and most KanR shoots that originated from
transgenic lines was − 8.2 °C and − 10.3 °C respectively, which explants transformed with pBI121 expressed the gus gene.
226 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

Field trials revealed that most transgenic lines which contained Jiménez-Bermúdez et al. (2002) transferred the antisense
the pat gene were resistant to the herbicide glufosinate- orientation of the PL sequence driven by the 35S promoter
ammonium. Subsequently, Zhang et al. (2001) obtained into strawberries. In most transformed ‘Apel’ lines, total yield
transgenic plants using the gus and bar genes via A. was significantly reduced. However, suppression of the PL
tumefaciens-mediated transformation by developing an efficient mRNA gene in strawberry by an antisense transformation
and stable regeneration system and genetic transformation significantly increased the firmness of ripe fruits and extended
system for the commercial strawberry cultivar ‘Tudla’. A high the post-harvest shelf-life without significantly affecting other
level of GUS activity was detected in five transgenic strawberry fruit characteristics such as color, size, shape and weight during
lines from 10 independent shoots putatively transformed with fruit ripening. In all six ‘Apel’ lines tested, expression of the PL
the gus gene. The transgenic strawberry plants transformed with gene in ripened fruit was 30% lower than that of the control, and
the bar gene were able to differentiate on medium containing three of them were completely suppressed. Compared to control
10 mg/L glufosinate and showed complete resistance to 450 mg/ fruit, transformed ripened ‘Apel’ fruit had a lower degree of in
L glufosinate after three applications of foliage spray in the vitro swelling and a lower amount of ionically bound pectins.
field. The transgenic plants flowered and set fruits normally The post-harvest softening of ‘Apel’ fruit was also diminished.
while the untransformed plant died after 10 d. Meanwhile, Recently, using an antisense sequence of a strawberry PL gene,
Morgan et al. (2002) obtained strawberry varieties tolerant to Sesmero et al. (2007) evaluated the effect of this transgenic
glyphosate following integration of the CP4.EPSP (5-enolpyr- modification on the texture of frozen and thawed fruits and the
uvylshikimate 3-phosphate) synthase gene through Agrobac- jam after strawberry processing. The mRNA transcript level of
terium-mediated transformation. Among the 73 independent PL was significantly reduced in two independent lines (90% in
transformants that were sprayed with commercial levels of ‘Apel 14’ and 99% in ‘Apel 23’). At harvest, the ripened fruits'
Roundup Ultra® in the nursery, a range of tolerance to the firmness from the two lines was significantly higher than that of
herbicide was shown in those transformants ranging from the controls. Transgenic fruits resisted the cooking process
complete tolerance to no resistance. Introduction of the CP4. better than the conventional ones in terms of having a higher
EPSP gene was confirmed by Southern blots and its expression amount of fruit berries in these jams and these processed fruits
was verified by Northern analysis. Preliminary assessment of were firmer than the control. The degree of firmness was posi-
fruit characteristics and yield data suggested that some tively correlated with the degree of PL silencing. In contrast,
glyphosate-resistant lines produced good quality fruit typical jams of these transgenic lines were similar in firmness but
of ‘Camarosa’ (Morgan et al., 2002). slightly less viscous than the control. These results indicated
that the PL gene has a great potential to be used for preventing
7. Transgenic strawberry for fruit quality improvement fruit softening in strawberry through biotechnology. Suppres-
sion or silencing of the PL gene in strawberry fruit can improve
Strawberry is a delicate fruit with a short shelf-life, mainly quality traits of strawberry jam, such as texture and content of
due to a rapid loss of firmness in texture. To prevent fruit whole berries.
softening in order to prolong shelf-life and to improve fruit Plant endo-β-(1,4)-glucanases (EGases) are hydrolytic
quality of strawberry, extensive studies have been carried out enzymes that are active against β-(1,4)-glucan links. Early
(Table 2). Antisense technology is a useful tool to prevent research indicated that EGase activity was involved in cell wall
strawberry fruit from softening by suppressing particular genes weakening, which ranged from cell wall expansion to fruit
involved in fruit softening without altering fruit quality ripening and disassembly (Trainotti et al., 1999a,b). Fruit
(Mathews et al., 1995; Woolley et al., 2001; Jiménez-Bermúdez softening during ripening is associated with the overlapping
et al., 2002; Palomer et al., 2006; Sesmero et al., 2007). presence of two divergent EGases, Cel1 and Cel2. Woolley et al.
Ethylene, a gaseous hormone, is produced in all higher plants (2001) studied the role of the Cel1 protein in fruit softening using
and can stimulate fruit ripening. The 1-aminocyclopropane-1- antisense technology and obtained transgenic plants with reduced
carboxylic acid (ACC) synthase is the limiting factor in ethylene cel1 mRNA levels. However, they found that the constitutive
production and it can be manipulated through biotechnology to antisense down-regulation of Cel1 transcripts in strawberry plants
delay fruit ripening. The control of ethylene production has did not significantly alter EGase activity and fruit firmness. To
been studied extensively. S-adenosylmethionine (SAM) is the further explore the role of Cel1 and Cel2 EGases in strawberry
metabolic precursor of ACC synthase, the proximal precursor to fruit softening, Palomer et al. (2006) obtained transgenic
ethylene. In recent years, biotechnology has been used to delay strawberry plants using antisense constructs for individual
the ripening of strawberry using the SAMase gene by down- silencing of Cel1 and Cel2 and a chimeric antisense Cel1/Cel2
regulating ACC synthase. Integration of the gene was under the control of a fruit-specific promoter (FBP7). The results
confirmed by Southern blot. However, no further report was showed that constant down-regulation of Cel1 expression
found regarding transgenic fruit ripening and shelf-life throughout ripening was accompanied by reduced Cel1 protein
(Mathews et al., 1995). accumulation. However, no difference in fruit firmness was found
Pectate lyase (PL) is an extracellular enzyme involved in cell between transgenic lines and control plants with a reduction of
wall disassembly and maceration during fruit ripening (Jimé- Cel1 protein level and EGase activity. Moreover, there was no
nez-Bermúdez et al., 2002; Marín-Rodríguez et al., 2002). To significant reduction of Cel2 protein accumulation in any of the
effectively control or delay fruit softening in strawberry, Cel2 transgenic or Cel1/Cel2 double-transgenic lines. Therefore,
 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232 227

they suggested that Cel1 alone is not the major determinant of responsible for catalyzing the breakdown of sucrose in many
strawberry fruit softening during ripening while Cel2 might be fruit species (Sturm, 1999). To study the role of invertase in
