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In vitro Studies of Strawberry - An Important Fruit Crop: A Review

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Jour Pl Sci Res 31 (2) 115-131 2015
In vitro Studies of Strawberry - An Important Fruit Crop: A Review

Suvalaxmi Palei1, Arun Kumar Das1 and Gyana Ranjan Rout2*

Department of Fruit Science, 2Department of Agriculture, Biotechnology, College of Agriculture,
1

Orissa University of Agriculture & Technology, Bhubaneswar- 751003, Odisha, India

*Email: grrout@rediffmail.com, suvalaxmipalei@gmail.com, das.arun@hotmail.com

Strawberry is the genus Fragaria under family Rosaceae is one of the most important fruit plants for
both fresh consumption and food processing in the temperate and subtropical countries. According
to Nutrient Database for Standard Reference (USDA 1999) the strawberry fruits are resources of the
nutritious elements essential for human being. Propagation of strawberry is achieved either through
runners or by in vitro micropropagation. In vitro p ropagation via. meristems or leaf explants of
strawberry has been discussed with Genetic transformation is a key process to sustain this demand
by permitting the potential enhancement of existing cultivars as well as the development of new
cultivars resistant to pest, diseases, and storage problems that occur in the major production areas.
This review summarizes the application of in vitro propagation techniques for production of mass
scale propagation, genetic stability of the micropropagated plants and advances of genetic engineering
applied to the improvement of strawberry with special reference to miraculin production and antifreeze
protein genes.
Keywords: In vitro, Micropropagation, Genetic transformation, Genetic fidelity.

INTRODUCTION and stigma attached to the receptacle. When

fertilization occurs the receptacle develops into a fleshy
Strawberry (Fragaria x ananassa Duch.) is a major
fruit. The fruit is called an achene which contains the
berry crop around the world. Production of
seeds. The edible part is an accessory type fruit. The
strawberries has been attempted to meet the
seeds are arranged on the outside of the receptacle
demands for improved yields, fruit size and quality
tissue. The growth of the receptacle is dependent on
traits. However, the narrow genetic base of the
successful fertilization of the ovules with its size and
cultivated strawberry, combined with the polyploid
shape dependent on the number of achiness formed.
nature of the crop constrain traditional breeding
Strawberry plants are day length dependent with
methods. In vitro approaches are an alternative
cultivars being long day, short day or day neutral. The
efficient strategy to implement strawberry
high degree of genetic heterozygosity present in
improvement. Strawberries are members of the
Fragaria spp. enabled the development of strawberry
family Rosaceae, subfamily Rosoideae, and genus
cultivars adapted to widely varying environment
Fragaria. Fragaria species are nine diploids, two
conditions and resistant to several diseases and pests.
tetraploids, one hexaploid, and four octoploids.
Not only the genetic variability, but also a high
Diploids (2n = 14) include F. vesca Duch., F. viridis
adaptability and plasticity of the strawberry plant itself
Duch., F. nilgerrensis Schlect., F. daltoniana. J.
give this crop such a remarkable range of adaptation
Gray, F. nubicola Lindl. Ex Lacaita., F. iinumae
(Darrow, 1966). That heterozygosity was explained
Makino, F. yezoensis Hara, F. nipponica Makino,
by as there are more than 20 Fragaria species
and F. mandschurica (Staudt,1962). Strawberry
worldwide, there are seven basic types of
blossoms contain many pistils, each with its own style
chromosomes that they all have in common (Gaafar
116 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout
and Saker, 2006). Some species are diploid, having two
sets of the seven chromosomes (14 chromosomes
total). Others are tetraploid (4x = 28), hexaploid (6x =
42), octoploid (8x = 56) or decaploid (10x = 70) (Darnell,
R., 2003). In vitro propagation of strawberry plants
was introduced about thirty years ago (Boxus 1974).
Most of the European nurseries producing several
millions plants per year through in vitro technique as it
gave a definitive answer to the problems of soil fungal
flora, causing a lot of damage to the strawberry fields
and by another way, tissue culture plants seemed to
produce more runners per mother plant in a short time
period (Mohan et al. 2005). Micropropagation has also
been widely used in the USA (Zimmerman 1981) in
commercial propagation of strawberries and in breeding
programs to produce many plants rapidly. The present
review highlighted the in vitro studies as well as genetic
improvement of strawberry.
STATUS OF NUTRITIONAL VALUE
According to Nutrient Database for Standard
Reference (USDA 1999) the strawberry fruits are
resources of the nutritious elements essential for
human being. Strawberries had been a favourite
among the fruits of the temperate world. They were
valued for delicious flavor and fragrance, for health-
restoring qualities and as harbinger of spring (Wilhelm
et al., 1974). Flavourful and nutritious, strawberries
are enjoyed by millions of people in all climates and
are predominantly used as fresh fruit. Their use in
processed forms such as cooked and sweetened
preserves, jams or jellies and frozen whole berries or
sweetened juice extracts or flavourings, and their use
in making a variety of other processed products made
them one of the most popular berry crops, more widely
distributed than any other fruit (Samir et al., 2005,
2006). Strawberry is cultivated all around the world,
not only for its digestive and tonic properties, but also
because of the nutritional value of its fruits, important
source of foliate, vitamin-C, fiber, potassium,
flavonoids, autocianidin, phytochemicals and
antioxidants.
PHYTOCHEMICALS ENRICHMENT
Although strawberry is not an essential component
of the diet, it’s delicious flavor and taste, attractive
The Journal of Plant Science Research
appearance and seasonal availability make this fruit
an excellent crop. Even more, strawberries are rich
in phytochemical compounds with potential
antioxidant compounds, mainly ellagic acid and
flavonoids, which can lower the risk of
cardiovascular events and tumorogenesis (Hannum,
2004). These qualities have ensured that the
economic importance of this crop has increased
throughout the world and, now-a-days, it remains as
