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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 appearance and seasonal availability make this fruit
sets of the seven chromosomes (14 chromosomes an excellent crop. Even more, strawberries are rich
total). Others are tetraploid (4x = 28), hexaploid (6x = in phytochemical compounds with potential
42), octoploid (8x = 56) or decaploid (10x = 70) (Darnell, antioxidant compounds, mainly ellagic acid and
R., 2003). In vitro propagation of strawberry plants flavonoids, which can lower the risk of
was introduced about thirty years ago (Boxus 1974). cardiovascular events and tumorogenesis (Hannum,
Most of the European nurseries producing several 2004). These qualities have ensured that the
millions plants per year through in vitro technique as it economic importance of this crop has increased
gave a definitive answer to the problems of soil fungal throughout the world and, now-a-days, it remains as
flora, causing a lot of damage to the strawberry fields a crop of primary interest for both research and fruit
and by another way, tissue culture plants seemed to production (Mercado, et al., 2007). Strawberry
produce more runners per mother plant in a short time (Fragaria x ananassa Duch.) fruits are rich
period (Mohan et al. 2005). Micropropagation has also sources of phytochemicals of which phenolic
been widely used in the USA (Zimmerman 1981) in compounds are predominating. Berry fruits are
commercial propagation of strawberries and in breeding reported to contain a wide variety of phenolics
programs to produce many plants rapidly. The present including hydroxybenzoic and hydroxycinnamic acid
review highlighted the in vitro studies as well as genetic derivatives, anthocyanins, flavonols, flavanols,
improvement of strawberry. condensed tannins (proanthocyanidins) and
hydrolyzable tannins (Machiex et al., 1990). In vitro
STATUS OF NUTRITIONAL VALUE studies indicate that berry phenolics have a wide
According to Nutrient Database for Standard range of biological properties such as anti-cancer,
Reference (USDA 1999) the strawberry fruits are antioxidant, anti-inflammatory, and cell regulatory
resources of the nutritious elements essential for effects (Seeram, 2006a,b; Seeram and Heber, 2006).
human being. Strawberries had been a favourite These anticancer effects are exerted through multi-
among the fruits of the temperate world. They were mechanistic means of action including the
valued for delicious flavor and fragrance, for health- antioxidant actions of the berries phenolic
restoring qualities and as harbinger of spring (Wilhelm constituents by protecting DNA from damage, and
et al., 1974). Flavourful and nutritious, strawberries also through effects exerted beyond antioxidation
are enjoyed by millions of people in all climates and (Seeram et al., 2006). The biological activities of
are predominantly used as fresh fruit. Their use in strawberry phytochemicals include the regulation of
processed forms such as cooked and sweetened phase-II enzymes and the modulation of gene
preserves, jams or jellies and frozen whole berries or expression and subcellular signalling pathways of cell
sweetened juice extracts or flavourings, and their use proliferation, angiogenesis and apoptosis
in making a variety of other processed products made (programmed cell death). Although there have been
them one of the most popular berry crops, more widely many published reports on the anticancer effects of
distributed than any other fruit (Samir et al., 2005, individual phenolics known to be present in the
2006). Strawberry is cultivated all around the world, strawberry fruit (Seeram 2006a,b). Strawberry
not only for its digestive and tonic properties, but also extracts have also been evaluated for their ability to
because of the nutritional value of its fruits, important inhibit mutation by the direct-acting mutagen methyl
source of foliate, vitamin-C, fiber, potassium, methane sulfonate, and the metabolically activated
flavonoids, autocianidin, phytochemicals and carcinogen, benzopyrene. Ethanol extracts from
antioxidants. freeze-dried fruits of several strawberry cultivars
were also evaluated and hydrolyzable tannin-
PHYTOCHEMICALS ENRICHMENT containing fractions from strawberries were found
Although strawberry is not an essential component to be most effective at inhibiting mutations (Hope et
of the diet, it’s delicious flavor and taste, attractive al., 2004).