responsible for fruit development prior to ripening, thus strawberry ripening, Bachelie et al. (1997) cloned two invertase
accounting for the lack of Cel2 protein down-regulation observed genes from potato, encoding cell wall and vacuolar forms
(Palomer et al., 2006). These studies paved the way for respectively, and integrated them into two strawberry cultivars
understanding the biochemical mechanisms of strawberry fruit ‘Symphony’ and ‘Senga Sengana’ via A. tumefaciens. The
softening. presence of the invertase genes in strawberry was confirmed by
Strawberry is a non-climacteric fruit and its ripening PCR analysis. However, no further study was found regarding
mechanism is unclear. Annexin plays an important role in transgenic fruit characteristics such as sugar balance, flavor and
fruit development and ripening (Wilkinson et al., 1995). In processing quality. Recently, Park et al. (2006) generated
order to elucidate the function of the annfaf (annexin of Fra- transgenic plants that incorporated an antisense cDNA of ADP-
garia × ananassa fruit) gene in ripening processes of strawberry, glucose pyrophosphorylase (AGPase) small subunit (FagpS)
Na et al. (2006) isolated the gene from strawberry fruit and driven by the strawberry fruit-dominant ascorbate peroxidase
transferred it into the strawberry genome using an antisense (APX) promoter, to evaluate the effects on carbohydrate
fusion annfaf gene. Southern blot confirmed that the gene was contents during fruit development. The results showed that
integrated into the strawberry genome as a single copy. Results the levels of AGPase mRNA were drastically reduced in the red
from this study provided the foundation for selecting deficient stage of fruits in all the transgenic plants. The suppression of the
transgenic plants, which could be beneficial for further studies AGPase small subunit in transgenic plants resulted in a 16–37%
on the mechanism of maturation of strawberry fruits and for increment of total soluble sugar content and a 27–47% decrease
breeding of traits related to freshness in strawberry (Wang et al., of the starch content in mature fruit without significantly
2001; Na et al., 2006). affecting other fruit characteristics such as color, weight and
Advanced knowledge on strawberry fruit productivity hardness. Results from previous studies suggested that, through
through genetic engineering is available. Auxin (IAA) produced biotechnological alternation, the AGPase gene might be used
by fertilized ovules is essential for strawberry fruit development for improving soluble sugar content and decreasing starch
and quality (Manning, 1994; 1998). To explore this role, content in strawberry fruits.
scientists have introduced the parthenocarpic chimeric gene Vitamin C is an essential component of human nutrition.
(DefH9-iaaM) into two strawberry species (F. ananassa cv. Agius et al. (2003) cloned a GalUR promoter from strawberry
‘AN93.231.53’ and F. vesca cv. ‘Alpina W. Original’) by A. that encodes an NADPH-dependent D-galacturonate reductase,
tumefaciens-mediated transformation (Mezzetti et al., 2002; an enzyme involved in the biosynthesis of vitamin C in
2004a,b; Mezzetti and Costantini, 2006). Transgenic strawberry strawberry fruit. The result of expression analysis showed that
lines showed significant increases in fruit size, number and fruit GalUR was correlated with changes in ascorbic acid content
yield. Compared with the conventional cultivars, fruit yields in strawberry fruit during ripening. Over-expression of the gene
were increased approximately 184% and 139% in transgenic in Arabidopsis thaliana enhanced vitamin C content 2–3 fold.
cultivated strawberry ‘AN93.231.53’ and ‘Alpina W. Original’, The study suggested that the gene might be useful for increasing
respectively. The total IAA contents of DefH9-iaaM transgenic vitamin C levels of strawberry fruit though genetic transforma-
young flower buds had increased about 1.5 times compared tion (Agius et al., 2003).
with the untransformed flower buds. Moreover, the increase in Recently a great stride has been taken in identifying and
fruit production did not reduce fruit total sugar content, an characterizing, through biochemical and molecular means, the
important parameter related to fruit quality. These results major enzymes and genes involved in flavonoid and proantho-
indicated that IAA plays an important role in plant fecundity in cyanidin biosynthesis during fruit development (Almeida et al.,
Rosaceae species. Meanwhile, Wawrzyńczak et al. (2005) 2007) and these findings would allow the potential cloning of
obtained transgenic strawberry plants with the maize IAA- strawberry-derived genes into strawberry, i.e. intra-generic
glucose synthase gene (iaglu) via the Agrobacterium-mediated genetic transformation, perhaps facilitating the improvement
method. Genomic integration and expression of transgenes was of color and flavor traits.
verified by PCR, Southern blot and RT-PCR analysis.
Compared to wt plants, there was a significant increase of 8. Problems and prospects
ester-conjugated IAA levels in the tissue of all transformants
harboring the iaglu gene while free IAA levels were Genetic engineering (GE), also termed transgenic biotechnol-
significantly decreased in two transgenic lines tested. The ogy, refers to the transfer of individual genes between unrelated
level of amide-conjugated hormone was not affected by species (animal or plant), through the use of recombinant DNA
transformation with iaglu. Compared to wt plants, all transgenic techniques. Through plant genetic engineering, a novel gene can
plants had a dwarfish genotype (i.e., shorter leaf petioles, be introduced from one plant species to another plant species to
smaller leaf laminas, less crown diameter and shorter runners) improve the later plant, a genetically modified organism (GMO).
even though they produced more roots in vitro. The application of GE in strawberry is an effective breeding
Carbohydrate content and balance play an important role in method to make strawberry plants more resistant to biotic and
determining the flavor and processing quality of the fruit. abiotic stresses, improve qualitative and quantitative fruit quality,
Invertases are known to exist in multiple forms which are increase yields, and better stress resistance, while also being
228 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

environmentally friendly. In past decades, great progress has been (Bachelie et al., 1997), rice (Asao et al., 1997, 2003), tomato
made in strawberry breeding and germplasm improvement (Chalavi et al., 2003), katemfe (Schestibratov and Dolgov, 2005),
through plant GE. Although, such approaches have demonstrated maize (Wawrzyńczak et al., 2005), kidney bean (Ricardo et al.,
a great promise and future, it is still at a nascent and experimental 2006), and tobacco (Ricardo et al., 2006). The challenge for
phase, requiring further development and testing in the field scientists is to expand the efforts to identify and understand
before any commercial application. These studies have also additional gene regulation and expression in strawberry growth
proposed many tough challenges for plant biologists and breeders and development.
