assessment of somatic embryogenesis potency in indian...
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Indian Journal of Experimental Biology
Vol. 51, October 2013, pp. 849-859
Assessment of somatic embryogenesis potency in Indian soybean
[Glycine max (L.) Merr.] cultivars
Thankaraj Salammal Mariashibu1, Kondeti Subramanyam, Muthukrishnan Arun,
Jeevaraj Theboral, Manoharan Rajesh, Sampath Kasthuri Rengan
1, Rajan Chakravarthy
1,
Markandan Manickavasagam & Andy Ganapathi*
1Department of Biotechnology and Genetic Engineering, School of Biotechnology,
Bharathidasan University, Tiruchirappalli 620 024, India
Received 10 October 2012; revised 22 July 2013
Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication
reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max.
Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic
embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos
for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM)
indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such
as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-
asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were
desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed
strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with
74.42% of response.
Keywords: Amino acids, Genotype, Immature cotyledon, Somatic embryogenesis, Soybean
The soybean (Glycine max [L] Merrill) is a species of
legume native to East Asia, widely grown for its
edible bean which has numerous uses. The plant is
classed as an oilseed rather than a pulse by the Food
and Agricultural Organization (FAO). The USA,
Argentina, Brazil, China, and India are the world's
largest soybean producers and represent more than
90% of global soybean production. India produces
10.12 million metric tons per year and is the fifth
largest producer in the world1. Soybeans contain
significant levels of omega-3-fatty acids, isoflavones,
and phytic acid which play an important role in
human health. The use of meat-based diets among the
growing world’s population has also increased the
demand for soybean protein for livestock and poultry
feed. Soybean cultivation in India was negligible until
1970, but it grew rapidly and surpassed over 6 million
tons in 2003. In India, more than 70 cultivars are
presently considered to be most promising as
germplasm. However, soybean cultivation is
threatened by various biotic and abiotic factors. In
India, the average yield is approximately one ton per
hectare, which is lower than the international average
of two tons per hectare. The existing cultivars should
be improved genetically to increase the yield without
extending the cultivation area. However, traditional
breeding practices have led to a limited success due to
the narrow genetic variation and the presence of
barriers to genetic crosses. During the past decade,
considerable achievements have been made in the
field of plant genomic research, which has helped in
the identification and cloning of genes controlling
desirable plant traits. However, the availability of
successful regeneration protocol is a prerequisite for
the transfer of desirable gene(s) into soybean to
improve this crop plant.
The induction of somatic embryogenesis for in
vitro plant regeneration provides several advantages
over the traditional organogenesis2. Somatic
embryogenesis provides an excellent morphogenetic
______________
*Correspondent author
Telephone: +91 431 2407086
Fax: +91 431 2407045, 240702
E-mail: [email protected]
Present address: 1Temasek Life Sciences Laboratory Limited, 1
Research Link, National University of Singapore, Singapore
117604, Singapore.
First two authors (TSM and KS) have contributed equally.
INDIAN J EXP BIOL, OCTOBER 2013
850
system for investigating the cellular and molecular
process underlying differentiation3. In addition,
somatic embryogenesis also provides the possibility to
produce artificial seeds and valuable tools for genetic
engineering and germplasm conservation via
cryopreservation4,5
. Somatic embryogenesis was first
reported in soybean by Christienson et al6. The
majority of soybean somatic embryogenesis protocols
are based on the use of immature cotyledons as the
explant7-13
. Even though there are reports on somatic
embryogenesis on soybean, only a limited number of
cultivars can be induced to produce somatic embryos.
In fact, most research on somatic embryogenesis of
soybean has been restricted to the highly regenerative
cultivar like Jack and Williams 82. Nevertheless,
somatic embryogenesis from immature cotyledons is
highly genotype dependent, and some genotypes are
recalcitrant in nature9,14-17
. Majority of the Indian
soybean cultivars are also recalcitrant to somatic
embryogenesis; to date, there is no information on the
specific somatic embryogenesis of Indian cultivars.
Hence, the present investigation undertaken to
standardise a rapid and reliable protocol for somatic
embryogenesis in Indian soybean cultivars and to study
the influence of the genotype on somatic embryo
induction. In addition to this we compared the effect of
solid and liquid medium on somatic embryogenesis of
Indian soybean cultivars.