a crop of primary interest for both research and fruit
production (Mercado, et al., 2007). Strawberry
(Fragaria x ananassa Duch.) fruits are rich
sources of phytochemicals of which phenolic
compounds are predominating. Berry fruits are
reported to contain a wide variety of phenolics
including hydroxybenzoic and hydroxycinnamic acid
derivatives, anthocyanins, flavonols, flavanols,
condensed tannins (proanthocyanidins) and
hydrolyzable tannins (Machiex et al., 1990). In vitro
studies indicate that berry phenolics have a wide
range of biological properties such as anti-cancer,
antioxidant, anti-inflammatory, and cell regulatory
effects (Seeram, 2006a,b; Seeram and Heber, 2006).
These anticancer effects are exerted through multi-
mechanistic means of action including the
antioxidant actions of the berries phenolic
constituents by protecting DNA from damage, and
also through effects exerted beyond antioxidation
(Seeram et al., 2006). The biological activities of
strawberry phytochemicals include the regulation of
phase-II enzymes and the modulation of gene
expression and subcellular signalling pathways of cell
proliferation, angiogenesis and apoptosis
(programmed cell death). Although there have been
many published reports on the anticancer effects of
individual phenolics known to be present in the
strawberry fruit (Seeram 2006a,b). Strawberry
extracts have also been evaluated for their ability to
inhibit mutation by the direct-acting mutagen methyl
methane sulfonate, and the metabolically activated
carcinogen, benzopyrene. Ethanol extracts from
freeze-dried fruits of several strawberry cultivars
were also evaluated and hydrolyzable tannin-
containing fractions from strawberries were found
to be most effective at inhibiting mutations (Hope et
al., 2004).
 In vitro Studies of Strawberry - An Important Fruit Crop: A Review
Anthocyanins are natural pigments providing scarlet
to blue colors in flowers, fruits, leaves and storage
organs. The recent interest in the field of anthocyanin
chemistry has been generated by restriction and
limitation of the use of synthetic dyes as food
ingredients. Because of low toxicity of anthocyanins,
they have a high potential as a food colorant as the
substitute of synthetic red dyes. Recently, these
anthocyanins have been thought to have
pharmacological effects, such as lowering the
atherogenic index (Igarashi et al., 1990) and decreasing
triglyceride and free fatty acid levels (Igarashi et al.,
1990). Moreover, Kamei et al. (1995) reported that
anthocyanin was more effective to inhibit the growth
of tumor cells than other flavonoids. Studies concerned
with anthocyanin production using plant tissue cultures
have therefore become very important. Masayuki et
al. (1999) demonstrated that Fragaria ananassa
(strawberry) callus, which produced high amounts of
anthocyanin in the dark which accumulated more than
1000 μg of anthocyanin per gm fresh cell (Igarashi and
Inagaki,1991). Sato et al. (1996) reported that in the
suspension cultures of F. ananassa cells, anthocyanin
content increased with the intensity of light irradiation
from 2500 to 8000.
DISEASE IN STRAWBERRIES
Several soil pathogens damage strawberry roots,
resulting in vigour declines and ultimately death. Two
very common problems across the world are red stele
or red core caused by Phytophthora fragariae
Hickman and Verticillium wilt caused by Verticillium
albo-atrum and V. dahlia. Black root rot is also
widespread and is caused by a complex of organisms
including Pythium, Rhizoctonia and the root lesion
nematode (Pratylenchus penetrans) (Hancock et al.,
2008). Fusarium wilt or Fusarium yellows (Fusarium
oxysporum) is of major importance in Japan, Korea
and Australia. Fumigation has been widely employed
to control soil pathogens, but the impending ban on
methyl bromide fumigation has stimulated increased
interest in developing resistant cultivars. Without
fumigation, cultivars yield 50% less fruit on average
(Hancock et al., 2008). Among the foliar diseases,
three are very widespread and can cause serious
damage including, leaf blight [Phomopsis obscurans],
117
Ramularia leaf spot, [Mycosphaerella fragariae] and
leaf scorch [Diplocarpon earliana ]. Alternaria leaf
spot or black leaf spot (Alternaria alternata (FR.)
Keissler) causes serious damage in Europe, New
Zealand and Korea (Hancock et al., 2008). Powdery
mildew (Sphaerotheca macularis) is also found across
most of the strawberry range, although it rarely does
economic damage. Angular leaf spot, Xanthomonas
fragariae, is a rapidly growing problem in strawberries
all across the world (Hancock et al., 2008).
Anthracnose is a common problem in strawberries,
causing a wide array of symptoms including fruit rot,
crown rot, and lesions of the stolons, petioles and
leaves. Anthracnose diseases of strawberry are caused
by Colletotrichum fragariae, C. Acutatum.
IN VITRO STUDIES OF STRAWBERRY
One of the important goals of the agricultural policy in
the world is to increase the acreage of strawberry to
meet the demand of local fresh market, processing and
export. Healthy stocks used for propagation through
conventional methods are not available.
Micropropagation of strawberry was established in
1974 because of two important factors i.e. soil fungi
causing a lot of damage to the strawberry fields and
in vitro raised plants seemed to produce more runners
per plant in a short time. However, strawberry tree is
difficult to propagate by seed due to genetic variation
and specific requirements of seed germination. In
addition, rooting percentage of cuttings is relatively low
(Mohan et al., 2005). Micropropagation of strawberry
through axillary buds has been studied intensively for a
long time (Boxus, 1989; 1992). On a commercial scale,
tissue culture-derived strawberry plants are estimated
to cost more than plants produced by conventional
propagation. But, micropropagated strawberry has
several advantages, such as its ability to multiply virus-
free stock rapidly and, the improved capacity of these
plants to produce runners for planting in the field
(Lopez-Aranda et al., 1994). Meristem tips, generally
obtained from runners of virus-free plants, are
commonly used to establish in vitro cultures, which
are employed for mass propagation or as a source of
plant material for regeneration and transformation
experiments (Mercado et al., 2007). The shoot
multiplication of strawberry (Fragaria x ananassa
The Journal of Plant Science Research
118 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout

Dutch.) Cv. Chandler was achieved in MS medium IBA and 2% sucrose within 2 weeks of culture (Fig.1b)
supplemented with 2.0 mg/l BAP and 0.25 mg/l NAA and establish in the soil (Fig. 1c, d). The details in
(Fig. 1a). The root induction from in vitro shoots were vitro study of strawberry (Fragaria x ananassa
achieved in MS medium supplemented with 0.25 mg/l Dutch.) is presented in Table 1.

Fig. 1. Micropropagation of strawberry (Fragaria x ananassa) Cv. Chandler by using apical meristem on
MS medium supplemented with 2.0 mg/l BA, 025 mg/l NAA after 3 weeks of culture (Fig. 1A), Induction
of rooting on MS medium supplemented with 0.25 mg/l IBA after 2 weeks of culture (Fig. 1B). The in
vitro raised plantlets established in soil mixture with normal growth (Fig. 1C & D).

The Journal of Plant Science Research

 In vitro Studies of Strawberry - An Important Fruit Crop: A Review 119

Table 1. In vitro studies of strawberries (Fragaria × ananassa Duch.).

Source of Explant Culture Medium Response References

Leaf disk, shoot tips, MS + 2 mg/l of BAP + 2 mg/l IAA. Shoot bud initiation Madani et al., 2013
hypocotyls
Immature leaves MS + 3.0 mg/l 2,4-D + 0.5 mg/l BAP Callus induction Karim et al., 2011
MS + 1.5 mg/l BAP + 0.75 mg /l NAA Shoot bud initiation,
multiplication, rooting
Shoot bud MS + 0.5 mg/l Kinetin + 1.0 mg/l Shoot multiplication Kaur et al., 2005;
BAP + 2.0 mg/l GA 3 + commercial sugar Kaur et al., 2012
MS + 1.0 mg/l IBA + 200 mg/l Root induction
Charcoal + 20g/l commercial sugar
Nodal segments MS + 4.0 μM 6- benzyladenine + Shoot multiplication Bhatt & Dhar, 2000
0.1 μM NAA
Meristem N6 medium + 1.0 mg/l BAP + 0.05 mg/l Shoot multiplication Ali-Akbar Mozafari &
IBA + 0.05 mg/l GA3, Mohammad Gerdakareh, 2012
Runner tip MS + 1.5 mg/l BA with 0.5 mg/l Kinetin. Shoot multiplication Tanziman Ara, et al., 2013
Nodal segment MS containing 1.5 mg/l BAP + Shoot multiplication Shaila harugade et al., 2014
0.5 mg/l Kinetin
MS + 1.0 mg/l IBA Induction of rooting
Nodal segment MS + 1.5 mg/l BAP + Shoot multiplication Sakila et al., 2007
0.5-0.1 mg/l Kinetin
MS + 1.0 mg/l IBA Root multiplication
Adventitious shoot MS + 0.5 mg/l BAP + 0.1 mg/l GA3 + Proliferation of axillary Litwinczuk & Zubeta, 2005
IBA 0.1 mg/l + 40 g/l sucrose shoot, development of runner
Leaf disc of mature leaf MS + 1.0 mg/l 2,4-D + 0.1 mg/l BAP Induction of callus Tanziman Ara et al., 2012
MS + 2.0 mg/l 6- BAP + 0.5 mg/l NAA Shoot induction
Fresh nodes MS + 1.5 mg/l BAP + 0.1 mg/l Kinetin Shoot bud, shoot Rama Bhat et al., 2012
multiplication, induction of
rooting, field establishment
Leaf tissue LS + 3 mg/l BAP + 0.1 mg/l IBA + Shoot bud regeneration Liu et al., 1988
400 - 600 mg/l Casein hydrolysate
leaf blade, nodal explant, MS + 4.0 mg /l NAA+ 1.5 mg/l IBA Callus proliferation Biswas et al., 2010
runner segments MS + 3 mg/L BA. Shoot regeneration
Leaf disc, Petiole, MS + 1.0 mg/l BAP + 0.1 mg/l Callus Proliferation Passey et al., 2003
Stipule, Root segment IBA + 0.2 mg/l GA3
MS + 3.0 mg/l IBA Induction of rooting
MS + 2.0 mg/l BAP + 0.5 mg/l Shoot bud regeneration
TDZ + 0.2 mg/l 2,4-D
Nodal segment 1.0 mg/l 2,4-D + 0.5 mg/l Development of Biswas et al., 2007
BAP + 50% Proline somatic embryo
Leaf disk MS + B5 vitamins + 2.3 mg/l Adventitious shoot Nehra et al., 1989
BAP + 1.8 mg/l IAA bud regeneration
Leaf disk N30K macrosalts + MS microsalts and Adventitious shoot Barceló et al., 1998
vitamins (2 BA + 0.5 IBA) regeration
Leaf disk, petiole, MS + 1 mg/l TDZ + 0.2 mg/l 2.4-D, Adventitious shoot bud Passey et al., 2003
root, MS + 2 mg/l BA + 0.5 mg/l TDZ + regeneration
stipule explant 0.2 mg/l 2,4-D, 1 mg/l TDZ + 0.2 mg/l
NAA, 2 mg/l BA + 0.2 mg/l 2,4-D

The Journal of Plant Science Research

120 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout

Leaf disk ½MS+ 1.0 mg/l BAP + 1.0 mg/l IAA Adventitious shoot bud Singh et al., 2004
regeneration
Leaf, Petiole segment MS + 0.5 mg/l BA + 0.1 mg/l Adventitious shoot bud Litwiñczuk, 2004
GA3 + 0.1 mg/l IBA regeneration
Leaf, Petiole segment MS + B5 vitamins + 2.2 μM Adventitious shoot bud Zhao et al., 2004
TDZ + 0.3 uM IBA regeneration
leaf disk, Petiole segment MS + 0.44-0.88 mg/l TDZ Adventitious shoot bud Debnath, 2005
regeneration
Leaf disk segment MS + 1.5 mg/l TDZ + 0.4 mg/l IBA Adventitious shoot bud Qin et al., 2005a
regeneration
Leaf disk segment MS + 1.0 mg/l AgNO3 + 1.5 mg/l Adventitious shoot bud Qin et al. 2005b
TDZ + 0.4 mg/l IBA regeneration
Leaf segment MS + 2 mg/l TDZ + 0.5 mg/l IBA Adventitious shoot bud Hanhineva et al. 2005
regeneration
Leaf, petiole, MS + 0.11 μM BA + 0.011 μM Adventitious shoot bud Folta et al., 2006
segment Stolon 2,4-D + 1 μM TDZ regeneration
Runner segment MS + 0.5 mg/l BAP Shoot multiplication Ashrafuzzaman et al., 2013
MS+ 0.5 mg/l IBA Root intiation

Several parameters used during the in vitro study times more than that produced by conventional
which affect the behaviour of micropropagated plant material.
in the nursery, e.g. plant genotype, mineral
One of the main problems encountered with in vitro
formulation, type and concentration of cytokinin in
propagation was the massive bacterial contamination
the medium and the number of subcultures. It is
at the initiation and multiplication stages. After
generally recommended not to exceed four to five
transfer of the bud on to solid sterile medium, whitish
subcultures to avoid loss of trueness-to type of the
exudates of bacteria was observed around the base
propagated material (Faedi et al., 2002). This
of the explant after 2-3 d. In some cases, the
technique is useful for introduction of new cultivars.
contaminants appeared upon the sixth or seventh
Moreover, the storage of tissue cultured propagules
subculture. Contamination at the initiation stage
requires less space than traditional runner plant and
caused rotting of the bud whereas at the multiplication
the in vitro storage can be initiated at any time during
stage, the rate of tillering bore an average three- fold
the production cycle (Swartz et al., 1981). Prior
decrease with subsequent death of the plantlets in
experiences with strawberry micropropagation
about one month (Moutia and Dookum, 1999).
indicate that vitro plants are more uniform, produce
Contaminants in the xylem vessel which are protected
higher number of runners, have better survival in the
from surface sterilization are endophytic bacteria
field, and the fruit yield increases in 24% than plants
detected even in meristem-tip explants. Endophytic
propagated by the traditional method (Kikas et al.,
bacteria have probably evolved a close relationship
2006). Another advantage of micro-propagation is the
with their host plant through co-evolutionary processes
elimination of pest and pathogen stress during the
and may influence plant physiology in ways that have
production cycle, assuming that the initial stock plant
not yet been elucidated.
is free of diseases. Therefore tissue culture technique
was applied to evaluate its feasibility for a wider use. Role of phenolic during in vitro growth
Boxus (1983) reported that each m2 of growing area
of strawberry can produce 40000 plantlets per year. Tissue blackening occurs due to action of copper-
These plants were vigorous and after transplanting in containing oxidase enzymes: polyphenoloxidases like
the soil some produced up to 500 new runner plants tyrosinases, which are released or synthesized in
and it also indicate that new runner plants were 10 oxidative conditions after tissue wounding and they