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In vitro Studies of Strawberry - An Important Fruit Crop: A Review 117

Anthocyanins are natural pigments providing scarlet Ramularia leaf spot, [Mycosphaerella fragariae] and
to blue colors in flowers, fruits, leaves and storage leaf scorch [Diplocarpon earliana ]. Alternaria leaf
organs. The recent interest in the field of anthocyanin spot or black leaf spot (Alternaria alternata (FR.)
chemistry has been generated by restriction and Keissler) causes serious damage in Europe, New
limitation of the use of synthetic dyes as food Zealand and Korea (Hancock et al., 2008). Powdery
ingredients. Because of low toxicity of anthocyanins, mildew (Sphaerotheca macularis) is also found across
they have a high potential as a food colorant as the most of the strawberry range, although it rarely does
substitute of synthetic red dyes. Recently, these economic damage. Angular leaf spot, Xanthomonas
anthocyanins have been thought to have fragariae, is a rapidly growing problem in strawberries
pharmacological effects, such as lowering the all across the world (Hancock et al., 2008).
atherogenic index (Igarashi et al., 1990) and decreasing Anthracnose is a common problem in strawberries,
triglyceride and free fatty acid levels (Igarashi et al., causing a wide array of symptoms including fruit rot,
1990). Moreover, Kamei et al. (1995) reported that crown rot, and lesions of the stolons, petioles and
anthocyanin was more effective to inhibit the growth leaves. Anthracnose diseases of strawberry are caused
of tumor cells than other flavonoids. Studies concerned by Colletotrichum fragariae, C. Acutatum.
with anthocyanin production using plant tissue cultures
have therefore become very important. Masayuki et IN VITRO STUDIES OF STRAWBERRY
al. (1999) demonstrated that Fragaria ananassa One of the important goals of the agricultural policy in
(strawberry) callus, which produced high amounts of the world is to increase the acreage of strawberry to
anthocyanin in the dark which accumulated more than meet the demand of local fresh market, processing and
1000 μg of anthocyanin per gm fresh cell (Igarashi and export. Healthy stocks used for propagation through
Inagaki,1991). Sato et al. (1996) reported that in the conventional methods are not available.
suspension cultures of F. ananassa cells, anthocyanin Micropropagation of strawberry was established in
content increased with the intensity of light irradiation 1974 because of two important factors i.e. soil fungi
from 2500 to 8000. causing a lot of damage to the strawberry fields and
in vitro raised plants seemed to produce more runners
DISEASE IN STRAWBERRIES
per plant in a short time. However, strawberry tree is
Several soil pathogens damage strawberry roots, difficult to propagate by seed due to genetic variation
resulting in vigour declines and ultimately death. Two and specific requirements of seed germination. In
very common problems across the world are red stele addition, rooting percentage of cuttings is relatively low
or red core caused by Phytophthora fragariae (Mohan et al., 2005). Micropropagation of strawberry
Hickman and Verticillium wilt caused by Verticillium through axillary buds has been studied intensively for a
albo-atrum and V. dahlia. Black root rot is also long time (Boxus, 1989; 1992). On a commercial scale,
widespread and is caused by a complex of organisms tissue culture-derived strawberry plants are estimated
including Pythium, Rhizoctonia and the root lesion to cost more than plants produced by conventional
nematode (Pratylenchus penetrans) (Hancock et al., propagation. But, micropropagated strawberry has
2008). Fusarium wilt or Fusarium yellows (Fusarium several advantages, such as its ability to multiply virus-
oxysporum) is of major importance in Japan, Korea free stock rapidly and, the improved capacity of these
and Australia. Fumigation has been widely employed plants to produce runners for planting in the field
to control soil pathogens, but the impending ban on (Lopez-Aranda et al., 1994). Meristem tips, generally
methyl bromide fumigation has stimulated increased obtained from runners of virus-free plants, are
interest in developing resistant cultivars. Without commonly used to establish in vitro cultures, which
fumigation, cultivars yield 50% less fruit on average are employed for mass propagation or as a source of
(Hancock et al., 2008). Among the foliar diseases, plant material for regeneration and transformation
three are very widespread and can cause serious experiments (Mercado et al., 2007). The shoot
damage including, leaf blight [Phomopsis obscurans], 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).

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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

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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

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In vitro Studies of Strawberry - An Important Fruit Crop: A Review 121

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

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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.