in creating new and better strawberry varieties. The major
challenges ahead include: 8.3. Unpredictability and variability

8.1. Efficiency of transformation Unpredictable and variable expression of a foreign gene is a

major problem that hinders the success of biotechnology
Fundamental research is still needed to improve transforma- programs in strawberry research. With the development of
tion frequency in delivering genes, as well as in developing a several novelties in plant GE (Teixeira da Silva, 2006a), many
highly-efficient and stable regeneration system, although great economically valuable heterogeneous genes have been trans-
strides have already been achieved. Progress in Agrobacterium- ferred into strawberry plants (Hokanson and Maas, 2001; Folta
mediated genetic transformation of strawberry had been and Dhingra, 2006). However, the subsequent results have
hampered greatly by their generally poor response to tissue shown that almost all transgene expression remains largely
culture, although several novel methods and genotype-inde- unpredictable and a great variation of expression level among
pendent protocols have now started to emerge (Debnath and transgenic lines exists. A number of strategies are available to
Teixeira da Silva, 2007), which will be a massive boost for the target the expression to specific tissues and/or to provide a high
GE protocols. Though a few strawberry transformation systems and stable level of gene expression using a specific promoter
have been developed for several strawberry cultivars by many (Agius et al., 2005; Zhao et al., 2004) or by an induced promoter
research groups since 1990s, most of these methods were (Li et al., 2005), or an expression vector harboring matrix
inefficient and were mostly not repeated (Finstad and Martin, attachment regions (Allen et al., 1996; Abranches et al., 2005),
1995; Barceló et al., 1998; Hokanson and Maas, 2001; Folta and or a small subunit leader and transit peptide (Wong et al., 1992;
Dhingra, 2006). The most important aspect of transformation Janette et al., 2001) or even site-specific recombination systems
efficiency is the choice of species and cultivars. Genotype is an (Lyznik et al., 2003; Djukanovic et al., 2006; D'Halluin et al.,
important factor that significantly influences the efficiency of 2008). Within the next few years, undoubtedly, the most
transformation (Tables 1 and 2). High variability of efficiency beneficial conditions for transformation will be categorized to
was observed among strawberry cultivars with different genetic allow for maximization and stabilization of gene expression
backgrounds, even within the same cultivar (Bachelie et al., levels in strawberry.
1997; Barceló et al., 1998; Cordero de Mesa et al., 2000, 2004;
Jiménez-Bermúdez et al., 2002; Abdal-Aziz et al., 2006; 8.4. GMO safety issues
Lunkenbein et al., 2006a,b; Sesmero et al., 2007). The door is
beginning to open for identifying strawberry germplasm from Risk assessment for human health and environment related
different genetic backgrounds that can be easily transformed to transgenic plants is another serious obstacle for public
(Oosumi et al., 2006; Folta et al., 2006). On a new and exciting acceptance and commercialization (Van den Eede et al., 2004;
front Hanhineva and Kärenlampi (2007) proposed the use of Kulikov, 2005). Recently, regulatory authorities and consumers
temporary immersion bioreactor systems to mass-propagate are concerned about the environmental safety of GMO and are
hygromycin-resistant strawberries. demanding for commercial transgenic plants to be free of
unnecessary genes, such as marker genes (antibiotic resistance)
8.2. Gene targeting and isolation or vector backbone sequences (Ebinuma et al., 1997; Endo
et al., 2002; Schaart et al., 2004; Abdal-Aziz et al., 2006;
Identification and isolation of interesting genes for transferring Ballester et al., 2006; Ludmila et al., 2006; Mihály et al., 2006).
into targeted strawberry varieties are one of the major goals for a Schaart et al. (2004), for example, used an inducible site-
strawberry biotechnology program. It is difficult to isolate an specific recombinase to obtain marker-free transgenic straw-
interesting gene for a strawberry plant since strawberries are berry plants using a bifunctional selectable marker gene for the
polyploid, and also most desirable traits are controlled by multiple initial positive selection of transgenic tissue. When shoots had
genes (Faedi et al., 2002; Folta and Davis, 2006). Isolation and regenerated on Kan, leaf explants from KanR shoots were treated
transformation of genes correlated with fruit softening, yield and with dexamethasone, which induces the recombinase gene, and
quality are still at the exploratory stage (Harpster et al., 1998; subsequently subjected to a second round of regeneration in the
Llop-Tous et al., 1999; Trainotti et al., 1999a,b; Palomer et al., presence of 5-fluorocytosine (5-FC). The codA gene converted
2004; Manning, 1994, 1998). Genes identified and transferred in the non-toxic 5-FC to cytotoxic fluorouracil (Stougaard, 1993),
most of the studies discussed so far are still from other crops, such leading to the death of cells that still contain the marker genes.
as barley (Wang et al., 2004), cowpea (James et al., 1992; Graham After negative selection, they successfully obtained 30 putative
et al., 1995, 1997, 2001, 2002), wheat (Houde et al., 2004), potato marker-free transgenic plants. In another practical success story,
 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232 229

Hoffmann et al. (2006) optimized RNAi silencing by agroinfil- Allen GC, Hall Jr GE, Michalowski S, Newman W, Spiker S, Weissinger AK, et al.
trating developing fruits attached to plants with a construct High-level transgene expression in plant cells: effects of a strong scaffold
attachment region from tobacco. Plant Cell 1996;8:899–9l3.
containing a sense and its corresponding antisense sequence of a Almeida JRM, D'Amico E, Preuss A, Carbone F, Ric de Vos CH, Deiml B, et al.
chalcone synthase gene (CHS) separated by an intron. Silencing Characterization of major enzymes and genes involved in flavonoid and
of the CHS gene could be detected by the absence of red proanthocyanidin biosynthesis during fruit development in strawberry
coloration in ripened fruits following agroinfiltration. Lunkenbein (Fragaria × ananassa). Arch Biochem Biophys 2007;465:61–71.
Alsheikh MK, Suso HP, Robson M, Battey NH, Wetten A. Appropriate choice
et al. (2006a,b) obtained transgenic plants containing an antisense
of antibiotic and Agrobacterium strain improves transformation of
CHS gene to assess the impact of down-regulation of this gene on antibiotic-sensitive Fragaria vesca and F. v. semperflorens. Plant Cell Rep
pigment accumulation in ripened fruit, while other transgenic 2002;20:1173–80.
plants harboring a down-regulated O-methyltransferase gene Arenas G, Marshall S, Espinoza V, Ramírez I, Peña-Cortés H. Protective effect
almost completely depleted the levels of DMMF (2,5-dimethyl-4- of an. antimicrobial peptide from Mytilus edulis chilensis expressed in Ni-
methoxy-3(2H)-furanone, the product of methylation of the most cotiana tabacum L. Electron J Biotechnol 2006;9:144–51.