Materials and Methods Explant source and preparation—Indian soybean
cultivar Pusa 16, a commercially important cultivar in
India, was preferred for standardizing somatic
embryogenesis. Plants were grown in an experimental
garden at the Department of Biotechnology,
Bharathidasan University, Tiruchirappalli
(10°40'58.9469''N; 78°44'28.608''E) between October
and February. The date of flowering was marked by
tagging, and immature pods were collected 15 days
after anthesis. These pods, containing immature
cotyledons of 2–7 mm in length, were surface sterilised
by immersion in 70% (v/v) ethanol for 30 sec followed
by 0.1% (w/v) HgCl2 for 5 min; the pods were then
washed thrice with sterile distilled water. The immature
seeds were aseptically collected from the pods, and the
embryonic axis was cut away and discarded. The
resulting immature cotyledon halves were used as
explants to induce somatic embryogenesis.
Somatic embryo induction—The embryogenic
potential of the immature cotyledon explants was
tested using solid somatic embryo induction medium
(SSEIM) containing Finer and Nagasava lite (FNL)
macro salts8, Murashige and Skoogs (MS) micro salts
18,
B5 vitamins19
, 87.64 mM sucrose, 2,4 dichlorophenoxy
acetic acid [2,4-D (13.57–361.92 µM)], and naphthalene
acetic acid [NAA (16.11–80.55 µM)]; the pH of the
medium was adjusted to 5.6–5.8 before solidifying with
0.2% phytagel. A pair of cotyledons was cultured in a
culture tube (15 × 150 mm) containing 10 mL of solid
medium by placing the adaxial side of the cotyledons on
the SSEIM medium. In case of liquid somatic embryo
induction medium (LSEIM), 10 immature cotyledon
explants were cultured on an orbital shaker (Orbitek,
Chennai, Tamil Nadu, India) at 100 rpm in a 150 mL
Erlenmeyer flask containing 35 mL of LSEIM,
composed of the following: FNL macro salts, MS micro
salts, B5 vitamins, 29.21 mM sucrose, 2,4-D (13.57–
361.92 µM), and NAA (16.11–80.55 µM). The media
were autoclaved at 121 °C for 15 min. All of the cultures
were incubated at 25±2 °C with a 23 h photoperiod at a
light intensity of 5–10 µEm−2
s−1
.
Somatic embryo proliferation—Somatic embryo
proliferation was performed in liquid somatic embryo
proliferation medium (LPM) containing FNL macro
salts, MS micro salts, B5 vitamins, 29.21 mM sucrose
with different concentrations of 2,4-D
(13.57–180.96 µM), and NAA (5.37–53.70 µM). Fifty
globular-shaped somatic embryos were transferred
into flasks containing LPM and maintained as above
for the somatic embryo induction. After two weeks of
culture in this proliferative medium, 50 globular
embryos were again transferred to fresh LPM for
further proliferation. This proliferation process was
continued for up to three cycles.
Histodifferentiation and maturation of somatic
embryo—Early-stage globular embryos from LPM were
transferred to 35 mL of liquid histodifferentiation
medium (LHM) containing FNL macro salts, MS micro
salts, B5 vitamins, 87.64 mM sucrose with different
concentrations of NAA (5.37–53.70 µM) for the
differentiation of globular into cotyledonary stage
embryos. After differentiation in LHM, green-coloured
cotyledonary-staged embryos were transferred to liquid
maturation medium (LMM) containing FNL macro
salts, MS micro salts, B5 vitamins, 164 mM D-sorbitol,
and 87.64 mM sucrose and cultured for two weeks. All
the cultures were incubated on an orbital shaker at
100 rpm at 25±2 °C with a 23 h photoperiod at a light
intensity of 5–10 µEm−2
s−1
.
Desiccation, germination, and acclimatization—
The fully matured cream-coloured cotyledonary stage
MARIASHIBU et al.: SOMATIC EMBRYOGENESIS IN INDIAN SOYBEAN (GLYCINE MAX) CULTIVARS
851
embryos were desiccated by the air-drying method of
Parrott et al20
. Cream-coloured, well-matured
embryos (25) were transferred to sterile empty petri
plates (120 mm diameter) and sealed with parafilm.
Humidity was maintained by placing 1 cm3 MS basal
medium near the embryos. The petri plates were
placed in the dark for 0–7 days at 25±2 °C.
The desiccated embryos were transferred to MS
basal semi-solid medium for conversion into plantlets.