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124 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout

PVPP to eliminate inhibitory substances from in vitro enhancing both root number and length/plantlet,
cultures. Excised shoots rooted on MS medium with plantlet height and leaf number/plantlets.
2 mg/l IAA and 0.3 mg/l BA.
In vitro regeneration via. callus culture
Sakila et al. (2007) showed that nodal segments of
strawberry gave rise to multiple shoots when cultured Plant regeneration from callus tissue cultured in vitro
on MS medium supplemented with different can play an important role in the propagation and
concentration of BA with KIN or GA3. The highest improvement of crop plants (Yeoman and Macleod
response of shoot multiplication was obtained in MS 1997) Plant regeneration from strawberry callus was
containing 1.5 mg/l BA + 0.5 mg/l Kinetin. The reported for first time by (Jones et al. 1988). (Nehra
regenerated shoots were rooted on MS basal medium et al. 1990a,b) reported that callus culture of F. ×
with different concentrations IBA and IAA. The ananassa Cv. Redcoat after 24 weeks, complete lost
maximum frequency of rooting and highest number its regeneration capacity. Adventitious shoot
of roots was produced on medium containing 1.0 regeneration has been achieved in several cultivars
mg/l IBA. The plantlets, thus developed were using a broad range of explants such as leaf explants,
hardened and successfully established in soil. The petioles, stipules, stem tissue, runners, mesophyll
plants raised through tissue culture exhibited normal protoplasts, anthers, cotyledons, roots and immature
growth, flowering and fruit setting. Emarah 2008, embryos (Mercado et al., 2007). Jemmali et al.
demonstrated that an efficient method for shoot (1992) focused that adventitious buds of strawberry
regeneration, root formation from runner tips and arises between the two leaf stipule points proliferating
acclimatization of strawberry plantlets was with very fast with axillary branching. They concluded
developed. Runner tips of 1-2 cm long were used as that the performances of the plants regenerated from
source of explants. After surface sterilization apical stipular shoots are similar to the control ones. They
meristems of 3-5 mm long were isolated and used noted that all roots of stipular plantlets were
as explants. At multiplication stage, results indicated exclusively white, whereas the control ones were more
that the highest shoot number, shoot length and leaf or less colored with anthocyanin. Rugini and Orlando
number were observed when MS medium (1992) studies the adventitious shoot regeneration
supplemented with 1 mg/l BA followed by the by using different explants and they found that there
medium contained 1 mg/l BA and 0.1 mg/l IBA. is great differences in shoot regeneration ability from
However control treatment showed a significant calluses derived from leaf, petiole and root tissues.
similar result in shoot length only. Results of this However, these differences disappeared when whole
study indicated that, BA was more effective in leaves, including stipules, were used as explants.
enhancement the growth of strawberry in vitro Passey et al. (2003) studied seven commercial
compared to Kin and thidiazuron. He also cultivars of strawberry using leaf disks, petioles, roots,
demonstrated that at rooting stage, it was clear that and stipules as explant material. The leaf disks had
MS medium at full strength containing 30 g/l sucrose the highest regeneration rates for all cultivars with
significantly surpassed all other combinations of MS greater than 90% of explants producing shoots. Samir
strengths and sucrose concentrations in increasing and Debnath (2005) reported an efficient system to
root number and length per plantlet and fresh weight/ regenerate shoots on excised sepals (calyx) of
plantlet. The same treatment enhanced the shoot greenhouse-grown strawberry through in vitro.
length but without significant difference compared They found that sepal cultures produced multiple buds
to some other combinations. The treatment and shoots without an intermediary callus phase on
contained 3 g/l agar with 6 g/l perlite significantly 2.4 mM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea
enhanced root formation as well as shoot length, (thidiazuron, TDZ)-containing shoot induction medium
fresh weight/plantlet, and leaf number/plantlets. The within 4 - 5 wk of culture initiation. He also observed
plantlets were successfully acclimatized and the soil that young expanding sepals with the adaxial side
mixture with high percentage of survival (80%) with touching the culture medium and kept for 14 days in