Asao HG, Nishizawa Y, Arai S, Sato T, Hirai M, Yoshida K, et al. Enhanced
important aroma (4-hydroxy-2,5-dimethyl-3(2H)-furanone) dur- resistance against a fungal pathogen Sphaerotheca fumuli in transgenic
ing strawberry fruit ripening. These studies would most likely strawberry expressing a rice chitinase gene. Plant Biotechnol 1997;14:145–9.
represent the state of the art in strawberry genetic engineering at Asao HG, Arai S, Nishizawa Y. Environmental risk evaluation of transgenic
present. strawberry expressing a rice chitinase gene. Seibutsu Kogakkaishi 2003;81:
Legislations and regulations towards genetically modified 57–63 (in Japanese with English Abstract).
Awang YB, Atherton JG, Taylor AJ. Salinity effects on strawberry plants grown
plants (GMPs) vary among countries, particularly in the EU, in rockwool. II. Fruit quality. J Hortic Sci 1993;68:791–5.
which are not allowing deliberate release of GMPs carrying Bachelie C, Graham J, Machray G, DuManoir J, Roucou JF, McNicol RJ, et al.
antibiotic resistance genes into the environment or permitting Integration of an invertase gene to control sucrose metabolism in strawberry
their commercialization. Therefore, developing procedures to cultivars. Acta Hortic 1997;436:161–3.
avoid the use of antibiotic selection or to allow elimination of Ballester A, Cervera M, Peña L. Efficient production of transgenic citrus plants
using isopentenyl transferase positive selection and removal of the marker
marker genes from a GMP (GM-gene-deletor) will be a high gene by site-specific recombination. Plant Cell Rep 2006;26:39–45.
research priority in the coming years (Luo et al., 2007). Barceló M, El-Mansouri I, Mercado JA, Quesada MA, Alfaro FP. Regeneration
The applications of biotechnology that permits particular and transformation via Agrobacterium tumefaciens of the strawberry
genes to be expressed in a specific manner can accelerate the cultivar chandler. Plant Cell, Tissue Organ Cult 1998;54:29–36.
process of breeding new and better strawberry varieties. Broekaert WF, Terras FR, Cammue BP, Osborn RW. Plant defensins: novel
antimicrobial peptides as components of the host defense system. Plant
Ongoing efforts to improve strawberry germplasm using Physiol 1995;108:1353–8.
molecular approaches will be greatly speeded up with the Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitor in
development of biotechnology and the increased understanding bacteria? Nat Rev Microbiol 2005;3:238–50.
of basic biological sciences. To obtain broad-spectrum Cesar B, Berta S, Fernando R. Relationship between concentrations of Botrytis
cinerea conidia in air, environmental conditions, and the incidence of grey
resistance plus good fruit quality, the introduction of multiple
mould in strawberry flowers and fruits. Eur J Plant Pathol 2006;114:
genes simultaneously will be extensively applied to strawberry 415–25.
breeding. Although such products are still a long way from Chalavi V, Tabaeizadeh Z, Thibodeau P. Enhanced resistance to Verticillium
commercialization, the next decade promises to be an exciting dahliae in transgenic strawberry plants expressing a Lycopersicon chilense
one for strawberry biotechnology. chitinase gene. J Am Soc Hortic Sci 2003;128:747–53.
Chen WP, Chen PD, Liu DJ, Kynast R, Friebe B, Velazhahan R, et al.
Development of wheat scab symptoms is delayed in transgenic wheat plants
Acknowledgement that constitutively express a rice thaumatin-like protein gene. Theor Appl
Genet 1999;99:755–60.
The authors sincerely thank Dr. Ye Grace Chen at Harvard Close TJ. Dehydrins: emergence of a biochemical role of a family of plant
University for her critical review and comments on this dehydration proteins. Physiol Plant 1996;97:795–803.
manuscript, and Dr. Lanlan Zhang at Zhejiang University for Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K.
Plant chitinase. Plant J 1993;3:31–40.
reference collection. Cordero de Mesa M, Jimnez-Bermudez S, Pliego-Alfaro F, Quesada MA,
Mercado JA. Agrobacterium cells as microprojectile coating: a novel
References approach to enhance stable transformation rates in strawberry. Aust J Plant
Physiol 2000;27:1093–100.
Cordero de Mesa M, Santiago-Doménech N, Pliego-Alfaro F, Quesada MA,
Abdal-Aziz SA, Pliego-Alfaro F, Quesada MA, Mercado JA. Evidence of Mercado JA. The CaMV 35S promoter is highly active on floral and pollen
frequent integration of non-T-DNA vector backbone sequences in transgenic of transgenic strawberry plants. Plant Cell Rep 2004;23:32–8.
strawberry plant. J Biosci Bioeng 2006;101:508–10. Danyluk J, Houde M, Rassart É, Sarhan F. Differential expression of a gene
Abranches R, Shultz RW, Thompson WF, Allen GC. Matrix attachment regions encoding an acidic dehydrin in chilling sensitive and freezing tolerant
and regulated transcription increase and stabilize transgene expression. Plant gramineae species. FEBS Lett 1994;344:20–4.
Biotechnol J 2005;3:535–43. Danyluk J, Perron A, Houde M, Benhamou N, Sarhan F. Accumulation of a
Agius F, González-Lamothe R, Caballero JL, Muñoz-Blanco J, Botella MA, major plasma membrane-associated protein during cold acclimation of
Valpuesta V. Engineering increased vitamin C levels in plants by overexpression wheat. Plant Cell 1998;10:623–38.
of a D-galacturonic acid reductase. Nat Biotechnol 2003;21: 177–81. Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, et al. Overexpression of
Agius F, Amaya I, Botella MA, Valpuesta V. Functional analysis of homologous the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants
and heterologous promoters in strawberry fruits using transient expression. enhances environmental friendly resistance to Rhizoctonia solani causing
J Exp Bot 2005;56:37–46. sheath blight disease. Theor Appl Genet 1999;98:1138–45.