The Journal of Plant Science Research

 In vitro Studies of Strawberry - An Important Fruit Crop: A Review
oxidize o-diphenols released due to cellular wounding
to O-quinones (Scalbert et al., 1988; Marks and
Simpson,1990). The onset of tissue browning has been
found to be associated with changes in protein pattern,
amino acid content, ethylene production and the
occurrence of saccharose and accumulation of starch
(Lindfor et al., 1990). These changes eventually lead
to growth inhibition or death of explants. Other types
of phenolic exudates appear at the end of incubation
period and are apparently products of dying cells
(Seneviratne and Wijesekara, 1996). The phenolic
exudation is aided by light and is autocatalytic. It is
possible, that some external factors trigger stress
symptoms such as browning in plant tissue. These
factors may be pathogens or in some cases even agar.
High concentrations of macrosalts, auxins, and sucrose
concentration in the culture medium have caused
browning as well. In addition, there are some
substances including 5, 6-Cl2-IAA, yeast extracts, and
phloridzin which directly enhance the production of
phenolic compounds (Marjo, 1999).
Accumulation of phenolics is most obviously associated
with the developmental stage of the plant and season,
which was shown in woody species. Explants collected
from the month of November to February had produced
low browning percentages in vitro, whereas the
browning was at its maximum if explants were collected
in month of April to August (Wang, et al., 1994).
Differences in browning between varieties and in
relation to the size of the explant were also observed.
Many medium components have been used to eliminate
tissue browning such as inclusion of nitrate as a source
of nitrogen; increased concentration of phytagel or
using gelrite instead of agar. Many authors have also
tested phenol traps such as activated charcoal,
adsorbent resin, citric acid, cysteine, PVPP and
antioxidants such as ascorbic acid or glutathione
(GSH). In addition, culturing in vitro plantlets under
low light intensity has given significant results (Marjo,
1999). Oxidized products, such as quinones, are known
to be highly reactive and inhibit enzyme activity leading
to the death of the explants. As a consequence of
browning, tissue senescence, recalcitrance in
embryogenesis and regeneration has been observed
(Marjo, 1999; Bhat and Chandel, 1991).
121
Effect of explants source on micro- propagation
There are numerous substantive phenotypic traits
associated with juvenility, but they vary considerably
among species. Commonly, the leaves on young plants
are of a different shape than those on mature parts
and may be simple rather than compound (or
occasionally the reverse); juvenile leaves may also
have a special type of cuticle and be arranged with a
distinct phyllotaxy. Compared to their adult counterparts,
young plants may have a modified resistance to pests
and diseases. Juvenility in woody plants is often
manifested by prolonged vigorous shoot growth. Young
healthy tissues that are rich in nutrients, and possibly
endogenous hormone, are the best choices for the
induction of cell division. While the woody plant material
is generally a poor choice. Also, plant tissues that are
high in oxidase activity pose a special problem since
enzymatic browning retards cell division. The browning
results from the activity of wound-induced copper
oxidases (polyphenoloxidase). This may be suppressed,
to some extent, by the use of an antioxidant compound
(John et al., 1995). The juvenile explants had pre-
existing competent cells that were able to respond to
auxin and become determined to form roots. However,
the mature explants appeared to lack cells with pre-
existing competence to form roots, but competence was
acquired by some callus cells once they had been
initiated. Explants taken from mature shoots are
frequently more liable than juvenile material to suffer
necrosis, especially when surface disinfested and placed
in culture, and juvenile explants are usually more readily
established in vitro and grow and proliferate at a more
rapid rate than adult material. This is particularly true
with tree species where micropropagation of adult
material is often difficult. Juvenile plants frequently
grow more rapidly and have stronger apical dominance
than adult forms. However, shoot explants from juvenile
plants generally proliferate more axillary shoots than
shoot explants from adult forms. A safer and more
promising alternative would be to use other plant parts
such as apical buds and apical meristems for
establishment of cultures. The apical bud is located
immediately below the apical meristem and enclosed
within the leaf sheath. Selecting these young tissues
makes it possible to reduce infection since the apical
zone displays better aseptic conditions because of the
The Journal of Plant Science Research
122 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout
reduced size of the explants and the small area exposed
to the external environment (Moutia and Dookum,
1999).
In vitro Organogenesis
The concentration and combination of auxins and
cytokinins in the nutrient medium is usually a key factor
which determines successful plant regeneration. The
use of activated charcoal as a culture component for
adsorption of toxic plant metabolites is known.
Activated charcoal is able to absorb high concentrations
of the growth regulators BAP, IAA, IBA, NAA and
Kinetin, in both liquid and solid media (Nissen and Sutter,
1990). Ethylene is a gaseous hormone produced by
plant tissue and/or the medium. More ethylene was
produced by Anemone caronaria seedlings than agar
alone (Mensuali-Sodi et al.,1993). Takayarna and
Misawa, 1980) reported that root formation and growth
were inhibited by higher concentration of BA, but this
inhibition was completely reversed by the addition of
activated charcoal. Root formation and growth were
better in BA-free medium containing activated charcoal
than in the medium without activated charcoal. Similar
results were observed in bulb formation. This could be
due to activated charcoal regulating internal
physiological processes (Pan and van Staden., 1998).
Meristem from the runner tip is mostly used as explant
for micropropagation of strawberry. The explant is
placed on a medium containing no or low levels of
auxins and higher levels of cytokinins to promote axillary
budding while preventing callus formation. The
cytokinins are used to overcome apical dominance and
enhance the branching of lateral buds from the leaf
axis. Additional shoots are produced through further
axillary bud growth (Debnath, 2003). Boxus (1974)
experimented with BA to determine its influence on
shoot and root production in vitro. He established that
shoots would proliferate in the presence of the cytokinin
but roots would not form until the explant was without
cytokinin. He concluded that micro-propagated
strawberry plants would replace traditional methods
of propagation for the commercial trade. Mullin et al.
(1974) grew strawberries with strawberry mild yellow
edge (SMYE) viruses for 6 weeks in a 36°C growth
chamber before excising 0.3 to 0.8 mm meristematic
tips with leaf primordia. The result was that 33% to
The Journal of Plant Science Research
75% of the resulting plants were SMYE free according
to leaf insert graft indexing to indicator strawberry
plants. Liu and Sanford (1988) developed the methods
for efficient shoot regeneration from leaf and runner
tissues of the strawberry Fragaria x ananassa Duch.
cultivars. Allstar. and. Heneoye. They showed that
optimal regeneration condition differed for .Allstar
depending on the leaf tissue was derived from plantlets
grown in vitro or from plants grown in the greenhouse.
Leaf tissues of Allstar derived from in vitro culture
regenerated shoots most efficiently in a Linsmaler Skoog
(LS) medium containing 2.5 mg/l BA and 0.5 mg/l IBA.
Leaf tissues of, Allstar derived from greenhouse plants
regenerated shoots significantly on LS medium
containing 3.0 mg/l BA and 0.1 mg/l IBA. Addition of
casein hydrolysate (CH) at either 400 or 600 mg/l
stimulated shoot production. A supplement of KNO3
at 2000 mg/l also enhanced regeneration efficiency of
greenhouse - grown leaf tissues. Shoots from runner
tissues of both cultivars were best obtained using (LS)
medium containing 10 mg/l BA, 2.0 mg/l IAA, and 500
mg/l CH. Shoots proliferation from runners was
dependent on the diameter of the runner, with diameters
of more than 2.0 mm having poor regeneration
potential. Boxus (1989) reported that this clonal
propagation process proceeded exclusively through
axillary branching which normally leads to true-to-type
progenies. Jelenkovic et al. (1991) used different
cultivars with various explants (hypocotyls, runners,
petioles and lamina0 for in vitro studies. They observed
that BA and 2, 4-D were the most effective
phytohormones to regeneration of shoot bud. Lopez -
Aranda et al. (1994) studied in vitro culture of
strawberry cvs. .Douglas. and .Chandler by using
mineral salts and growth regulators for induction of
regeneration ability. The best regeneration frequency
was achieved with a formulation of N30K salts
formulation for both cultivars. In vitro rooting were
obtained with either the Knop or N30K salts formulation
or addition of low concentration of auxin. Addition of
500 mg/l activated charcoal also enhanced rooting; in
contrast, high BA levels during multiplication decreased
the rooting capacity of shoots. They noted that
micropropagated plants showed a higher runner
production capacity than their control counterparts.
They also concluded that salts formulation, BAP levels
 In vitro Studies of Strawberry - An Important Fruit Crop: A Review 123