The germinated embryos were transferred to small
pots containing vermiculite, sand, and soil (2:1:1).
The plantlets were covered with polythene bags to
maintain a high humidity (80%). After one week of
hardening, the bags were removed gradually, and the
plantlets were transferred to earthen pots.
Effect of amino acids on somatic embryo induction,
proliferation, and histodifferentiation—Amino acids,
such as L-alanine (10.0–80.0 mM), L-asparagine
(1.0–20.0 mM) and L-glutamine (3.0–40.0 mM), were
tested for any role in the somatic embryo induction,
proliferation and histodifferentiation. The immature
cotyledon explants were inoculated into LSEIM
containing different concentrations of different amino
acids. Each treatment comprised of fifty explants in
replicates of five. The number of embryos induced
was counted after 3 weeks of culturing. In the similar
way globular embryos were inoculated into LPM and
LHM containing different concentrations of different
amino acids. Proliferated and histodiffrentiated
embryos were counted after 2 weeks of culturing.
Genotypic effects on somatic embryogenesis—After
standardising the media composition, growth regulator
requirements and supplements (amino acids) for
somatic embryo induction using cultivar Pusa 16,
immature cotyledon explants from seventy Indian
cultivars were cultured in LSEIM to test their somatic
embryo induction response with reference to their
genotypic features. The genotypes tested were as
follows: ADT−1, Alankar, Ankur, Birsa soy 1, Bragg,
DS 228, Co 1, Co Soya 2, DS 97−12, Durga, Gaurav,
Gujarat soybean 1, Gujarat soybean 2, Hardee, Hara
soy, Indira soy 9, Improved pelican, Palam Soya, JS 2,
JS 71−05, JS 75−46, JS 76−205, JS 79−81, JS 80−21,
JS 90−41, JS 93−05, JS 335, Kalitur, KB−79, KHSb 2,
Lee, Lsb 1, MACS 13, MACS 57, MACS 58, MACS
124, MACS 450, MAUS 1, MAUS 2, MAUS 32,
MAUS 47, MAUS 61, MAUS 61−2, MAUS 71,
MAUS 81, PK 416, Pusa 20, Pusa 22, Pusa 40, Punjab
1, RAUS 5, PK 262, PK 308, PK 327, VL Soya 1, VL
Soya 21, PK 471, PK 472, PK 564, PS 1024, PS 1029,
PS 1042, PS 1092, PS 1347, Punjab 1, Pusa 16, Pusa
24, Pusa 37, NRC 12, and NRC 37.
Histology and photomicrography—For histological
studies, immature cotyledon explants inoculated in
LSEIM at various time intervals, proliferating
embryogenic clumps, and embryos at different
developmental stages were fixed in formalin, acetic acid,
and 50% ethyl alcohol (0.5:0.5:90, v/v/v) for 48 h and
then dehydrated through graded series of ethyl alcohol
and tertiary butyl alcohol and were finally embedded in
paraffin (58–60 °C). Serial sections of 8 µm thickness
were cut with a rotary microtome (2035 BIOCUT,
Germany), stained in Toludene blue orange and
observed under bright field microscope. Organization
and other features of cells and tissues were
photomicrographed using Nikon optihit microscope and
Nikon sterio microscope with photographic unit Nikon
FX – 35 camera (Nikon, Japan).
Results and Discussion
Explant age and size—Immature cotyledon
explants that were 2–5 mm in size (most of these
explants were collected between 12–15 days after
anthesis) (Fig. 1a) responded well to the somatic
embryo induction and produced more embryos than
larger (>6 mm) and older explants; the latter did not
respond well to somatic embryo induction and
produced non-embryogenic calli in both liquid and
solid somatic embryo-induction medium. Immature
cotyledons from field-grown soybean plants are more
suitable explants for somatic embryogenesis21,7
.
Indeed, Lazzeri et al.22
have reported that the size and
age of immature cotyledon explants are crucial factors
for somatic embryo induction in soybean.