230 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

Debnath SC, Teixeira da Silva JA. Strawberry culture in vitro: applications in Hanhineva KJ, Kärenlampi SO. Production of transgenic strawberries by
genetic transformation and biotechnology. Fruit, Veg Cereal Sci Biotechnol temporary immersion bioreactor system and verification by TAIL-PCR.
2007;1:1–12. BMC Biotechnol 2007;7:11–22.
Djukanovic V, Orczyk W, Gao HR, Sun XF, Garrett N, Zhen SF, et al. Gene Harpster MH, Brummell DA, Dunsmuir P. Expression analysis of a ripening-
conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP specific, auxin-repressed endo-1,4-β-glucanase gene in strawberry. Plant
site-specific recombination systems. Plant Biotechnol J 2006;4:345–57. Physiol 1998;118:1307–16.
D'Halluin K, Vanderstraeten C, Stals E, Cornelissen M, Ruiter R. Homologous Haymes KM, Davis TM. Agrobacterium-mediated transformation of ‘Alpine’
recombination: a basis for targeted genome optimization in crop species such Fragaria vesca, and transmission of transgenes to R1 progeny. Plant Cell
as maize. Plant Biotechnol J 2008;6:93–102. Rep 1998;17:279–83.
du Plessis HJ, Brand RJ, Glyn-Woods C, Goedhart MA. Efficient genetic Hoffmann T, Kalinowski G, Schwab W. RNAi-induced silencing of gene
transformation of strawberry (Fragaria × ananassa Duch.) cultivar Selekta. expression in strawberry fruit (Fragaria × ananassa) by agroinfiltration: a
Acta Hortic 1997;447:289–94. rapid assay for gene function analysis. Plant J 2006;48:818–26.
Dziadczyk P, Bolibok H, Tyrka M, Hortyñski J. In vitro selection of strawberry Hokanson SC, Maas JL. Strawberry biotechnology. Plant Breed Rev 2001;21:
(Fragaria × ananassa Duch.) clones tolerant to salt stress. Euphytica 139–80.
2003;132:49–55. Horowitz S, Freeman S, Sharon A. Use of green fluorescent protein-transgenic
Dziadczyk P, Kiszczak W, Tyrka M, Laba M, Diufer K. Evaluation of strains to study pathogenic and nonpathogenic lifestyles in Colletotrichum
strawberry (Fragaria × ananassa Duch.) cultivar's salt stress tolerance on acutatum. Phytopathology 2002;92:743–9.
the basis of S1 seeds in vitro germination. J Food Agric Environ 2005;3: Houde M, Dallaire S, N'Dong D, Sarhan F. Overexpression of the acidic
275–81. dehydrin WCOR410 improves freezing tolerance in transgenic strawberry
Ebinuma H, Sugita K, Matsunaga E, Yamakado M. Selection of marker-free leaves. Plant Biotechnol J 2004;2:381–7.
transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci Huang Y, Nordeen RO, Di M, Owens LD, McBeath JH. Expression of an engineered
USA 1997;94:2117–21. cecropin gene cassette in transgenic tobacco plants confers disease resistance to
El-Mansouri I, Mercado JA, Valpuesta V, López-Aranda JM, Pliego-Alfaro FP, Pseudomonas syringae pv. Tabaci. Phytopathology 1997;87:494–9.
Quesada MA. Shoot regeneration and Agrobacterium-mediated transforma- James DJ, Passey AJ, Barbara DJ. Agrobacterium-mediated transformation of
tion of Fragaria vesca L. Plant Cell Rep 1996;15:642–6. the cultivated strawberry (Fragaria × ananassa Duch) using disarmed binary
Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H. Single-step transformation vectors. Plant Sci 1990a;69:79–94.
for generating marker-free transgenic rice using the ipt-type MAT vector James DJ, Passey AJ, Barbara DJ. Agrobacterium-mediate transformation of apple
system. Plant J 2002;30:115–22. and strawberry using disarmed Ti-binary vectors. Acta Hortic 1990b;280:
Faedi W, Mourgues F, Rosati C. Strawberry breeding and varieties: situation and 495–502.
perspectives. Acta Hortic 2002;567:51–9. James DJ, Passey AJ, Esterbrook MA, Solomon MG, Barbara DJ. Progress in
FAOSTAT. http://www.fao.org/waicent/portal/statistics_en.asp, 2007. the introduction of transgenes for pest resistance in apples and strawberry.
Finstad K, Martin RR. Transformation of strawberry for virus resistance. Acta Phytoparasitica 1992;20(suppl):83–7.
Hortic 1995;385:86–90. Janette, V.O., Seema, D., Maud, A.W.H., Jeanne, G.L., Methods for strawberry
Firsov AP, Dolgov SV. Agrobacterial transformation and transfer of the antifreeze transformation using Agrobacterium tumefaciens. US Patent 6274791, 2001.
protein gene of winter flounder to the strawberry. Acta Hortic 1999;484:581–6. Jiménez-Bermúdez S, Redondo-Nevado J, Muñoz-Blanco J, Caballero JL,
Folta KM, Davis TM. Strawberry genes and genomics. Crit Rev Plant Sci López-Aranda JM, Valpuesta V, et al. Manipulation of strawberry fruit
2006;25:399–415. softening by antisense expression of a pectate lyase gene. Plant Physiol
Folta KM, Dhingra A. Transformation of strawberry: the basis for translational 2002;128:751–9.
genomics in Rosaceae. In Vitro Cell Dev Biol-Plant 2006;42:482–90. Jin WM, Yin SP, Lu RQ, Yuan WF, Dong J, Wang GX. GO gene transformation
Folta KM, Dhingra A, Howard L, Stewart PJ, Chadler CK. Characterization of of strawberry and its resistance to gray mold. Mol Plant Breed
LF9, an octoploid strawberry genotype selected for rapid regeneration and 2005;3:797–800 (in Chinese with English Abstract).
transformation. Planta 2006;224:1058–67. Kafkas E, Koch-Dean M, Shabtai S, Tanaami Z, Salts Y, Barg R. Development
Galletta GJ, Maas JL. Strawberry genetics. HortScience 1990;25:871–9. of methods for transformation of strawberry in Israel, with the aim of
Georges F, Saleem M, Cutler AJ. Design and cloning of a synthesis gene for the improving fruit development. Acta Hortic 2002;567:109–11.
flounder antifreeze protein and its expression in plant cells. Gene 1990;91: Karasuda S, Tanaka S, Kajihara H, Yamamoto Y, Koga D. Plant chitinase as a
159–65. possible biocontrol agent for use instead of chemical fungicides. Biosci
Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow Biotechnol Biochem 2003;67:221–4.
