Content uploaded by Pravej Alam
Author content
All content in this area was uploaded by Pravej Alam on Nov 28, 2014
Content may be subject to copyright.
1783
AJCS 5(13):1783-1789 (2011) ISSN:1835-2707
High frequency somatic embryogenesis in mustard crop (Brassica juncea L. cv. Pusa Jai
kisan): Microscopic and histological analyses
M. Akmal
1
, T. Nafis
1
, K. J. Mirza
1
, P. Alam
1
, A. Mohammad
1
, A. Mujib
2
and M. Z. Abdin
1
*
1
Department of Biotechnology, Hamdard University, New Delhi-110062, India
2
Department of Botany, Hamdard University, New Delhi-110062, India
*Corresponding author: mzabdin@rediffmail.com
Abstract
A high frequency somatic embryogenesis has been established in mustard crop (Brassica juncea L. cv. Pusa Jai kisan), in which
embryogenic calli were induced from hypocotyls and cotyledons of in vitro germinated seedlings. The hypocotyl derived
embryogenic calli (HEC) were transparent and whitish, while cotyledon derived embryogenic calli (CEC) were creamy yellow in
colour. Highest embryogenic callusing frequency (98%) was obtained in cotyledons on 2 mg/l 2, 4-D added MS medium. Hypocotyls
and cotyledons derived calli were differentiated into somatic embryos at high frequency (90-100%) on 2 mg/l 2ip or 2 mg/l BAP
amended medium. Embryo maturation occurred on the same embryo development medium, and germination was best achieved on
2.6 mg/l ABA amended medium. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and histological
studies revealed that the embryos had bipolar structure and developed mainly from the epidermis of explants. Furthermore, the
embryonic tissues have stored bodies and numerous cell organelles. Various embryological stages are presented in this short
communication. This protocol is much faster and took just six weeks to obtain complete plantlets.
Keywords: Embryogenesis, cotyledon, hypocotyl, callus, cytokinin, MS medium.
Abbreviations: BAP-6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid, TDZ-thidizouron, 2ip-2-isopentenyladenine,
SEM-scanning electron microscopy, Kin-Kinetin, TEM-transmission electron microscopy, ABA-abscisic acid, HEC-hypocotyl
derived embryogenic calli, CEC-cotyledon derived embryogenic calli, DMRT-Duncan multiple range test, PGR-plant growth
regulator, ANOVA-analysis of variance, MS-Murashige and Skoog’s (1962) medium, SE-somatic embryogenesis.
Introduction
Brassica juncea L. belongs to the Brassicaceae family. It is a
major oil seed crop of the Indian subcontinent and is an
alternative source of canola quality oil (Stoutjesdijk et al.
1999). The crop is the best source of edible oils, having
essential fatty acids with lowest amount of saturated fat. It
also provides oil free meal to animals. B. juncea is rich in
protein with well balanced aminogram (Agnihotri et al.,
2004). It is highly susceptible to a variety of fungal diseases
like white and yellow rusts and leaf rot that reduce yield and
affect crop quality. Further, the crop is sensitive to various
abiotic and biotic stresses. Hence, the primary objectives of
research in this crop are to develop B. juncea plant tolerant /
resistant to stresses and also produce desirable oil quality and
yield. A large number of research reports are available on the
genetic improvement of oilseed crops (Stoutjesdijk et al.,
1999; Potts et al., 1999; Das et al., 2006). Somatic
embryogenesis, a useful in vitro technique is often used in
genetic engineering programme as it offers fast and efficient
way of gene transfer. The same embryogenic system also
allows recovery of whole plant in one step as opposed to
organogenesis, where regenerated shoots need to be
subsequently rooted (Thorpe, 1995). Moreover, somatic
embryos (SEs) have often been used in developing synthetic
seeds that are later used for storage, transport and
transplantation (Mujib and Samaj, 2006). Earlier, attempts on
induction of SEs in Brassica juncea L. were made by several
workers (Kirti and Chopra, 1989; Sharma et al., 1991;
Kumari et al., 1995, 2000) by using different explants viz.
immature zygotic embryos, hypocotyls, young leaves,
protoplasts and microspores. However, to the best of our
knowledge, there is no report available on high frequency
somatic embryogenesis and germination of SE in B. juncea.
In this investigation, a detailed study of B. juncea
embryogenesis was conducted in which the role of PGR and
carbohydrate was evaluated. TEM, SEM and histological
investigations were also conducted at different stages
regarding in vitro embryogenesis.