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In vitro Studies of Strawberry - An Important Fruit Crop: A Review 125

dark produced the high rate of differentiation. He also observed that the organic potential of explants from in
reported that the explant like sepals proved more vitro culture shoots compared with those taken from
effective than the leaf discs and petiole segments for shoot glasshouse grown plants was significantly different for
regeneration on medium having TDZ (0.5 – 4.0 mg/l) the same media. The calli induced from in vitro grown
promoted more callus formation and suppressed shoot plant exhibited high regeneration as compared to those
elongation. TDZ-initiated cultures transferred into the induced from glasshouse grown plants. They also
medium containing 2.4 mM zeatin, produced usable reported that the maximum shoot regeneration was
shoots after one additional subculture and subsequently observed on media containing 2.25 mg/l BAP and 1.0
rooted on medium without growth regulators. Qin et mg/l IBA whereas, glasshouse grown explants gave
al. (2005) reported that the leaf explants cultured for maximum shoots on same medium. Wojciech et al.
10 days in shoot regeneration medium in the presence (2005) demonstrated that the growth of in vitro cultures
of AgNO3 not only enhanced shoot regeneration of strawberry (Fragaria × ananassa) depending on
efficiency but also expedited the formation of different photoperiods. Wang et al. (1984) reported
adventitious buds. Samir and Debnath (2006) reported that the somatic embryogenesis was achieved from
that the inclusion of 2, 4 or 8 uM thidiazuron (TDZ) in cotyledons explants on MS medium supplemented with
the growth medium enhanced the rate of shoot 22.6 uM 2, 4-D, 2.2 uM BA and 500 mg/l casein
regeneration from young expanding sepals, leaf disks, hydrolysate whereas few of the embryogenic tissues
petiole without an intermediary callus phase. Sepals developed into somatic embryos. Morphologically,
proved more effective than leaf discs and petiole normal plants were obtained from somatic embryos
segments for regenerating shoots. that were transferred to MS medium containing 2.89
uM GA3 or 2.22 uM BA + 0.54 uM NAA. Maintenance
Kartha et al. (1980) studied the effects of light intensity
of the embryogenic cultures was, however,
and culture media on shoot bud regeneration of
unsuccessful. Donnoli et al. (2001) achieved somatic
Fragaria var. Redcoat. Calli developed from leaf disk
embryogenesis on MS medium supplemented with 4.88
of in vitro raised shoot has low regeneration frequency
uM BA and 4.90 uM IBA in strawberry var.Clea. Murti
than the green-house grown plants. Narender et al.
and Yeoung (2013) used meristems excised from
(1990) compared the regeneration frequency from
runners of Camarosa and Redpearl strawberry cultivars
immature leaf explants derived from in vitro shoots
for micropropagation on MS media enriched with
and greenhouse grown plants of the strawberry cv.
different concentrations of IBA (0.2, 0.3, 0.4, 0.5, 0.6
Redcoat. Both types of explants formed callus and
ppm) and BA (0.5, 0.6, 0.7, 0.8, 0.9 ppm). The result
multiple shoots at various frequencies in the presence
showed that all explants in IBA produced one plantlet
of benzyladenine (BA) and 2,4-dichlorophenoxyacetic
and 0.2 ppm produced the best plantlet performance,
acid at different concentrations. Highest frequency of
while all of BA concentration induced multiple shoots
shoot regeneration occurred with the calli derived
and 0.6 ppm produced 7-26 shoots/explant. MS media
from leaf explants at 5 μM each of BA and 2,4-D within
without IBA was recommended for rooting induction.
24 weeks. Improved regeneration (50%) with vigorous
In both of hormones, Cv.Camarosa in vitro plantlet
shoot proliferation was achieved in 10 weeks by
performance was better than Cv. Redpearl, but the
transferring the 5 week old green compact calli derived
performance of Cv. Redpearl plantlet was better than
from leaf explants on MS basal medium supplemented
Cv. Camarosa after acclimatized.
with 10 μM BA and 1 μM 1-naphthaleneacetic acid
(NAA) and incubating the cultures at 16 h light (62.5
Transplantation of in vitro grown plants
μE. m -2 s-l). Saifullah and Spoor (2000) demonstrated
that the potential of callus culture and regeneration was A substantial number of micropropagated plants do not
evaluated in strawberry (Fragaria ananassa). The survive transfer from in vitro conditions to greenhouse
effect of different hormonal combinations and explant or field environment. The greenhouse and field have
sources was studied in- order -to produce maximum substantially lower relative humidity, higher light level
number of plant in shortest possible time. They and septic environment that are stressful to