MF. Low temperature regulation of the Arabidopsis CBF family of AP2 Kaya C, Ero B, Higgs D, Murillo-Amador B. Influence of foliar-applied calcium
transcriptional activators as an early step in cold-induced COR gene nitrate on strawberry plants grown under salt-stressed conditions. Aust J Exp
expression. Plant J 1998;16:433–42. Agric 2002a;42:631–6.
Graham J, McNicol RJ, Grieg K. Towards genetic based insect resistance in Kaya C, Kirnak H, Higgs D, Saltali K. Supplementary calcium enhances plant
strawberry using the cowpea trypsin inhibitor gene. Ann Appl Biol 1995;127: growth and saline medium and fruit yield in strawberry cultivars grown at
163–73. high (NaCl) salinity. Sci Hort 2002b;93:65–74.
Graham J, Gordon SC, McNcol RJ. The effect of the CpTi gene in strawberry against Keutgen AJ, Keutgen N. Influence of NaCl salinity on stress fruit quality in
attack by vine weevil (Otiorhynchus sulcatus F. Coleotera: Curculionidae). Ann strawberry. Acta Hortic 2003;609:155–7.
Appl Biol 1997;131:133–9. Khammuang S, Dheeranupattana, Hanmuangjai P, Won-groung S. Agrobac-
Graham J, Gordon SC, McNcol RJ, McNcol JW. The effect of genetically terium-mediated transformation of modified antifreeze protein gene in
modified strawberries expressing CpTi under field conditions. J Hortic Sci strawberry. Songklanakarin J Sci Technol 2005;27:693–703.
Biotechnol 2001;77:33–40. Ki WK, Warmund MR. Low temperature injury to strawberry floral organs at
Graham J, Gordon SC, Smith K, McNcol RJ, McNcol JW. The effect of the several stages of development. HortScience 1992;27:1302–4.
cowpea trypsin inhibitor in strawberry on damage by vine weevil under field Kim KK, Fravel DR, Papavizas GC. Identification of a metabolite produced by
conditions. J Hortic Sci Biotechnol 2002;77:33–40. Talaromyces flavus as glucose oxidase and its role in biocontrol of Verti-
Gruchala A, Korbin M, Zurawicz E. Conditions of transformation and cillium dahliae. Phytopathology 1988;78:488–92.
regeneration of ‘Induka’ and ‘Elista’ strawberry plants. Plant Cell Tissue Kulikov AM. Genetically modified organisms and risks of their introduction.
Organ Cult 2004;79:153–60. Russ J Plant Physiol 2005;52:99–111.
Hancock JF. Strawberries. New York: CABI Publishing; 1999. Legard DE, Ellis M, Chandler CK, Price JF. Integrated management of strawberry
Hancock JF, Luby JJ. Genetic resources at our doorstep: the wild strawberries. diseases in winter fruit production areas. In: Childers N, editor. The strawberry:
BioScience 1993;43:141–7. a book for growers. Institute of Food and Agricultural Sciences, Horticultural
 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232 231

Sciences Department, University of Florida, Gainesville. Norm Childers Mihály Kondrák, Ingrid MM, Zsófia Bánfalvi. Generation of marker- and
Publications; 2003. p. 111–24. backbone-free transgenic potatoes by site-specific recombination and a bi-
Li J, Gong X, Lin H, Song Q, Chen J, Wang X. DGP1, a drought-induced guard functional marker gene in a non-regular one-border Agrobacterium transfor-
cell-specific promoter and its function analysis in tobacco plants. Sci China mation vector. Transgenic Res 2006;15:729–37.
C Life Sci 2005;48:181–6. Monticelli S, Gentile A, Damiano C. Regeneration and Agrobacterium-mediated
Liu FH, Guo Y, Gu DM, Xiao G, Chen ZH, Chen SY. Salt tolerance of transformation in stipules of strawberry. Acta Hortic 2002;567:105–7.
transgenic plants with BADH cDNA. Acta Genet Sin 1997;24:54–8 (in Morgan A, Baker CM, Chu JSF, Lee K, Crandall BA, Jose L. Production of
Chinese with English Abstract). herbicide tolerant strawberry through genetic engineering. Acta Hortic
Llop-Tous I, Domínguez-Puigjaner E, Palomer X, Vendrell M. Characterization 2002;567:113–5.
of two divergent endo-β-1,4-glucanase cDNA clones highly expressed in Na J, Wang GL, Xia R, Yang HY. Construction of anti-sense gene of annfaf and
the nonclimacteric strawberry fruit. Plant Physiol 1999;119:1415–22. genetic transformation of strawberry. Sci Agric Sin 2006;39:582–6 (in
Ludmila M, Anthony J, Jan-Peter N. Directed microspore-specific recombina- Chinese with English Abstract).
tion of transgenic alleles to prevent pollen-mediated transmission of Nehra NS, Chibbar RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C.
transgenes. Plant Biotechnol J 2006;4:445–52. Genetic transformation of strawberry by Agrobacterium tumefaciens using a
Lunkenbein S, Coiner H, Ric de Vos CH, Schaart JG, Boone MJ, Krens FA, et al. leaf disk regeneration system. Plant Cell Rep 1990a;9:293–8.
Molecular characterization of a stable antisense chalcone synthase phenotype Nehra NS, Chibbar RN, Kartha KK, Datla RSS, Crosby WL, Stushnoff C.
in strawberry (Fragaria ×ananassa). J Agric Food Chem 2006a;54:2145–53. Agrobacterium-mediated transformation of strawberry calli and recovery
Lunkenbein S, Salentijn EMJ, Coiner HA, Boone MJ, Krens FA, Schwab W. of transgenic plants. Plant Cell Rep 1990b;9:10–3.
Up- and down-regulation of Fragaria × ananassa O-methyltransferase: Nyman M, Wallin A. Transient gene expression in strawberry (Fragaria
impacts on furanone and phenylpropanoid metabolism. J Exp Bot ananassa Duch.) protoplasts and the recovery of transgenic plants. Plant
2006b;57:2445–53. Cell Rep 1992;11:105–8.