(1.48 μM - 4.44 μM) in the culture medium and the Their multiplication rate and cytokinin content were
number of the subcultures (1 - 8) seemed not to be also higher than for axillary buds. No significant
affected the behaviour of plants in the nursery. No difference was observed in auxin content. Meria et al.
adverse effects on total fruit production or fruit size (2002) demonstrated that the actively growing shoots
could be associated with the mineral formulation used of potted greenhouse – grown strawberry tree
during the in vitro phase. The authors concluded that (Arbutus unedo L.) were grown in basal woody plant
the micropropagated plants are greatly influenced by medium containing 11.1 μM BA. Optimum shoot
their in vitro culture. proliferation was achieved on a basal WPM containing
MS vitamins, sucrose, agar and 22.2 μM BA. They
Nehra et al. (1994) studied the field performance of
observed in preliminary testes with MS basal salts
in vitro grown plantlets of two strawberry cultivars
instead of WPM basal salts resulted in extensive
‘Redcoat’ and ‘Veestar’. Flowering and fruiting
oxidative browning and failure of explants after culturing
behaviour of ‘Veestar’ was not appreciably influenced
for 3 weeks, This browning was due to a high
by in vitro propagation methods. However, in vitro
concentration of salts or, in particular, due to high
propagated plants of ‘Redcoat’ flowered earlier and
NH4NO3 concentration in MS medium. Microshoots
produced more flowers and fruits than SR plants, but
rooted successfully in basal medium containing 10 μM
still maintained normal berry weight. Among in vitro
IBA or IAA, but their survival rate during
propagated plants, daughter plants of Redcoat were
acclimatization was low. Addition of a mixture 1 part
the earliest to flower, whereas MC plants produced
peat: 4 parts perlite in the basal in vitro rooting medium
more flowers and fruits. The field performance of the
(1:1 v/v) containing 10 μM IAA resulted in high rooting
first daughter plants derived from the in vitro
percentage and plantlets with branched roots. These
propagated plants was consistent with their respective
plantlets were successfully acclimatized. This novel
mother plants. They also observed that leaf shape of
rooting medium can bexploited further due to its
both cultivars was not altered by in vitro propagation.
potential in commercial applications. Zhou et al. (2005)
Phenotypic abnormalities were mainly confined to
observed that rooting strawberry plantlets under
occurrence of yellow leaf variants in MC and CC plants
photoautotrophy conditions significantly increased plant
and occasional appearance of plants with irregular
height and weight after acclimatization. This effect was
flowering and growth habit among CC plants. Jemmali
related to a better photosynthetic performance of in
et al. (2002) demonstrated that adventitious stipular
vitro plants. Biswas et al. (2007) studied about the
bud formation occurred in vitro in many strawberry
effects of colour illumination on multiple shoot
cultivars during the proliferation phase on medium
regeneration from runner tip explants of strawberry.
containing Knop macronutrients, MS micronutrients,
Proliferated shoots showed 100% rooting in half
vitamins, amino acids, 2.22 μM BAP, 2.46 μM IBA
strength of MS media. Plantlets were established
and 0.29 μM GA3. As described previously for cultivar
successfully in soil. Madany and Hosseini (2007)
Gorella and Elsanta also showed adventitious stipular
suggested that an efficient method of micropropagation
buds developing on the abaxial median zone between
based on an increased percentage survival of explants
the stipule tips. To compare the shoots produced from
and reduced phenol-induced browning in strawberry
both types of buds, clonal propagation was initiated from
(Fragaria x ananassa Duch.) cv. Camorosa and Selva
stipular buds and from axillary buds on the Knop
has been developed. The effect of hormone
macronutrients and MS micronutrients medium. Stipular
concentration growth regulator balance and kind of
buds were separated from the meristem-tip initiated
antioxidant in medium on the direct shooting of meristem
plantlet and cultivated in the presence of a lower BAP
culture was studied. They showed that the best results
concentration (1.33 μM) to prevent further stipular bud
were obtained on MS basal medium with B-5 vitamins
formation. During proliferation cycles, stipular
supplemented with 1 mg/l IAA and 2 mg/l BA for
originated propagules were very easily distinguished
Camorosa and 2 mg/l IAA and 2 mg/l BAP for Selva.
by their specific leaf phenotype and light green colour
Use of 0.2 % activated charcoal was better than 2%
in comparison to plantlets cloned for an axillary bud.