Results
Callus induction
In present study, two different explant types (hypocotyls and
cotyledons) were used. Both the cotyledon and hypocotyl
explants showed embryogenic calli (98 and 88%) on MS,
when fortified with 2,4-D (2 mg/l) and added with 0.5 or
1.5% sucrose (Table 1, Fig. 1a and b). Low frequency calli
formation was also induced at higher concentration of 2,4-D
(3, 5mg/l).The HEC were compact, transparent and white in
colour, while CEC were compact and creamy-yellow in
colour. The cut end of both the explants produced more
embryogenic calli compared to the other parts of explants.
Uncut sides became brownish and failed to produce calli.
Embryo development
The embryos (globular and heart shaped) were induced on
both HEC and CEC within ten days of culture on embryo
development medium. The frequency of SE development was
very high (99.6%) in HEC on 2 mg/l 2ip amended MS
medium. High embryo development frequency was also
1784
observed in CEC (i.e.100%), when the embryogenic calli
were cultured on MS and supplemented with BAP (2mg/l).
The ultrastructural features provided by TEM observations
revealed that the embryogenic cells contained smaller
vacuoles, large nucleus (Nu) with numerous organelles and
stored bodies. These features observed are very common,
when a cell starts to convert into an embryo (Fig. 2A). In B.
juncea, the most distinct feature is the presence of stored
lipid bodies. The SEM studies showed the SEs as distinct
bipolar structures with radicular and cotyledonary poles and
these developed on the entire surface of the embryogenic
calli. The globular and heart shaped embryos were developed
in higher frequency than the torpedo and cotyledonary
embryos on HEC, while the number of heart shaped embryos
was more on CEC (Fig. 2B, C). Histological studies revealed
that on upper epidermis of hypocotyl, SEs were mostly
induced and appeared as bulging structures in which the cells
of embryos were smaller and had densely stained cytoplasm.
In the developing embryos, inner mass of cells were
surrounded by an epidermis. This developing embryo
outgrowth was observed on hypocotyl surface from all
directions (Fig. 2D, E). The proliferation of SE was
continued on these cytokinin containing medium, the
globular embryos progressed to heart and then into torpedo.
The torpedo embryos subsequently developed into
cotyledonary embryos. Cotyledonary, torpedo and advanced
heart shaped embryos were more frequently developed on
0.5mg/l TDZ and 2ip containing medium (Table 2 and 3, Fig
c, d, e and f).
Embryo maturation and germination
The maturation of SEs occurred on the same embryo
induction medium, when induced embryos were regularly
sub-cultured after an interval of fifteen days. Maturation
percentage of SE developed from HEC was 80% at 0.5mg/l
BAP. Maturation percentage was also nearly the same on
kinetin and TDZ amended medium. In CEC developed
somatic embryos, the maximum maturation percentage was
85% and was noted on medium containing 0.1mg/l BAP or
0.1mg/l kinetin (Table 4, Fig. 1g). On cyokinins omitted
medium, loose callusing occurred and induced embryos
neither matured nor germinated at all (data not shown). At
lower concentration of ABA, the embryo germination
frequency was very low, while at higher concentrations
embryo desiccation occurred. On 2.6mg/l ABA added
medium, the germination frequency was quite high. Hence, it
was selected and amended in germination medium. Best
embryo germination (58%) was observed on 0.5mg/l TDZ +
2.6mg/l ABA in case of HEC, while the germination
percentage of CEC embryos was even higher (60%) in the
same medium. Embryos started to germinate within two
weeks of culture on embryo germination medium (Table 5,
Fig. 1h to m). The radicular ends gave rise to the roots, while
shoots were developed from the cotyledonary ends.
Discussion
In the present study, a high frequency somatic embryo
induction was reported in hypocotyl and cotyledon explants
from five days old in vitro grown seedlings of B. juncea. It
offers an excellent and fast method of plant regeneration of
this valuable oilseed crop. Two different embryogenic calli
were induced from two different explants of B. juncea, which
could be differentiated with non-embryogenic calli in several
respects. Embryogenic calli showed organized growth
resulting in bipolar structure. Their protein profiles also
differed and generally had embryo specific proteins (Sung
and Okimoto, 1983). Reprogramming was also noted to be
operative during embryogenesis for the development of
whole plant from a single cell, thus demonstrating and
reconfirming the concept of totipotency in higher plants
(Nolan et al., 2003; Imin et al., 2004, 2005). In the present
investigation of B. Juncea, embryogenic callus was induced
on 2,4-D added medium. The use of the synthetic auxin 2,4-
dichlorophenoxyacetic acid (2,4-D) for the induction of SEs
on cultured explants was earlier reported and reviewed in
angiospermic plants (Yantcheva et al., 1998; Raghavan et al.,
2004; Raemakers et al., 2005) including B. juncea (Eapen et
al., 1989) and other members of Brassicaceae i.e. B.
