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Research Article
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Comparison of Somatic Embryogenesis in Medicago sativa and Medicago truncatula |
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F. Hoori,
A.A. Ehsanpour
and
A. Mostajeran
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ABSTRACT
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In this study, the regeneration through embryogenesis of two species
of Medicago were studied. Seeds of Medicago sativa cv. Rehnani
and M. truncatula line A17 were grown on MS medium. After 4-6 weeks,
segments of leaf and stem from two species were transferred to MS medium
containing 2 mg L-1 NAA, 2,4-D and Kinetin. The results indicated
that callus formation from leaf explants of M. sativa was higher
than M. trancatula. In the next stage, media with different combinations
of auxin, cytokinin or ethinyl estradiol were provided for regeneration.
Then in two stages, explants of leaf and stem of two species were transferred
on these media. Results after 3-6 weeks showed that in medium containing
NAA and TDZ, stem pieces of M. sativa produced shoots while leaf
pieces on NAA and ethinyl estradiol formed roots. Leaf explants of
M. truncatula in the medium containing NAA and BAP, produced somatic
embryos. Also in media with auxin and ethinyl estradiol, somatic embryos
were formed on calli of two species. Ethinyl estradiol and auxin together
can induce somatic embryogenesis and root production on calli and stem
or leaf explants.
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INTRODUCTION
Medicago truncatula Gaertn. (known as barrel medic because
of the shape of its seeds pods) is an annual species (Fabaceae) that is
native to the Mediterranean area and is widely grown as a pasture legume
and crop rotation in a number of regions throughout the world (Crawford
et al., 1989). This species is an autogamous diploid species (2n
= 2X = 16) with a small genome (Neves et al., 1999). This plant
species is self fertile and has short life cycle of about 3 months (Barker
et al., 1990; Araujo et al., 2004). These characteristics
enables this species to be used in molecular genetic studies like analysis
of gene expression, promoter functional analysis, T-DNA mutagenesis and
expression of genes for crop improvement (Somers et al., 2003).
It is also a model plant for the study of legume-rhizobium symbiosis (Barker
et al., 1990).
Medicago sativa L. is not annual species but is closely related
to M. truncatula. This species has high degree of heterozygosity
and large genome size (2n = 32) (Iantcheva et al., 1999).
Legumes have been regarded as recalcitrant to transformation and their
in vitro regeneration is highly genotype dependent and only rarely
cultivated varieties are amenable to regeneration (Somers et al.,
2003).
Regeneration through somatic embryogenesis is frequently of single cell
origin, resulting in a low frequency of chimera and a high number of regenerants
(Neves et al., 1999). These characteristics render somatic embryogenesis
an attractive system for the introduction of gnomic traits of interest
by genetic engineering (Neves et al., 1999). Usually an auxin is
required to induce somatic embryogenesis and subsequent auxin withdrawal
or lowering of the auxin concentration is required for embryo maturation
(Dudtis et al., 1991; Nolan et al., 2003; Von Arnold et al.,
2002). Auxin play critical roles in the major growth responses during
plant development. At cellular level, auxin acts as a signal for division,
expansion and differentiation throughout the plant life cycle. By contrast
less attention has been directed to effect of steroids on plants. In addition
to the hormone regime, key variables are explant, developmental stage,
nutrition regime and genotype (Rose and Nolan, 1995). Regeneration via
organogenesis or embryogenesis are the basis of tissue culture methods
and without regeneration, it is impossible to produce transgenic plants.
In this study, we considered the effect of several growth regulators
including ethinyl estradiol on somatic embryogenesis of M. truncatula
line A17 and M. sativa cv. Rehnani.
MATERIALS AND METHODS
Seeds of M. truncatula line A17 were obtained from State
Agriculture and Biotechnology Center ( SABC) WA. Seeds of M. sativa
cv. Rehnani were supplied from Seed and Seedling Research Center
of Isfahan.
Table 1: |
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Seeds of M. truncatula first scarified by sands for germination. Then seeds
of both species were surface sterilized for 30 sec with ethanol (70% v/v)
followed by 30% (v/v) sodium hypochlorite for 15 min. After 3-4 rinses
by sterile distilled water, seeds of M. sativa were cultured on
MS medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose
and solidified with 1% (w/v) agar while, seeds of M. truncatula
first were cultured on 1% (w/v) water agar medium and after two weeks
seedlings were transferred to MS medium with the same combination as described
already. The pH of all media was adjusted to 5.8 before autoclaving (15
min at 121°C).
For callus induction, leaf and stem segments of in vitro grown
seedlings were transferred to callus proliferation, C medium.
Cultures were maintained in the culture room with 16/8 light-dark photoperiod
at 25±2°C. The experiments were carried out with 10 replications
and 4 explants in each replication. In the next stage, after two subcultures
calli were transferred to MS medium supplemented with different growth
regulators according to Table 1.
Embryogenic calli then were transferred to hormone free medium to develop
somatic embryos.
All data were analyzed according to Duncan or Chi-Square tests using
SPSS and Sigma Stat programs.
RESULTS
Stem and leaf explants of M. sativa and M. truncatula
on C medium produced high percentage of soft calli as Table
2 showing fresh weight of callus resulted from stem is relatively
lower than leaf.
When calli obtained from either stem or leaf were transferred to 7 different
media for somatic embryogenesis, a few media induced somatic embryos in
both Medicago species. For instance, leaf and stem callus produced
the highest percentage of embryos in R1 medium (Table 3),
while, in Medicago truncatula leaf and stem callus produced the
highest percentage of somatic embryo in R3 and R1 media, respectively
(Table 4). In other media with different combination
of plant growth regulators we observed different percentage of somatic
embryo initiation within 20-30 days after calli were transferred to the embryogenesis media.