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126 vSuvalaxmi Palei, Arun Kumar Das and Gyana Ranjan Rout

micropropagated plants compared to in vitro transpiration rates. Several studies have focused
conditions. The benefit of any on VA mycorizal formation during acclimatization
micropropagationsystem can, however, only be fully of cultural plantlets (Abdelmalek et al. 1995)
realized by the successful transfer of plantlets from
tissue-culture vessels to the ambient conditions Somaclonal variation
found ex vitro. Most species grown in vitro require Strawberry plants spread vegetatively using runners
an acclimatization process in order to ensure that and this enables them to be easily transplanted and
sufficient number of plants survive and grow propagated as clones. Commercial production of
vigorously when transferred to soil. The transfer strawberry using micropropagation processes bears
of in vitro plantlets to ex vitro conditions is one of several risks. Plant off-types, i.e. non true-to-type and
the most critical factors of the micro-propagation genetically not identical to the mother plant (Gaafar
process and a cause of higher production costs. and Saker, 2006). Nevertheless, these auxins are known
High mortality is often observed upon transfer to to be associated with genetic instability in plants, a
ex vitro conditions as the cultured plants have a phenomenon called somaclonal variation. Although,
poorly developed cuticle, non-functional stomata somaclonal variation may be used as a source for
and a weak root system. In order to increase growth variation to get superior clones, it could be also a very
rate and reduce mortality of plantlets at the stage serious problem in the plant tissue culture industry
of acclimatization, recent research has focused on resulting in the production of undesirable plant.
control of the environmental conditions. One However, through micro-propagation of strawberries,
approach has been to modify the environmental several morphological abnormalities were detected as
conditions during acclimatization by increasing light somaclonal variation. Changes include leaf color
intensity or both increasing the light levels and variants and dwarf plants, among others (Adel El-Sawy
altering the CO 2 concentration. Another approach Mohammed, 2007). Callus-derived strawberry Var.
has been to change the environment during the Redcoat plants differ significantly from standard runner
multiplication and rooting stages, including plant with several vegetative characteristics (Nehra et
increasing light intensity and CO2 concentration in al., 1992). These variants pose a problem for
culture tubes, and decreasing the sugar production of uniform, true-to-type plants. Nehra et
concentration. All were found to be beneficial for al. (1994) observed that two cultivars of strawberry
plantlet growth in the later stages of responded differently to various forms of in vitro
micropropagation (Abdelmalek et al.,1995). A propagation and in both cases variants were found in
biological approach to reducing the stress of callus-derived plantlets, but not those derived from
acclimatization and providing faster growth of meristems or via direct regeneration from leaf disk.
micropropagated plantlets is the establishment of Some studies have shown that modified characteristics
vesicular-arbuscular mycorrhizae (VAM) on are epigenetic and disappear over time. Numerous
micropropagated plantlets during acclimatization. authors have reported that genetic changes including
VAM colonization of horticultural plant roots can insertions, deletions, point mutations and
improve growth by increased uptake of rearrangements occur during tissue culture, but few of
phosphorus, zinc and other minerals and may reduce the phenotypic symptoms found are heritable (Karp,
the incidence of disease. Moreover, colonization 1995 and Kumar et al., 1999). Damiano et al. (1997)
with VAM fungi may increase transplant uniformity reported the somaclonal variation and in vitro
and reduce both transplant mortality and injury. regeneration of strawberry cultivar “Teodora’ and
Recent work has also shown improvement in water ‘Clea’ on MS medium. They shown that petioles and
relations of the host plant using VAM. These fungi laminas, produced poor callus only, but the stipules are
may also improve drought tolerance by decreasing highly competent for organogenesis, and the BAP alone
leaf water potential, by reducing stomatal and root is sufficient to induce regeneration, while 2, 4-D is
hydraulic resistances, and by increasing strongly inducing callus formation. Most somaclonal