Effect of growth regulators on somatic embryo
induction—The immature soybean cotyledons started
to produce small green-coloured protuberances from
the margin and abaxial surfaces after five weeks of
culture on SSEIM, whereas the immature cotyledons
placed in LSEIM begin to produce such protuberances
(Fig. 1b) after 14 dyas (2 weeks) of culture. Within 21
days (3 weeks), the protuberances developed into
globular stage embryos (Fig. 1d), which were clearly
visible in LSEIM. The induced globular somatic
embryos started to detach from the explant after 25
days culture (Fig. 1e). The rate of response and the
number of somatic embryos varied significantly with
the physical nature of medium: the liquid medium
(LSEIM) produced better results than the
solid medium (SSEIM) (Table 1). Further, the
type of auxins in the medium played a vital role in the
INDIAN J EXP BIOL, OCTOBER 2013
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MARIASHIBU et al.: SOMATIC EMBRYOGENESIS IN INDIAN SOYBEAN (GLYCINE MAX) CULTIVARS
853
induction of somatic embryos from immature
cotyledon explants. The auxins, 2,4-D and NAA, were
tested at different concentrations to assess their
efficiency in somatic embryo induction. Although both
auxins induced somatic embryogenesis, 2,4-D
produced better results than NAA in terms of the
percentage of response and the number of somatic
embryos per explant, where 45.24 µM 2,4-D in LSEIM
resulted a 52.43% response and an average of 5.23
globular somatic embryos per cotyledon. NAA
produced an average of 2.83 globular stage somatic
embryos and a 17.81% response at 26.85 µM in
LSEIM (Table 1). The somatic embryos induced on
2,4-D were friable, translucent, yellowish-green in
colour and globular- to torpedo-shaped (Fig. 1d, e, and
f). In contrast, the somatic embryos induced on NAA
were compact, opaque and pale green in colour, with
an advanced morphology, forming cotyledon-like
structures. Somatic embryos induced by NAA exhibited
normal morphology and started to differentiate into the
cotyledonary stage and produced adventitious roots
before attaining physiological maturity.
The auxin type, concentration, and exposure time are
important for the initiation of somatic embryogenesis in
legumes20
. Previous studies have demonstrated that
either 2,4-D or NAA was required to induce somatic
embryos from immature cotyledon explants, and most of
these research articles have recommended the use of 181
µM 2,4-D in solid medium to induce somatic
embryogenesis16,21,23-27
. In the present study also 180.96
µM 2,4-D evoked somatic embryo induction in a solid
medium (SSEIM). However, a lower concentration,
45.24 µM, of 2,4-D in LSEIM produced a higher
number of somatic embryos with a higher percentage of
response. Several studies have also included NAA at
lower concentrations20,22,28,29
, but in the present study a
lower number of somatic embryos with NAA was
observed. It is interesting to note that the embryo
induction from explants required only 2–3 weeks in
LSEIM, whereas induction in the solid medium required
5−6 weeks26
. It seems likely that the liquid medium
allowed a better distribution of the nutrients, which may
be an important factor for embryogenic tissue, in which
there is a significant competition for nutrients. Hence,
the liquid based medium may provide better selection
regime during the transgenic somatic embryo recovery30
.
Effect of growth regulator on embryo
proliferation—To increase the number of somatic
embryos, the globular stage embryos were separated
from the explants and sub-cultured in LPM containing
Table 1Effect of 2,4-D and NAA on somatic embryo induction
from immature cotyledon explants of soybean cv. Pusa16 in solid
and liquid medium
[Values are mean ± SE from 5 independent experiments]
Percentage of
response
Mean no. of globular
embryos/cotyledon*
Hormone
con (µM)
SSEIM LSEIM SSEIM LSEIM
Control NR NR ND ND
2,4-D
13.57 07.81±0.2i 20.23±0.6d 1.24±0.04k 2.06±0.03g
22,62 10.64±0.2f 37.66±0.7b 1.92±0.04g 3.64±0.02c
45.24 11.65±0.1e 52.43±1.0a 2.54±0.03d 5.23±0.02a
90.48 17.43±0.3b 21.26±0.4c 2.83±0.04c 4.26±0.03b
180.96 27.27±0.5a 15.65±0.3f 3.65±0.02a 3.04±0.02d
271.44 13.26±0.1c 13.20±0.3i 3.15±0.01b 2.34±0.01f
361.92 12.83±0.2d 10.05±0.2k 2.03±0.02e 1.42±0.01j
NAA
16.11 05.82±0.2m 14.23±0.3h 1.00±0.02m 1.40±0.01k
26.85 06.63±0.2l 17.81±0.3e 1.41±0.02i 2.83±0.02e
37.59 07.41±0.4j 15.25±0.2g 1.62±0.03h 2.00±0.01h
53.70 08.52±0.4g 11.43±0.2j 2.01±0.04f 1.83±0.01i
64.44 08.23±0.2h 09.67±0.2l 1.26±0.02j 1.23±0.01l
80.55 07.24±0.3k 07.62±0.1m 1.02±0.02l 0.83±0.01m
SSEIM–Solid somatic embryo induction medium; LSEIM–Liquid
somatic embryo induction medium; NR–No response; ND–Not
determined due to no response. *Results were recorded after 21
days (3 weeks) of culturing in case of LSEIM and after 5 weeks in
case of SSEIM. Each treatment comprised of 50 explants in
replicates of five. Percentage of response = No. of immature
cotyledons responded for somatic embryo induction/Total No. of
immature cotyledons cultured X 100. Values with the same letter
within columns are not significantly different according to
Duncan’s Multiple Range Test (DMRT) at a 5% level.
