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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 Mariashibu 1 , Kondeti Subramanyam, Muthukrishnan Arun, Jeevaraj Theboral, Manoharan Rajesh, Sampath Kasthuri Rengan 1 , Rajan Chakravarthy 1 , Markandan Manickavasagam & Andy Ganapathi* 1 Department 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 world 1 . 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 organogenesis 2 . Somatic embryogenesis provides an excellent morphogenetic ______________ *Correspondent author Telephone: +91 431 2407086 Fax: +91 431 2407045, 240702 E-mail: [email protected] [email protected] Present address: 1 Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore 117604, Singapore. First two authors (TSM and KS) have contributed equally.

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Page 1: Assessment of somatic embryogenesis potency in Indian ...nopr.niscair.res.in/bitstream/123456789/21454/1/IJEB 51(10) 849-859...Assessment of somatic embryogenesis potency in Indian

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]

[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.

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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

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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

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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.

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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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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.

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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.

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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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