The Journal of Plant Science Research

124 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout
PVPP to eliminate inhibitory substances from in vitro
cultures. Excised shoots rooted on MS medium with
2 mg/l IAA and 0.3 mg/l BA.
Sakila et al. (2007) showed that nodal segments of
strawberry gave rise to multiple shoots when cultured
on MS medium supplemented with different
concentration of BA with KIN or GA3. The highest
response of shoot multiplication was obtained in MS
containing 1.5 mg/l BA + 0.5 mg/l Kinetin. The
regenerated shoots were rooted on MS basal medium
with different concentrations IBA and IAA. The
maximum frequency of rooting and highest number
of roots was produced on medium containing 1.0
mg/l IBA. The plantlets, thus developed were
hardened and successfully established in soil. The
plants raised through tissue culture exhibited normal
growth, flowering and fruit setting. Emarah 2008,
demonstrated that an efficient method for shoot
regeneration, root formation from runner tips and
acclimatization of strawberry plantlets was
developed. Runner tips of 1-2 cm long were used as
source of explants. After surface sterilization apical
meristems of 3-5 mm long were isolated and used
as explants. At multiplication stage, results indicated
that the highest shoot number, shoot length and leaf
number were observed when MS medium
supplemented with 1 mg/l BA followed by the
medium contained 1 mg/l BA and 0.1 mg/l IBA.
However control treatment showed a significant
similar result in shoot length only. Results of this
study indicated that, BA was more effective in
enhancement the growth of strawberry in vitro
compared to Kin and thidiazuron. He also
demonstrated that at rooting stage, it was clear that
MS medium at full strength containing 30 g/l sucrose
significantly surpassed all other combinations of MS
strengths and sucrose concentrations in increasing
root number and length per plantlet and fresh weight/
plantlet. The same treatment enhanced the shoot
length but without significant difference compared
to some other combinations. The treatment
contained 3 g/l agar with 6 g/l perlite significantly
enhanced root formation as well as shoot length,
fresh weight/plantlet, and leaf number/plantlets. The
plantlets were successfully acclimatized and the soil
mixture with high percentage of survival (80%) with
The Journal of Plant Science Research
enhancing both root number and length/plantlet,
plantlet height and leaf number/plantlets.
In vitro regeneration via. callus culture
Plant regeneration from callus tissue cultured in vitro
can play an important role in the propagation and
improvement of crop plants (Yeoman and Macleod
1997) Plant regeneration from strawberry callus was
reported for first time by (Jones et al. 1988). (Nehra
et al. 1990a,b) reported that callus culture of F. ×
ananassa Cv. Redcoat after 24 weeks, complete lost
its regeneration capacity. Adventitious shoot
regeneration has been achieved in several cultivars
using a broad range of explants such as leaf explants,
petioles, stipules, stem tissue, runners, mesophyll
protoplasts, anthers, cotyledons, roots and immature
embryos (Mercado et al., 2007). Jemmali et al.
(1992) focused that adventitious buds of strawberry
arises between the two leaf stipule points proliferating
with very fast with axillary branching. They concluded
that the performances of the plants regenerated from
stipular shoots are similar to the control ones. They
noted that all roots of stipular plantlets were
exclusively white, whereas the control ones were more
or less colored with anthocyanin. Rugini and Orlando
(1992) studies the adventitious shoot regeneration
by using different explants and they found that there
is great differences in shoot regeneration ability from
calluses derived from leaf, petiole and root tissues.
However, these differences disappeared when whole
leaves, including stipules, were used as explants.
Passey et al. (2003) studied seven commercial
cultivars of strawberry using leaf disks, petioles, roots,
and stipules as explant material. The leaf disks had
the highest regeneration rates for all cultivars with
greater than 90% of explants producing shoots. Samir
and Debnath (2005) reported an efficient system to
regenerate shoots on excised sepals (calyx) of
greenhouse-grown strawberry through in vitro.
They found that sepal cultures produced multiple buds
and shoots without an intermediary callus phase on
2.4 mM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea
(thidiazuron, TDZ)-containing shoot induction medium
within 4 - 5 wk of culture initiation. He also observed
that young expanding sepals with the adaxial side
touching the culture medium and kept for 14 days in
 In vitro Studies of Strawberry - An Important Fruit Crop: A Review
dark produced the high rate of differentiation. He also
reported that the explant like sepals proved more
effective than the leaf discs and petiole segments for shoot
regeneration on medium having TDZ (0.5 – 4.0 mg/l)
promoted more callus formation and suppressed shoot
elongation. TDZ-initiated cultures transferred into the
medium containing 2.4 mM zeatin, produced usable
shoots after one additional subculture and subsequently
rooted on medium without growth regulators. Qin et
al. (2005) reported that the leaf explants cultured for
10 days in shoot regeneration medium in the presence
of AgNO3 not only enhanced shoot regeneration
efficiency but also expedited the formation of
adventitious buds. Samir and Debnath (2006) reported
that the inclusion of 2, 4 or 8 uM thidiazuron (TDZ) in
the growth medium enhanced the rate of shoot
regeneration from young expanding sepals, leaf disks,
petiole without an intermediary callus phase. Sepals
proved more effective than leaf discs and petiole
segments for regenerating shoots.
Kartha et al. (1980) studied the effects of light intensity
and culture media on shoot bud regeneration of
Fragaria var. Redcoat. Calli developed from leaf disk
of in vitro raised shoot has low regeneration frequency
than the green-house grown plants. Narender et al.
(1990) compared the regeneration frequency from
immature leaf explants derived from in vitro shoots
and greenhouse grown plants of the strawberry cv.
Redcoat. Both types of explants formed callus and
multiple shoots at various frequencies in the presence
of benzyladenine (BA) and 2,4-dichlorophenoxyacetic
acid at different concentrations. Highest frequency of
shoot regeneration occurred with the calli derived
from leaf explants at 5 μM each of BA and 2,4-D within
24 weeks. Improved regeneration (50%) with vigorous
shoot proliferation was achieved in 10 weeks by
transferring the 5 week old green compact calli derived
from leaf explants on MS basal medium supplemented
with 10 μM BA and 1 μM 1-naphthaleneacetic acid
(NAA) and incubating the cultures at 16 h light (62.5
μE. m -2 s-l). Saifullah and Spoor (2000) demonstrated
that the potential of callus culture and regeneration was
evaluated in strawberry (Fragaria ananassa). The
effect of different hormonal combinations and explant
sources was studied in- order -to produce maximum
number of plant in shortest possible time. They
125
observed that the organic potential of explants from in
vitro culture shoots compared with those taken from
glasshouse grown plants was significantly different for
the same media. The calli induced from in vitro grown
plant exhibited high regeneration as compared to those
induced from glasshouse grown plants. They also
reported that the maximum shoot regeneration was
observed on media containing 2.25 mg/l BAP and 1.0
mg/l IBA whereas, glasshouse grown explants gave
maximum shoots on same medium. Wojciech et al.
(2005) demonstrated that the growth of in vitro cultures
of strawberry (Fragaria × ananassa) depending on
different photoperiods. Wang et al. (1984) reported
that the somatic embryogenesis was achieved from
cotyledons explants on MS medium supplemented with
22.6 uM 2, 4-D, 2.2 uM BA and 500 mg/l casein
hydrolysate whereas few of the embryogenic tissues
developed into somatic embryos. Morphologically,
normal plants were obtained from somatic embryos
that were transferred to MS medium containing 2.89
uM GA3 or 2.22 uM BA + 0.54 uM NAA. Maintenance
of the embryogenic cultures was, however,
unsuccessful. Donnoli et al. (2001) achieved somatic
embryogenesis on MS medium supplemented with 4.88