Fig. 1—Somatic embryogenesis from immature cotyledon explant
of Indian soybean cv. Pusa 16. a: Immature cotyledon explants
cultured in LSEIM containing 45.24 µM 2,4-D, 20 mM
L-glutamine, and 29.21 mM sucrose (bar = 1.0 mm), b: Somatic
embryo induction from edges of immature cotyledon explant in
LSEIM containing 45.24 µM 2,4-D, 20 mM L-glutamine, and
29.21 mM sucrose after 12 days of culture (bar = 1.0 mm), c:
Immature cotyledon explant with embryos after 14 days (2 weeks)
of culture (bar = 1.0 mm), d: Globular embryos in LSEIM
containing 45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM
sucrose after 21 days (3 weeks) of culture (bar = 1.0 mm), e:
Globular embryos separated out of explant in LSEIM containing
45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM sucrose
after 25 days of culture (bar = 1.0 mm), f: Proliferated
embryogenic clumps showing globular embryos in LPM
containing 22.62 µM 2,4-D, 10 mM L-asparagine, and 29.21 mM
sucrose (bar = 1.0 mm), g: Histodifferentiated somatic embryos in
LHM containing 10.74 µM NAA, 30 mM L-glutamine, and
87.64 mM sucrose. h: Matured somatic embryos in LMM
containing 164 mM D-sorbitol and 87.64 mM sucrose, i:
Desiccated somatic embryos, j: Germinated somatic embryo in
MS basal medium, k: Hardened soybean plantlet, l: Acclimatised
soybean plant surviving in greenhouse.
INDIAN J EXP BIOL, OCTOBER 2013
854
various concentrations of either 2,4-D or NAA. The
LPM without auxins did not show any proliferation.
The medium containing 2,4-D promoted the
proliferation (Fig. 1f) within two weeks and also an
increase in the size of the somatic embryos. A highest
number of globular stage embryos was observed with
the use of 2,4-D at 22.62 µM (Table 2), a proliferation
trend that was observed for up to two successive
cycles, although increasing the concentration of 2,4-D
did not yield any further positive effect. The
proliferation experiments conducted using NAA
failed to show a positive response, indeed it favoured
the conversion of globular embryos to cotyledonary
stage embryos (unpublished data not shown).
The proliferation of somatic embryos has a great
advantage in transgenic recovery. Therefore, it is
desirable to have secondary embryo proliferation
from primary embryos. Embryo initiation in presence
of 2,4-D is preferable for the maintenance of
proliferation in embryogenic soybean cultures24
, and a
higher level of 2,4-D apparently prevents the
development and maturation of the embryo while still
allowing proliferation7,21
. However, some reports have
indicated that a high level of 2,4-D (82.4 µM) was
required for the multiplication of somatic embryos on
solid medium31
, whereas a low level of 2,4-D (20.6
µM) was beneficial in liquid culture8,9
. In agreement
with the latter, the present observations indicated the
requirement of a lower concentration of 2,4-D in
liquid medium for the proliferation of somatic
embryos.