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In vitro Studies of Strawberry - An Important Fruit Crop: A Review 127

variations occur in plants regenerated from cultures chosen for RAPD analysis, all of them indicating
that have undergone a differentiation phase. Some genetic stability for micropropagated plants of the
studies indicating that a strong genetic component investigated varieties of ornamental strawberry. A
determines the success of adventitious regeneration comparative study was conducted based on
(Tian et al., 2003). Yonghua et al. (2005) found that morphological parameters as well as genetic
shoot regeneration was enhanced when explants were assessments using ISSR markers. The in vitro
cultured under red or green plastic films, and this was generated strawberry plants exhibited significantly
correlated with an increase in activity of antioxidant vigorous morphological growth and earlier flower
enzymes and endogenous hormone concentration. induction when compared to the plants propagated
Presently, there are various methods available which through planting of runners. Genetic assessment
can be used to detect and monitor tissue culture-derived through ISSR showed no polymorphism in banding
plants and varietal identification. The most reliable pattern and thus it was revealed that, there was no
methods are the molecular marker techniques that significant variation between micropropagated and
identify the variance depending on the plant proteins, conventional propagated plants at molecular level. Sutan
which are expressed from defined regions of DNA, or et al. (2009) studies the genetic stability of plants
DNA polymorphisms. ISSR (Inter Simple sequence obtained from tissue culture propagation of ornamental
repeats) and RAPD (random amplified polymorphic strawberry “Serenata”. Ten primers (from 48
DNAs) are the molecular technique for identification previously tested) were selected and used in RAPD
of genetic variation. It has the distinct advantage of analysis to prove the clonal fidelity of the tissue culture-
being technically simple and quick to perform, requiring derived ornamental strawberry plants. They observed
only small amounts of DNA compared to restriction that there are similar banding patterns of the RAPD
fragment length polymorphism (RFLP) analysis. profiles obtaining from in vitro plants, regenerated via
Strawberries (Fragaria × ananassa Duch.) have organogenesis or meristem culture. There are no
been extensively analyzed using randomly generated differences with regard to different explants source,
markers for clone identification and diversity studies number of subcultures or use of different basal medium
(Adel El-Sawy, 2007). Polymorphisms also appear in with growth regulators.
acid-phosphatase, glutamate-dehydrogenase and
peroxidase of regenerated plants. Adel El-Sawy (2007) GENETIC TRANSFORMATION STUDY
studied the somaclonal variation in micro-propagated The Rosaceae ranks third among families of
strawberry detected at the molecular level. Meristem economically important cultivated crops in temperate
tips of three strawberry cultivars, namely Chandler, regions. Genetic improvement of most rosaceous crops
Sweet Charlie and Gaviota were excised and cultured is hampered by cumbersome genome size, intolerance
for 5 weeks on the starting medium. Subculture was to inbreeding, and lengthy life cycle. Propagation of
carried out for five weeks on shooting medium, and strawberry is achieved either by runners or by in vitro
finally shoots transferred to the rooting medium. DNA micropropagation. Several improvements of the
extracted from in vitro-derived plantlets and technology have been proposed by authors working
conventionally propagated plants were analyzed by with strawberry but the highest genotypic, physiological
RAPD to detect possible drift in genetic stability of and morphological quality of micropropagated plants is
micro-propagated plants. He observed that the banding produced by the method described by Boxus and co
pattern of in vitro-derived plantlets were similar with workers (Boxus, 1983). Tissue culture stock is used
the fingerprints of mother plants, demonstrating no by most researchers as the source plant material for
variation in the pattern obtained with DNAs from the regeneration and transformation studies. Genetic
two sources of strawberry plants. It is concluded that engineering of strawberry has already been reported
mass propagation via meristem tip culture is reliable in (Jame et al., 1989; Nehra et al., 1990a,b). However,
producing genetically similar plants to the mother ones. transformation frequencies are greatly influenced by
Ten deca-nucleotide primers (among 48 tested) were 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 on MS medium containing 4 mg/ l hygromycin. Using
cultivar Rapella (Jame et al., 1990) and 6.5% for the this protocol we achieved 100% transformation