campestris (Bhattacharya et al., 1980), B. nigra (Narasimhulu
et al., 1992), B. oleracea (Pareek and Chandra, 1978) and B.
napus (Turgut et al., 1998). In most cases embryo formation
has primarily been observed by culturing explants on high or
low concentration of 2, 4-D, either alone or with cytokinins
(Gaj, 2004). In Brassica, the combined influence of auxins
and cytokinins on induction of callus and somatic embryo
was earlier described (Kirti and Chopra, 1989, 1990; Sharma
et al., 1991; Kumari et al., 1995, 2000). Here, we evaluated
the influence of various cytokinins on in vitro
embryogenesis. We also studied the effect of sucrose on the
induction of embryogenic calli in the medium supplemented
with 2,4-D. The embryogenic calli obtained from hypocotyls
and cotyledons in the above medium produced large numbers
of SEs in a short period of time (3 weeks) on cytokinin
containing medium. The use of cytokinin alone to obtain SEs
from zygotic embryos was also established in a wide range of
angiosperms (Maheswaran and Williams, 1984), where
exogenous cytokinin was found to enhance the number of
embryos in culture (Thorpe, 1995). The polarity once
established in the cells, cytokinin present in the medium
triggers cell division. Continuous divisions of cells produce
different forms of embryos like globular, heart and torpedo.
In our experiments, we noted that the presence of different
cytokinins is essential for progression of embryo
development (from induction to germination phase). In the
absence of these cytokinins, a loose organized callus
development occurred (data not shown) as the cytokinins are
responsible for the establishment and maintenance of apical
meristems of embryos (Sugiyama, 1999). We also observed
that the embryo proliferation and maturation behaviour of
SEs from two different explants, hypocotyls and cotyledons
were nearly the same. The germination of cotyledon derived
SEs was however, higher as compared to the hypocotyl
derived SEs. It was reported that the developed embryos
started to reserve metabolic deposition, and these reserve
substances are good indicators of maturation (Bewley and
Black, 1985). Availability of carbohydrate in medium also
appears to be important for both embryo quality and embryo
number during embryo development (Thorpe, 1995) and its
range usually varies between 3-6%. In the present
investigation, 3% sucrose was used in maturation as well as
germination medium. In medium, ABA was added for
inducing embryo’s tolerance to desiccation. In the
experiments, ABA promoted germination in embryos derived
from both HEC and CEC. Senaratna et al., (1991) used 10-
200 µM ABA pretreatment for inducing the tolerance to
1785
Table 1. Callusing frequency of Brassica juncea L. cv. Pusa jai kisan from hypocotyls and cotyledons explants.
Callusing frequency (%) 2,4-D
(mg/l) Sucrose (%) Hypocotyl Cotyledon
0.5 80.8a 90.4a
1.0 77.2b 80.3d
1 1.5 76.2c 85.6c
2.0 70.5d 87.4b
2.5 70.7d 80.3d
3.0 65.4e 80.2d
0.5 88.3c 90.0c
1.0 85.9f 95.0b
2 1.5 86.7e 98.2a
2.0 75.4b 85.5d
2.5 77.1a 80.2e
3.0 75.4d 80.2e
0.5 75.2a 80.3a
1.0 67.4d 80.4a
3 1.5 66.6e 75.3b
2.0 62.7c 75.2b
2.5 70.8b 60.1d
3.0 75.2a 63.5c
0.5 60.5c 80.0a
1.0 55.2e 75.5d
4 1.5 55.5e 77.5b
2.0 56.3d 76.1c
2.5 62.5b 75.5d
3.0 66.2a 70.4e
0.5 50.2e 80.2b
1.0 57.4d 85.2a
5 1.5 60.2c 75.3e
2.0 63.5b 77.5c
2.5 63.5b 76.6d
3.0 65.4a 66.3f
Means with common letters within each column are not significantly different at p ≤ 0.05 according to DMRT
Fig 1. Cotyledon derived embryogenic callus (A), Hypocotyl derived embryogenic callus (B), Globular embryo (C), Torpedo shaped
embryo (D), Heart shaped embryo (E), Globular stalked embryo (F), Heart shaped mature embryo (G)(arrow indicating distinct shoot
and root pole), Heart shaped embryo with cotyledons and long radical (H), Cotyledonary embryo (I), Germinated seedling like
cotyledonary embryo (J), development of emblings (K and L), plantlets developed from the emblings (M).