Somatic embryo histodifferentiation and
maturation—It was observed in the present study that
the proliferated somatic embryos need to be transferred
to the medium with NAA or without growth regulators
for histodifferentiation. However, the somatic embryos
induced with NAA did not require any change in
medium, and a simple sub-culture in medium with the
same composition favoured histodifferentiation. The
globular embryos initiated from immature cotyledon
explants and the proliferating early-staged embryos
cultured with 2,4-D progressed through all of the stages
of development (globular–heart–torpedo) and reached
the cotyledonary stage (Fig. 1g) when cultured in
histodifferentiation medium containing NAA. The
liquid histodifferentiation medium (LHM) without
plant growth regulator also exhibited differentiation
(Table 3). The lack of a requirement of growth
regulators for the differentiation into the cotyledonary
stage is in agreement with previous studies, where it
has been suggested that the globular embryos may be
capable of producing their own hormones to support
early development8,32
. However, the differentiation rate
(37.43 embryos per 50 embryos) and quality of the
embryos were significantly higher in the
histodifferentiation medium containing 10.74 µM
NAA, where 72.05% of the globular stage embryos
progressed to the cotyledonary stage. The somatic
embryos differentiated on NAA showed advanced
stages of embryo morphology, when compared with
the embryos differentiated on basal medium. The
cotyledonary stage embryos differentiated in presence
Table 2Effect of 2,4-D on proliferation of globular embryos initiated from immature cotyledon
explants of soybean cv. Pusa 16 in LSEIM containing 45.24 µM 2,4-D, and 29.21 mM sucrose
[Values are mean ± SE from 5 independent experiments]
2,4-D conc
(µM)
Response
(%)
Mean no. of globular
embryos in first cycle of
proliferationA
Mean no. of globular
embryos in second cycle
of proliferationB
Mean no. of globular
embryos in third cycle
of proliferationC
13.57 70.21±2.0c 076.42±2.0e 75.86±1.6e 73.42±1.2e
22.62 76.83±2.2a 101.63±2.2a 99.02±1.4a 96.45±1.8a
45.24 72.46±1.9b 090.41±2.4b 91.06±2.1b 88.04±1.8b
90.48 68.47±1.4d 087.64±2.1c 85.82±1.9c 85.02±1.6c
135.72 60.08±1.6e 081.83±1.9d 80.46±1.8d 79.23±1.9d
180.96 41.65±0.9f 070.27±1.8f 69.65±1.6f 68.84±1.8f
AFifty fresh globular stage embryos were inoculated per treatment in replicates of five. BFifty
embryos from the first cycle of proliferation were sub-cultured in fresh medium. CFifty embryos
from second cycle proliferation were sub cultured in fresh medium. Percentage of response = No. of
embryos responded for first proliferation cycle/Total No. of embryos cultured X 100. Values with the
same letter within columns are not significantly different according to Duncan’s Multiple Range Test
(DMRT) at a 5% level.
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855
of NAA had expanded cotyledons of a compact nature
and exhibited typical bipolarity with a distinct radical
and hypocotyl (Fig. 1g) whereas, the embryos
differentiated in the basal medium displayed narrow
cotyledons and were light green in colour. Somatic
embryo differentiation in LHM was faster than the
basal medium and required only two weeks to reach
the cotyledonary stage. Further maturation of the
somatic embryos in liquid medium was achieved
without any growth regulator, and the somatic
embryos lost their green colour when they reached
maturation and were ready for germination. In the
present study, the histodifferentiation and maturation
process required 4 weeks.
Effect of amino acids on somatic embryo induction,
proliferation and histodifferentiation—Amino acids
are most commonly used as a nitrogen source33
.
Nitrogen is indispensable for somatic embryogenesis
since it is required as key components in plant
structure, functions and for building blocks such as
proteins, nucleic acids, and plant hormones. Amino
acids, including L-alanine, L-asparagine, and
L-glutamine, were tested for their role in somatic
embryo induction, proliferation, and
histodifferentiation. Although all the three amino acids
showed positive response to somatic embryo induction,
L-glutamine at 20 mM was found to be optimum with
maximum percentage of response (74.42%) and mean
number of somatic embryos (8.45 embryos per
cotyledon). Among the three amino acids tested for
somatic embryo proliferation, L-asparagine at a
concentration of 10 mM favoured the somatic embryo
proliferation with 85.64% of response and produced
170.24 globular somatic embryos. The highest
percentage of response (80.82%) to somatic embryo
histodifferentiation and mean number of cotyledonary
stage embryos (56.62) was noticed when the LHM
supplemented with 30 mM concentration of
L-glutamine. It has been proved that, the amino acids,
such as L-asparagine and L-glutamine, played a
positive role in the development of soybean
embryogenic cell suspensions8,34
. It is established
beyond doubt that addition of L-glutamine during
embryo development increased the size of the somatic
embryos35,36
. Formation of embryogenic clumps was
improved in cell suspensions of soybean when
L-asparagine was added to the culture medium37
.