uM BA and 4.90 uM IBA in strawberry var.Clea. Murti
and Yeoung (2013) used meristems excised from
runners of Camarosa and Redpearl strawberry cultivars
for micropropagation on MS media enriched with
different concentrations of IBA (0.2, 0.3, 0.4, 0.5, 0.6
ppm) and BA (0.5, 0.6, 0.7, 0.8, 0.9 ppm). The result
showed that all explants in IBA produced one plantlet
and 0.2 ppm produced the best plantlet performance,
while all of BA concentration induced multiple shoots
and 0.6 ppm produced 7-26 shoots/explant. MS media
without IBA was recommended for rooting induction.
In both of hormones, Cv.Camarosa in vitro plantlet
performance was better than Cv. Redpearl, but the
performance of Cv. Redpearl plantlet was better than
Cv. Camarosa after acclimatized.
Transplantation of in vitro grown plants
A substantial number of micropropagated plants do not
survive transfer from in vitro conditions to greenhouse
or field environment. The greenhouse and field have
substantially lower relative humidity, higher light level
and septic environment that are stressful to
The Journal of Plant Science Research
126 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout
micropropagated plants compared to in vitro
conditions. The benefit of any
micropropagationsystem can, however, only be fully
realized by the successful transfer of plantlets from
tissue-culture vessels to the ambient conditions
found ex vitro. Most species grown in vitro require
an acclimatization process in order to ensure that
sufficient number of plants survive and grow
vigorously when transferred to soil. The transfer
of in vitro plantlets to ex vitro conditions is one of
the most critical factors of the micro-propagation
process and a cause of higher production costs.
High mortality is often observed upon transfer to
ex vitro conditions as the cultured plants have a
poorly developed cuticle, non-functional stomata
and a weak root system. In order to increase growth
rate and reduce mortality of plantlets at the stage
of acclimatization, recent research has focused on
control of the environmental conditions. One
approach has been to modify the environmental
conditions during acclimatization by increasing light
intensity or both increasing the light levels and
altering the CO 2 concentration. Another approach
has been to change the environment during the
multiplication and rooting stages, including
increasing light intensity and CO2 concentration in
culture tubes, and decreasing the sugar
concentration. All were found to be beneficial for
plantlet growth in the later stages of
micropropagation (Abdelmalek et al.,1995). A
biological approach to reducing the stress of
acclimatization and providing faster growth of
micropropagated plantlets is the establishment of
vesicular-arbuscular mycorrhizae (VAM) on
micropropagated plantlets during acclimatization.
VAM colonization of horticultural plant roots can
improve growth by increased uptake of
phosphorus, zinc and other minerals and may reduce
the incidence of disease. Moreover, colonization
with VAM fungi may increase transplant uniformity
and reduce both transplant mortality and injury.
Recent work has also shown improvement in water
relations of the host plant using VAM. These fungi
may also improve drought tolerance by decreasing
leaf water potential, by reducing stomatal and root
hydraulic resistances, and by increasing
The Journal of Plant Science Research
transpiration rates. Several studies have focused
on VA mycorizal formation during acclimatization
of cultural plantlets (Abdelmalek et al. 1995)
Somaclonal variation
Strawberry plants spread vegetatively using runners
and this enables them to be easily transplanted and
propagated as clones. Commercial production of
strawberry using micropropagation processes bears
several risks. Plant off-types, i.e. non true-to-type and
genetically not identical to the mother plant (Gaafar
and Saker, 2006). Nevertheless, these auxins are known
to be associated with genetic instability in plants, a
phenomenon called somaclonal variation. Although,
somaclonal variation may be used as a source for
variation to get superior clones, it could be also a very
serious problem in the plant tissue culture industry
resulting in the production of undesirable plant.
However, through micro-propagation of strawberries,
several morphological abnormalities were detected as
somaclonal variation. Changes include leaf color
variants and dwarf plants, among others (Adel El-Sawy
Mohammed, 2007). Callus-derived strawberry Var.
Redcoat plants differ significantly from standard runner
plant with several vegetative characteristics (Nehra et
al., 1992). These variants pose a problem for
production of uniform, true-to-type plants. Nehra et
al. (1994) observed that two cultivars of strawberry
responded differently to various forms of in vitro
propagation and in both cases variants were found in
callus-derived plantlets, but not those derived from
meristems or via direct regeneration from leaf disk.
Some studies have shown that modified characteristics
are epigenetic and disappear over time. Numerous
authors have reported that genetic changes including
insertions, deletions, point mutations and
rearrangements occur during tissue culture, but few of
the phenotypic symptoms found are heritable (Karp,
1995 and Kumar et al., 1999). Damiano et al. (1997)
reported the somaclonal variation and in vitro
regeneration of strawberry cultivar “Teodora’ and
‘Clea’ on MS medium. They shown that petioles and
laminas, produced poor callus only, but the stipules are
highly competent for organogenesis, and the BAP alone
is sufficient to induce regeneration, while 2, 4-D is
strongly inducing callus formation. Most somaclonal
 In vitro Studies of Strawberry - An Important Fruit Crop: A Review
variations occur in plants regenerated from cultures
that have undergone a differentiation phase. Some
studies indicating that a strong genetic component
determines the success of adventitious regeneration
(Tian et al., 2003). Yonghua et al. (2005) found that
shoot regeneration was enhanced when explants were
cultured under red or green plastic films, and this was
correlated with an increase in activity of antioxidant
enzymes and endogenous hormone concentration.
Presently, there are various methods available which
can be used to detect and monitor tissue culture-derived
plants and varietal identification. The most reliable
methods are the molecular marker techniques that
identify the variance depending on the plant proteins,
which are expressed from defined regions of DNA, or
DNA polymorphisms. ISSR (Inter Simple sequence
repeats) and RAPD (random amplified polymorphic
DNAs) are the molecular technique for identification
of genetic variation. It has the distinct advantage of
being technically simple and quick to perform, requiring
only small amounts of DNA compared to restriction
fragment length polymorphism (RFLP) analysis.
Strawberries (Fragaria × ananassa Duch.) have
been extensively analyzed using randomly generated
markers for clone identification and diversity studies
(Adel El-Sawy, 2007). Polymorphisms also appear in
acid-phosphatase, glutamate-dehydrogenase and
peroxidase of regenerated plants. Adel El-Sawy (2007)
studied the somaclonal variation in micro-propagated
strawberry detected at the molecular level. Meristem
tips of three strawberry cultivars, namely Chandler,
Sweet Charlie and Gaviota were excised and cultured
for 5 weeks on the starting medium. Subculture was
carried out for five weeks on shooting medium, and
finally shoots transferred to the rooting medium. DNA
extracted from in vitro-derived plantlets and
conventionally propagated plants were analyzed by
RAPD to detect possible drift in genetic stability of
micro-propagated plants. He observed that the banding
pattern of in vitro-derived plantlets were similar with
the fingerprints of mother plants, demonstrating no
variation in the pattern obtained with DNAs from the
two sources of strawberry plants. It is concluded that
mass propagation via meristem tip culture is reliable in
producing genetically similar plants to the mother ones.
Ten deca-nucleotide primers (among 48 tested) were
127
chosen for RAPD analysis, all of them indicating
genetic stability for micropropagated plants of the
investigated varieties of ornamental strawberry. A
comparative study was conducted based on