Table 2: |
Callus production in C medium. Uncommon letters are
significant (p<0.05) |
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Table 3: |
The effects of different combination of growth regulators
on somatic embryogenesis of Medicago sativa callus |
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Table 4: |
The effects of different combination of growth regulators
on somatic embryogenesis of Medicago truncatula callus |
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Table 5: |
Comparing the number of somatic embryos formed per callus
of stems and leaves in M. sativa and M. truncatula in different
media based on Chi-Square analysis on 5% (comparisons are columnar). Uncommon
letters are significant (p<0.05) |
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Uncommon letter (s) are significant |
Development of somatic embryos
from leaf segments of M. sativa and M. truncatula are illustrated
in Fig. 1.
In R5 medium, calli obtained from stems and leaves of M. sativa
grew very well and became green and after 35 days formed shoot buds.
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Fig. 1: |
Different stages of somatic embryo development From M.
sativa (A, B, C, G, H), M. truncatula (D, E, F, I, J), letters
are: globular stage, heart stage, torpedo stage, plant let formation, mature
plant, respectively |
In
this medium, leaf segments produced green calli but no shoots were developed. Comparing
of somatic embryogenesis data for both Medicago species in different
media are illustrated in Table 5.
DISCUSSION
We present here an efficient procedure for somatic embryogenesis
using a suitable combination of plant growth regulators in particular
using ethanol estradiol for Medicago trucatula line A17 and Medicago
sativa cv. Rehnani. At the first step, calli were formed on MS medium
containing 2 mg L-1 of each three growth regulators (NAA, 2,4-D
and kinetin). In respect to R7 medium (NAA 0.5 mg L-1), we
concluded that NAA alone is not enough for callus formation but combination
of auxin and cytokinin and their possible interaction in the medium promote
callus formation. Our results agree with Saunders and Bingham (1972).
They reported that B-II basal medium supplemented with 2 mg L-1
of NAA, 2,4-D and kinetin produced calli from anther, cotyledons, hypocotyls
and internodes culture of M. sativa within 4 weeks. Nolan and Rose
(1998) has also showed that NAA alone can not induce callus formation
in M. truncatula. It has been well documented that 2,4-D (a strong
growth regulator) with combination of cytokinin and yeast extract as a
source of organic nitrate stimulates callus formation and somatic embryogenesis
in M. sativa (McKersie and Brown, 1997, Saunders and Bingham, 1972).
We found that this combination of plant hormones in MS medium promote
callus formation from M. truncatula.
The responses of various explants to the medium is depending to the type
of explant. For example, leaves were more responsive explants for dedifferentiation
and callus formation in Medicago sativa while, stems produced more
callus than leaves in M. truncatula.
Our data showed that NAA or ethinyl estradiol alone could not form callus
but rather induced root formation. Estradiol is the strongest form of
natural estrogen that resembles in central nucleus to cholesterol and
is synthesized from testosterone (Rawn, 1989). Ethinyl estradiol may have
auxin-like activity that its combination with NAA strengthened its effect
and cause root formation from leaf explants of M. sativa.
In R2 medium, 4.2% of leaf explants of M. truncatula developed
plantlets via indirect regeneration. It has been shown that NAA and BAP
in the medium are very effective for induction of somatic embryogenesis
of M. truncatula (Nolan and Rose, 1998; Nolan et al., 1989).
McKersie and Brown (1997) reported that IAA is not an effective auxin
in callus formation and induction of somatic embryogenesis in M. sativa.
Application of ethinyl estradiol alone (R6 medium) had no effects on embryo
development. Therefore, combination of IAA and ethinyl estradiol (R3)
had better effect on embryo formation.
Explants of leaves and stems of M. sativa in R5 medium formed
calli but only stem pieces produced shoots. The auxin/cytokinin ratio
is an important factor for regeneration. Increasing of cytokinin level
may lead to shoot formation. Our results indicated that in R1 medium root
and embryogenic masses formation may occur from interaction of auxin and
ethinyl estradiol in this medium. According to some datas, auxin acts
synergistically with estradiol (Goda et al., 2004; Bajguz and Tretyn, 2003). Cytokinin-like activity of ethinyl estradiol in combination
with auxin might be another reason for embryo formation (Bao et al.,
2004).
TDZ is a synthetic compound which is a derivative of phenylurea and has
been reported to possess strong cytokinin-like activity in a number of
plants (Huetteman and Preece, 1993). It has been used successfully for
shoot regeneration in diverse plant species including woody plants and
leguminous plants such as beans and peanut (Matand et al., 1994).
However, presence of TDZ probably prevented somatic embryogenesis in calli
derived from leaves and stems of M. sativa and M. trancatula.
Our data are in contrast with Iantcheva et al. (1999), they proposed
that high frequency of somatic embryogenesis induced by TDZ might influence
the endogenous level of cytokinins, auxins and abscisic acid so as induce the
positive embryogenic response of cultivated tissue.
It can be concluded that embryogenic potential of many Medicago
species is genotype specific. In addition, type of explant, its genetic
potential, combination of plant growth regulators in the medium and endogenous
level of hormone in tissues are the most effective factors on somatic
embryogenesis and plant regeneration.
ACKNOWLEDGMENT
Authors thank the Graduate Directorate of Isfahan University for
their support of this project.
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