Loganathan et al.38
have observed a doubling of the
embryogenic response when L-glutamine was added
to the somatic embryo induction medium. The present
study also indicated the positive influence of
L-glutamine and L-asparagine in somatic embryo
development.
Desiccation, germination, and acclimatization—
The embryos matured (Fig. 1h) in liquid maturation
medium did not germinate into plantlets without
desiccation. The differentiated embryos were
desiccated for different period of time (1−7 days).
During the desiccation process, the embryos lost their
water content, shrivelled up and reached physiological
maturity (Fig. 1i). Somatic embryos desiccated for
5 days showed a higher germination response (40.88 %),
whereas non-desiccated embryos failed to germinate
(data not shown). Embryos with well-defined shoot
apices germinated within 15 days
(Fig. 1j), whereas embryos lacking a defined shoot
apex required another two weeks for germination. The
plants germinated from somatic embryos were
transferred to plastic cups (Fig. 1k) containing
sand:soil:vermiculite (2:1:1 v/v/v). After the
emergence of a new leaf, the plantlets were
transplanted into pots and acclimatized in greenhouse
(Fig. 1l).
Maturation and germination is a significant rate-
limiting factor in most somatic embryogenesis
studies. In the present study, germination was also a
rate-limiting factor, thus, the somatic embryos were
subjected to an air-drying desiccation treatment to
enhance the germination. Indeed, it has been reported
that a physiological state permitting germination can
be rapidly induced by desiccating the somatic embryo
in a dry, sterile petri dish for a week20
, Jang et al.37
Table 3Effect of NAA on histodifferentiation of globular embryos
to cotyledonary stage in LHM containing 87.64 mM sucrose
[Values are mean ± SE from 5 independent experiments]
NAA conc
(µM)
Response
(%)
Mean no. of cotyledonary
stage embryo
0.00 46.04±1.2g 27.04±0.8c
5.37 68.26±1.4c 30.62±0.8b
10.74 72.05±1.6a 37.43±0.9a
16.11 65.83±1.3e 26.67±0.6d
26.85 70.65±1.8b 25.21±0.5e
37.59 67.41±1.2d 23.06±0.3f
53.70 57.26±1.1f 19.23±0.2g
Each treatment comprised of 50 globular embryos in replicates of
five. Mean values of five independent experiments (±) with standard
errors. Percentage of response = No. of globular embryos converted
into cotyledonary stage embryos/Total no. of globular embryos
cultured X 100. Values with the same letter within columns are not
significantly different according to Duncan’s Multiple Range Test
(DMRT) at a 5% level.
INDIAN J EXP BIOL, OCTOBER 2013
856
have recorded 90% germination of somatic embryos
after 4 days of desiccation. An air-drying method, in
which somatic embryos were desiccated in an empty
sealed petri dish for 3–5 days, has also been reported
to have resulted in the best germination efficiency
among four tested methods: fast, slow, air, and KCl
methods26
.
Genotypic effects on somatic embryogenesis—The
liquid-based somatic embryo induction medium (FNL
macro salts, MS micro salts, B5 vitamins, 45.24 µM
2,4-D, 20 mM L-glutamine and 29.21 mM sucrose)
developed in the present study was employed to
screen 71 Indian soybean cultivars. Six Indian
cultivars, namely Pusa 16, DS 97−12, Gujarat
soybean 1, PK 416, VL soya 1, and PK 472 responded
favourably, though, with varied efficiency (Table 4).
Among these, Pusa 16 showed a higher response rate
(74.42%), with an average of 8.45 embryos per
immature cotyledon explant in the liquid system. The
other Indian cultivars did not respond well or
produced fewer numbers of somatic embryos with
less percentage of response (unpublished).
Parrott et al.39
have observed that certain cultivars
exhibited strong genotypic specificity in North
American germplasm lines; the Manchu and AK
Harrow cultivars responded well in somatic
embryogenesis, and the other responding germplasm
lines were genetically related to these lines. Other
studies have also indicated that the genotype affected
somatic embryo induction, proliferation and
maturation14,16,40,41
. The Indian germplasm contains
more than 1500 lines, and only 70 popular genotypes
were selected in the present study to identify those
Indian cultivars/genotypes that responded the best to
treatment. The screening of Indian genotypes will be
useful in the selection of cultivars for genetic
transformation and subsequent breeding programmes.