Luo KM, Duan H, Zhao DG, Zheng XL, Deng W, Chen YQ, et al. ‘GM-gene- Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman JL, et al.
deletor’: fused loxP-FRT recognition sequences dramatically improve the High-efficiency transformation of the diploid strawberry (Fragaria vesca) for
efficiency of FLP or CRE recombinase on transgene excision from pollen functional genomics. Planta 2006;223:1219–30.
and seed of tobacco plants. Plant Biotechnol J 2007;5:263–74. Owens CL, Thomashow MF, Hancock JF, Iezzoni AF. CBF1 orthologs in sour
Lyznik LA, Gordon-Kamm WJ, Tao Y. Site-specific recombination for genetic cherry and strawberry and the heterologous expression of CBF1 in strawberry.
engineering in plants. Plant Cell Rep 2003;21:925–32. J Am Soc Hortic Sci 2002;127:489–94.
Manning K. Changes in gene expression during strawberry fruit ripening and Owens CL, Iezzoni AF, Hancock JF. Enhancement of freezing tolerance of
their regulation by auxin. Planta 1994;194:62–8. strawberry by heterologous expression of CBF1. Acta Hortic 2003;626:93–100.
Manning K. Isolation of a set of ripening-related genes from strawberry: their Palomer X, Domínguez-Puigjaner E, Vendrell M, Llop-Tous I. Study of
identification and possible relationship to fruit quality traits. Planta 1998;205: strawberry Cel1 endo-β-(1,4)-glucanase protein accumulation and char-
622–31. acterization of its in vitro activity by heterologous expression in Pichia
Mariana SC, Wagner F. Plant defense and antimicrobial peptides. Prot Peptide pastoris. Plant Sci 2004;167:509–18.
Letters 2005;12:11–6. Palomer X, Llop-Tous I, Vendrell M, Krens FA, Schaart JG, Boone MJ, et al.
Marín-Rodríguez MC, Orchard J, Seymour GB. Pectate lyases, cell wall degra- Antisense down-regulation of strawberry endo-β-(1,4)-glucanase genes
dation and fruit softening. J Exp Bot 2002;53:2115–9. does not prevent fruit softening during ripening. Plant Sci 2006;171:640–6.
Martinelli L, Rugini E, Saccardo F. Genetic transformation for biotic stress resistance Park JI, Lee YK, Chung WI, Lee IH, Choi JH, Lee WM, et al. Modification of
in horticultural plants. In Vitro Cell Dev Biol — Plant 1996;32:69–70. sugar composition in strawberry fruit by antisense suppression of an ADP-
Martinelli A, Gaiani A, Cella R. Agrobacterium-mediated transformation of glucose pyrophosphorylase. Molec Breed 2006;17:269–79.
strawberry cultivar Marmolada Onebor⁎. Acta Hortic 1997;439:169–73. Phillip AW. Improved regeneration and Agrobacterium-mediated transformation
Marta AE, Camadro EL, Diaz-Ricci JC, Castagnaro AP. Breeding barriers of wild strawberry (Fragaria vesca L.). MS Dissertation. Virginia Polytechnic
between the cultivated strawberry, Fragaria × ananassa, and related wild Institute and State University, 2005.
germplasm. Euphytica 2004;136:139–50. Potter D, Luby JJ, Harrison RE. Phylogenetic relationships among species of
Mass JL. Compendium of strawberry diseases. 2nd edn. St Paul, Minn: Fragaria (Rosaceae) inferred from non-coding nuclear and chloroplast
American Phytopathological Society; 1998. DNA sequences. Syst Bot 2000;25:337–48.
Mathews H, Wagoner W, Kellogg J, Bestwick R. Genetic transformation of Qin YH, Zhang SL. Factors influencing the efficiency of Agrobacterium-
strawberry: stable integration of a gene to control biosynthesis of ethylene. mediated transformation in strawberry cultivar Toyonoka. J Nucl Agric Sci
In Vitro Cell Dev Biol — Plant 1995;31:36–43. 2007;21:461–5 (in Chinese with English Abstract).
Mathews H, Dewey V, Wagoner W, Bestwick RK. Molecular and cellular Qin YH, Zhang SL, Syed A, Zhang LX, Qin QP, Chen KS, et al. Regeneration
evidence of chimaeric tissues in primary transgenic and elimination of mechanism of strawberry under different color plastic films. Plant Sci
chimaerism through improved selection protocols. Transgenic Res 1998;7: 2005a;168:1425–31.
123–9. Qin YH, Zhang SL, Zhang LX, Zhu DY, Syed A. Response of in vitro
Mercado JA, Martín-Pizarro C, Pascual L, Quesada MA, Pliego-Alfaro F, de los strawberry to silver nitrate (AgNO3). Hortic Sci 2005b;40:747–51.
Santos B, et al. Evaluation of tolerance to Colletotrichum acutatum in Qin YH, Zhang SL, Xu K, Wu YJ, Qin QP. A highly efficient system
strawberry plants transformed with Trichoderma-derived genes. Acta Hortic establishment of shoot regeneration from leaf explant of strawberry. Acta
2007;738:383–8. Hortic Sin 2005c;32:101–4 (in Chinese with English Abstract).
Mezzetti B, Costantini E. Strawberry (Fragaria × ananassa). Methods in Mol Ricardo VG, Coll Y, Castagnaro A, Ricci JCD. Transformation of a strawberry
Biol, vol. 344. Totowa, NJ: Humana Press Inc.; 2006. p. 287–95. Agro- cultivar using a modified regeneration medium. HortScience 2003;38:
bacterium protocols (Wang K). 277–80.
Mezzetti B, Landi L, Scortichini L, Rebori A, Spena A, Pandolfini T. Genetic Ricardo VG, Ricci JCD, Hernández L, Castagnaro AP. Enhanced resistance to
engineering of parthenocarpic fruit development in strawberry. Acta Hortic Botrytis cinerea mediated by the transgenic expression of the chitinase
2002;567:101–4. gene ch5B in strawberry. Transgenic Res 2006;15:57–68.
Mezzetti B, Costantini E, Chionchetti F, Landi L, Pandolfini T, Spena A. Samac DA, Hironaka CM, Yallaly PE, Shah DM. Isolation and characterization
Genetic transformation in strawberry and raspberry for improving plant of the genes encoding basic and acidic chitinase in Arabidopsis thaliana.
productivity and fruit quality. Acta Hortic 2004a;649:107–10. Plant Physiol 1990;93:907–14.