cultivar Red Coat (Nehra et al., 1990a). Barcelo efficiency for 6 of 14 F. vesca accessions tested.
et al. (1998) achieved the regeneration of shoot buds Accession PI 551572 was determined to be the best
from leaf disk explants of strawberry in presence of candidate for a model in F. vesca functional genomics
2.46 uM IBA and 8.88 uM BA, where 47% of the research, as it showed the greatest propensity for callus
cultures regenerated after 16 weeks with 2.9 shoot formation, transformation, shoot regeneration, ex vitro
colonies per regenerating leaf disk. Optimum incubation establishment, and plant growth, requiring only 14–15
conditions included two weeks in the dark with weeks to complete its life cycle in different seasons in
subsequent transfer to light with 16 h photoperiod. the greenhouse. Further, Toshiyuki et al. (2008)
Transformation was attempted using Agrobacterium introduced the transgenic plants having the gene
tumefaciens carrying the plasmid pBI121. Leaf disks encoding the taste-modifying protein miraculin was with
from in vitro cultures proliferating in the presence of 35S or El2 promoter into strawberry (Fragaria x
2.21 uM kinetin were best explants for transformation. ananassa) by Agrobacterium-mediated transformation
The transgenic nature of several shoots was also to produce transgenic plants. Although miraculin was
confirmed by the GUS assay and PCR analysis. detected in the leaves and fruits of the transgenic plants,
Previous studies on strawberry have clearly the level of accumulation among the transgenic lines,
demonstrated the importance of various factors such which ranged from 0.5 to 2.0 mg/g miracle fruits (145
as genotype (Liu and Sanford, 1988), type and source mg/g) fresh fruit, was not significantly different and
of explant (Nehra et al., 1989), hormonal balance and was lower than that in fresh fruit). High levels of
incubationconditions (Liu and Sanford, 1988). miraculin accumulation were detected in the mature
Khammuang et al (2005) reported the optimum fruits. The transgenic lines were subsequently
condition for shoot bud regeneration from leaf explants propagated via the runners for three vegetative
of strawberry cultivar Tiogar. It was found that the generations, and miraculin was detected at equal levels
best regeneration condition was MS medium containing in the leaves and fruits of the plants from each
1.0 mg/l BA and 0.2 mg/l 2,4-D. Antibiotics sensitivity generation.
test found that shoot regeneration from leaf explants
was inhibited more than 90% at the concentration of CONCLUSION
kanamycin as low as 5 mg/l . The modified gene In vitro propagation of strawberry has significant
encoding antifreeze protein isoform HPLC 6 was opportunities for commercial cultivation. To overcome
successfully constructed using codons which were this challenge, careful optimalisation of protocol for each
optimally expressed in the strawberry plant. The cultivar is utmost important. Suitable protocol can be
antifreeze protein genes, naturally in plasmid pSW1 and used for automation, using a bioreactor, is one of the
modified in plasmid pBB, were transformed to most effective ways to reduce the costs of
strawberry leaf explants by Agrobacterium micropropagation. The growth regulators like BA and
tumefaciens LBA 4404. The strawberry plants, IBA or NAA have effective for high scale of progation.
transformed with both AFP genes, were able to root in The in vitro raised plants produced maximum number
MS media containing 50 mg/l kanamycin, while no roots of runners as compared with conventional propagated
grew from nontransformed plant in this condition. plants. Liquid medium with less cytokinin concentration
Polymerase chain reaction indicated that the transgenes (1-2 mM BA or zeatin) produced more numbers of
were integrated in the genome of transformants. microshoots as compared with agar gelled medium.
Teruko et al. (2006) described a new transformation Clonal fidelity is one of the main concerns in strawberry
procedure that uses leaf explants from newly unfolded micropropagation. True-to-type propagules and genetic
trifoliate leaves obtained from stock plants 6–7 weeks stability are prerequisites for the application of
after seed germination, co-cultivation with micropropagation. The occurrence of somaclonal
Agrobacterium strain GV3101, and stringent selection

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In vitro Studies of Strawberry - An Important Fruit Crop: A Review 129

variation during micropropagation can be controlled by Biswas M Hossain MB Ahmed UK Roy R Karim MA Razvy M
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can be monitored by their morphological, biochemical, Colloques Scientifiques, Horticultural Abstracts 53
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Received : 08-07-13
Accepted: 09-02-15

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