Histology and photomicrography—Histological
studies show that globular structures were originated
from the basal cells (by sub-epidermal divisions) of
the cotyledonary mesophyll tissues and the
protuberance was covered by the single layer of the
epidermal cells (Fig. 2c). These globular structures
elongated and differentiated into somatic embryos.
The globular structures that formed somatic embryos,
exhibiting clear bipolarity, were observed at the sub-
marginal region of explants (Fig. 2d). Histological
studies of secondary somatic embryogenesis showed
that, the apical cells of the epidermal layer of the
primary embryos undergone division and produced
secondary somatic embryos (Fig. 2 e–i). Secondary
somatic embryos formed directly from the primary
embryos and they became clearly visible after 21 days
(3 weeks) of culture. The anatomical analysis of
cotyledonary stage somatic embryo differentiated on
LHM containing NAA (10.74 µM) showed regular
vascularisation along with hypocotyledonary axis and
the presence of root meristems and shoot meristems
(Fig. 2j)
In conclusion, an efficient somatic embryogenesis
protocol was developed for Indian soybean cultivars
and 71 Indian cultivars were screened for somatic
embryo induction. Of the 71 cultivars tested, six
cultivars showed better response to somatic embryo
induction than remaining cultivars and those 6
cultivars may be used to transfer the desirable genes
to improve the quality and quantity of the soybean.
Fig. 2—Histological analysis of somatic embryo development in
Indian soybean cv. Pusa 16. a: Cross section of immature
cotyledon explant showing epidermal layers (bar = 300 µm), b:
Immature cotyledon explant showing divisions of cells at the
epidermal layer after 3 days of culture (bar = 300 µm), c: Globular
structures resulted from sub-epidermal divisions after 7 days of
culture (bar = 300 µm), d: Globular embryos showing continuous
epidermis with explants (bar = 300 µm), e: Secondary embryo
originating from apical layer of primary embryos (bar = 300 µm),
f–i: Different stages of secondary somatic embryogenesis (bar =
300 µm), j: Longitudinal section of cotyledonary stage embryo
showing regular vascularisation along with hypocotyledonary axis
(bar = 300 µm).
Table 4Effect of genotype in somatic embryo induction in
LSEIM containing FNL macro salts, MS micro salts, B5 vitamins,
29.21 mM sucrose, 45.24 µM 2,4-D, and 20 mM L-glutamine (pH
5.8)
[Values are mean ± SE from 5 independent experiments]
Genotype tested Response (%) Mean no. of globular
embryos per cotyledon*
PK 472 59.42±1.2f 3.62±0.2f
Ds 97-12 67.24±1.4b 6.24±0.4b
Gujarat soybean 60.42±1.2e 5.62±0.3c
Pusa 16 74.42±1.5a 8.45±0.2a
PK 416 63.62±1.4c 5.06±0.2d
VL soya 1 62.21±1.2d 4.26±0.2e
Each cultivar comprised of 50 explants in replicates of five.
*Results were recorded after 21 days (3 weeks) of culturing.
Percentage of response = No. of immature cotyledons responded
for somatic embryo induction/Total No. of immature cotyledons
cultured X 100. Values with the same letter within columns are
not significantly different according to Duncan’s Multiple Range
Test (DMRT) at a 5% level.
MARIASHIBU et al.: SOMATIC EMBRYOGENESIS IN INDIAN SOYBEAN (GLYCINE MAX) CULTIVARS
857
INDIAN J EXP BIOL, OCTOBER 2013
858
Acknowledgement The authors are thankful to the Department of
Biotechnology (DBT) of Ministry of Science and
Technology, New Delhi, Government of India, for the
financial support (BT/PR9622/AGR/02/464/2007).
AG is thankful to University Grants Commission
(UGC), New Delhi, Government of India for
providing fellowship under UGC–BSR scheme. KS
thanks Council of Scientific and Industrial Research
(CSIR), New Delhi for the award of Senior Research
Fellowship (SRF). Thanks are due to Prof. Yong Eui
Choi, Division of Forest Resources, Kangwon
National University, Chunchon, South Korea for help
in histological sectioning.
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