morphological parameters as well as genetic
assessments using ISSR markers. The in vitro
generated strawberry plants exhibited significantly
vigorous morphological growth and earlier flower
induction when compared to the plants propagated
through planting of runners. Genetic assessment
through ISSR showed no polymorphism in banding
pattern and thus it was revealed that, there was no
significant variation between micropropagated and
conventional propagated plants at molecular level. Sutan
et al. (2009) studies the genetic stability of plants
obtained from tissue culture propagation of ornamental
strawberry “Serenata”. Ten primers (from 48
previously tested) were selected and used in RAPD
analysis to prove the clonal fidelity of the tissue culture-
derived ornamental strawberry plants. They observed
that there are similar banding patterns of the RAPD
profiles obtaining from in vitro plants, regenerated via
organogenesis or meristem culture. There are no
differences with regard to different explants source,
number of subcultures or use of different basal medium
with growth regulators.
GENETIC TRANSFORMATION STUDY
The Rosaceae ranks third among families of
economically important cultivated crops in temperate
regions. Genetic improvement of most rosaceous crops
is hampered by cumbersome genome size, intolerance
to inbreeding, and lengthy life cycle. Propagation of
strawberry is achieved either by runners or by in vitro
micropropagation. Several improvements of the
technology have been proposed by authors working
with strawberry but the highest genotypic, physiological
and morphological quality of micropropagated plants is
produced by the method described by Boxus and co
workers (Boxus, 1983). Tissue culture stock is used
by most researchers as the source plant material for
regeneration and transformation studies. Genetic
engineering of strawberry has already been reported
(Jame et al., 1989; Nehra et al., 1990a,b). However,
transformation frequencies are greatly influenced by
the cultivar and the procedures used, e.g. 0.95%
The Journal of Plant Science Research
128 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout
transformation frequency has been reported for the
cultivar Rapella (Jame et al., 1990) and 6.5% for the
cultivar Red Coat (Nehra et al., 1990a). Barcelo
et al. (1998) achieved the regeneration of shoot buds
from leaf disk explants of strawberry in presence of
2.46 uM IBA and 8.88 uM BA, where 47% of the
cultures regenerated after 16 weeks with 2.9 shoot
colonies per regenerating leaf disk. Optimum incubation
conditions included two weeks in the dark with
subsequent transfer to light with 16 h photoperiod.
Transformation was attempted using Agrobacterium
tumefaciens carrying the plasmid pBI121. Leaf disks
from in vitro cultures proliferating in the presence of
2.21 uM kinetin were best explants for transformation.
The transgenic nature of several shoots was also
confirmed by the GUS assay and PCR analysis.
Previous studies on strawberry have clearly
demonstrated the importance of various factors such
as genotype (Liu and Sanford, 1988), type and source
of explant (Nehra et al., 1989), hormonal balance and
incubationconditions (Liu and Sanford, 1988).
Khammuang et al (2005) reported the optimum
condition for shoot bud regeneration from leaf explants
of strawberry cultivar Tiogar. It was found that the
best regeneration condition was MS medium containing
1.0 mg/l BA and 0.2 mg/l 2,4-D. Antibiotics sensitivity
test found that shoot regeneration from leaf explants
was inhibited more than 90% at the concentration of
kanamycin as low as 5 mg/l . The modified gene
encoding antifreeze protein isoform HPLC 6 was
successfully constructed using codons which were
optimally expressed in the strawberry plant. The
antifreeze protein genes, naturally in plasmid pSW1 and
modified in plasmid pBB, were transformed to
strawberry leaf explants by Agrobacterium
tumefaciens LBA 4404. The strawberry plants,
transformed with both AFP genes, were able to root in
MS media containing 50 mg/l kanamycin, while no roots
grew from nontransformed plant in this condition.
Polymerase chain reaction indicated that the transgenes
were integrated in the genome of transformants.
Teruko et al. (2006) described a new transformation
procedure that uses leaf explants from newly unfolded
trifoliate leaves obtained from stock plants 6–7 weeks
after seed germination, co-cultivation with
Agrobacterium strain GV3101, and stringent selection
The Journal of Plant Science Research
on MS medium containing 4 mg/ l hygromycin. Using
this protocol we achieved 100% transformation
efficiency for 6 of 14 F. vesca accessions tested.
Accession PI 551572 was determined to be the best
candidate for a model in F. vesca functional genomics
research, as it showed the greatest propensity for callus
formation, transformation, shoot regeneration, ex vitro
establishment, and plant growth, requiring only 14–15
weeks to complete its life cycle in different seasons in
the greenhouse. Further, Toshiyuki et al. (2008)
introduced the transgenic plants having the gene
encoding the taste-modifying protein miraculin was with
35S or El2 promoter into strawberry (Fragaria x
ananassa) by Agrobacterium-mediated transformation
to produce transgenic plants. Although miraculin was
detected in the leaves and fruits of the transgenic plants,
the level of accumulation among the transgenic lines,
which ranged from 0.5 to 2.0 mg/g miracle fruits (145
mg/g) fresh fruit, was not significantly different and
was lower than that in fresh fruit). High levels of
miraculin accumulation were detected in the mature
fruits. The transgenic lines were subsequently
propagated via the runners for three vegetative
generations, and miraculin was detected at equal levels
in the leaves and fruits of the plants from each
generation.
CONCLUSION
In vitro propagation of strawberry has significant
opportunities for commercial cultivation. To overcome
this challenge, careful optimalisation of protocol for each
cultivar is utmost important. Suitable protocol can be
used for automation, using a bioreactor, is one of the
most effective ways to reduce the costs of
micropropagation. The growth regulators like BA and
IBA or NAA have effective for high scale of progation.
The in vitro raised plants produced maximum number
of runners as compared with conventional propagated
plants. Liquid medium with less cytokinin concentration
(1-2 mM BA or zeatin) produced more numbers of
microshoots as compared with agar gelled medium.
Clonal fidelity is one of the main concerns in strawberry
micropropagation. True-to-type propagules and genetic
stability are prerequisites for the application of
micropropagation. The occurrence of somaclonal
 In vitro Studies of Strawberry - An Important Fruit Crop: A Review
variation during micropropagation can be controlled by
numerous factors including genotype, the presence of
chimeral tissue, explant type and origin, media type, types
and concentrations of growth regulators, cultural
environment (temperature, light, etc.) and duration of
culture. In vitro organogenesis and somatic
embryogenesis were alternatives means of mass scale
propagation. Clonal fidelity of micropropagated plants
can be monitored by their morphological, biochemical,
physiological and DNA level. Molecular markers like
ISSR, RAPD, RFLP are powerful tools in genetic
identification of somaclonal variation. In the present
scenario, a high market demand of strawberry cultivars
with high productivity and berry quality, and a reduced
pesticide application. In recent advances in plant
biotechnology could help to satisfy this consumer
demand. Transformation technology in strawberry has
been achieved with taste-modifying protein miraculin
with 35S promoter. Although miraculin was detected in
the leaves and fruits of the transgenic plants, the level
of accumulation among the transgenic lines, which
ranged from 0.5 to 2.0 mg/g miracle fruits (145 mg/g)
fresh fruit. The development of a new “clean vector
technology”, the sequencing of gene controlling
agronomic traits, under the control of their own promoters
will allow to produce transformed plants with better
consumer acceptance. Applications of this technology
could support and complement the strawberry
improvement program and competitiveness in the world
market.
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