Mezzetti B, Landi L, Pandolfini T, Spena A. The defH9-iaaM auxin- Sargent DJ, Davis TM, Tobutt KR, Wilkinson MJ, Battey NH, Simpson DW. A
synthesizing gene increases plant fecundity and fruit production in straw- genetic linkage map of microsatellite, gene-specific and morphological
berry and raspberry. BMC Biotechnol 2004b;4:4–14. markers in diploid Fragaria. Theor Appl Genet 2004;109:1385–91.
232 Y. Qin et al. / Biotechnology Advances 26 (2008) 219–232

Schaart JG, Krens FA, Pelgrom KTB, Mendes O, Rouwendal GJA. Effective Van den Eede G, Aarts HJ, Buhk HJ, Corthier G, Flint HJ, Hammes W, et al. The
production of marker-free transgenic strawberry plants using inducible site- relevance of gene transfer to the safety of food and feed derived from
specific recombination and a bifunctional selectable marker gene. Plant genetically modified (GM) plants. Food Chem Toxicol 2004;42:1127–56.
Biotechnol J 2004;2:233–40. Wang GL, Yang HY, Xia R, Fang HJ, Jing SX. Cloning and sequencing the full-
Schestibratov KA, Dolgov SV. Transgenic strawberry plants expressing a length cDNA of annexin from strawberry fruit. Acta Bot Sin 2001;43: 874–6.
thaumatin II gene demonstrate enhanced resistance to Botrytis cinerea. Sci Wang JL, Ge HB, Peng SQ, Zhang HM, Chen PL, Xu JR. Transformation of
Hortic 2005;106:177–89. strawberry (Fragaria ananassa Duch.) with late embryogenesis abundant
Schlumbaum A, Mauch F, Vogel U. Plant chitinases are potent inhibitors of protein gene. J Hortic Sci Biotechnol 2004;79:735–8.
fungal growth. Nature 1986;25:365–7. Wawrzyńczak D, Sowik I, Michalczuk L. Agrobacterium-mediated transforma-
Sesmero R, Quesada MA, Mercado JA. Antisense inhibition of pectate lyase tion of five strawberry genotypes. J Fruit Ornam Plant Res 2000;8:1–8.
gene expression in strawberry fruit: characteristics of fruits processed into Wawrzyńczak D, Michalczuk L, Sowik I. Modification in indole-3-acetic acid
jam. J Food Eng 2007;79:194–9. metabolism, growth and development of strawberry through transformation
Sharma A, Sharma R, Imamura M, Yamakawa M, Machii H. Transgenic with maize IAA-glucose synthase gene (iaglu). Acta Physiol Plant 2005;27:
expression of cecropin B, an antibacterial peptide from Bombyx mori, 19–28.
confers enhanced resistance to bacterial leaf blight in rice. FEBS Lett Weretilnyk EA, Hanson AD. Betaine-aldehydrogenase polymorphism in spinach:
2000;484:7–11. genetic and biochemical characterization. Biochem Genet 1987;26:143–51.
Simpson DW. Resistance to Botrytis cinerea in pistillate genotypes of the Weretilnyk EA, Hanson AD. Molecular cloning of a plant betaine-aldehyde
cultivated strawberry Fragaria ananassa. J Hortic Sci 1991;66:719–23. dehydrogenase, an enzyme implicated in adaptation to salinity and drought.
Stougaard J. Substrate-dependent negative selection in plants using a bacterial Proc Natl Acad Sci 1990;87:2745–9.
cytosine deaminase gene. Plant J 1993;3:755–61. Wilkinson JQ, Lanahan MB, Conner TW, Klee HJ. Identification of mRNAs
Sturm A. Invertases. Primary structures, functions and roles in plant with enhanced expression in ripening strawberry fruit using polymerase
development and sucrose partitioning. Plant Physiol 1999;121:1–7. chain reaction differential display. Plant Mol Biol 1995;27:1097–108.
Sutton JC. Epidemiology and management of Botrytis leaf blight of onion and Wise MJ. LEAping to conclusions: a computational reanalysis of late embryogenesis
gray mold of strawberry: a comparative analysis. Can J Plant Pathol abundant proteins and their possible roles. BMC Bioinformatics 2003;4:52–70.
1990;12:100–10. Wong EY, Hironaka CM, Fischoff DA. Arabidopsis thaliana small subunit
Sutton JC, James TDW, Dale A. Harvesting and bedding practices in relation to leader and transit peptide enhance the expression of Bacillus thuringiensis
grey mould of strawberries. Ann Appl Biol 1988;113:167–75. proteins in transgenic plants. Plant Mol Biol 1992;20:8l–93.
Teixeira da Silva JA. Floriculture, ornamental and plant biotechnology: advances Woolley LC, James DJ, Manning K. Purification and properties of an endo-β-
and topical issues. Global Science Books, Isleworth, UK, 1st Edn, vol. 2.; 1,4-glucanase from strawberry and down-regulation of the corresponding
2006a. p. 571. gene, cel1. Planta 2001;214:11–21.
Teixeira da Silva JA. Floriculture, ornamental and plant biotechnology: advances Zhang HM, Wang JL. Establishment of genetic transformation system of
and topical issues. Global Science Books, Isleworth, UK, 1st Edn, vol. 3.; “Allstar” strawberry leaf. Biotech 2005;15:68–70 (in Chinese with English
2006b. p. 317–567. Abstract).
Theis T, StahI U. Antifungal proteins: targets, mechanisms and prospective Zhang ZH, Wu LP. Development of genetic transformation system of the
applications. Cell Mo1 Life Sci 2004;61:437–55. commercial strawberry cultivar “Tudla”. J Agric Biotechnol 1998;6:200–4
Trainotti L, Ferrarese F, Vecchia FD, Rascio N, Casadoro G. Two different endo- (in Chinese with English Abstract).
β-1,4-glucanase contribute to the softening of the strawberry fruits. J Plant Zhang ZH, Wu LP, Dai HY, Wang GY, Zhao TY, Bi XY, et al. Regeneration and
Physiol 1999a;154:355–62. transformation in vitro of the strawberry varieties. Acta Hortic Sin
Trainotti L, Spolaore S, Pavanello A, Baldan B, Casadoro G. A novel E-type 2001;28:189–93 (in Chinese with English Abstract).
endo-β-(1,4)-glucanase with a putative cellulose-binding domain is highly Zhao Y, Liu QZ, Davis RE. Transgene expression in strawberries driven by a
expressed in ripening strawberry fruits. Plant Mol Biol 1999b;40:323–32. heterologous phloem-specific promoter. Plant Cell Rep 2004;23:224–30.