1786
Table 2. Effect of various concentrations of four different cytokinins on development of somatic embryos from HEC and CEC.
PGR
(mg/l)
Somatic Embryogenesis
(%)
Number of Embryos /callus mass (100mg)
Hypocotyl Cotyledon HEC CEC
H G T C H G T C
BAP
0.5 80.2c 95.7c 55.2a 20.2c 12.5b 0.0c 67.2a 17.2c 0.0c 6.6c
1.0 82.0b 99.8b 45.3b 33.4b 6.2c 2.6b 58.3b 22.2b 3.4b 8.2b
2.0 98.6a 100.0a 38.5c 37.5a 14.4a 7.2a 55.2c 26.5a 4.4a 12.2a
Kinetin
0.5 81.5c 97.8b 50.5a 18.5c 0.0c 1.0c 58.8a 20.3b 0.0b 0.0c
1.0 85.2b 96.7c 44.3b 26.6b 12.2b 2.2b 54.5c 24.5a 0.0b 9.5b
2.0 96.6a 98.9a 35.2c 28.9a 18.4a 5.9a 55.5b 24.2a 5.5a 11.5a
TDZ
0.5 90.7b 85.2a 22.5b 54.3b 26.9a 2.5a 35.2a 54.2c 0.0b 0.0c
1.0 95.5a 77.2c 28.4a 49.5c 16.5b 0.0b 12.5c 56.6b 0.0b 0.0b
2.0 88.2c 78.9b 13.2c 59.2a 2.2c 0.0b 14.1b 58.4a 2.5a 1.5a
2ip
0.5 95.6b 92.9c 57.3a 34.4c 19.3a 7.6a 66.2a 16.5c 0.0c 0.0c
1.0 95.4b 98.0b 55.5b 40.4a 12.5b 5.2c 57.4b 26.5b 5.5b 5.3b
2.0 99.6a 98.9a 55.2b 38.2b 10.5b 6.5b 45.5c 33.5a 8.2a 10.5a
G-Globular, H-Heart, T-Torpedo, C-Cotyledon, Data represent s mean with common letters within each column are not
significantly different at p ≤ 0.05 according to DMRT.
Fig 2. TEM image of embryo cell having stored bodies (A), Cell contain smaller vacuole, large nucleus (Nu) with numerous
organelles (B), SEM image of a hypocotyl showing different shapes of embryos (C). SEM image of cotyledon embryos longitudinal
section of somatic embryo under Olympus system microscope the embryos formed from upper meristematic cells (D), T.S. of
developing embryos clear the presence of stored bodies (arrow) as observed under Olympus system microscope (E).
desiccation in embryos of B. napus. Recently, Angoshtari et
al., (2009) reported ABA induced somatic embryogenesis
and used the same for induction of desiccation tolerance in
SEs from Brassica napus. In our experiments, 2.6 mg/l of
ABA was observed to be very efficient for embryo
germination. At lower ABA levels, there was no germination,
while at higher concentrations embryos germinated but the
germinated emblings dried due to excessive desiccation. To
the best of our knowledge, this is the first ever report
describing high frequency plant regeneration in Brassica
juncea L. cv. Pusa Jai Kisan through somatic embryogenesis.
This protocol is more rapid than earlier reported methods. It
took about six weeks to get complete plantlets and
approximatly 2000 plantlets were obtained by employing this
method. This protocol can be used to raise transgenic B.
juncea en masse as it takes less time compared to other
methods available.
Materials and methods
Plant material and culture conditions
Seeds of mustard (Brassica juncea L. cv. Pusa Jai Kisan)
were procured from NRCPB, IARI, New Delhi-110012,
India. Mature seeds were thoroughly washed with tap water
for 20 minutes and surface sterilized for 8 minutes in 3%
1787
Table 3. Maturation percentage of somatic embryo on various cytokinins containing MS medium.
Somatic embryo maturation (%)
PGR (mg/l)
Hypocotyl Cotyledon
BAP
0.5 80.2a 75.5d
1.0 72.5d 85.6a
2.0 70.6e 70.8f
Kinetin
0.5 80.2a 78.2c
1.0 78.5b 85.5a
2.0 75.5c 80.5b
TDZ
0.5 80.5a 78.6c
1.0 75.7c 76.3e
2.0 70.5e 76.2e
2ip
0.5 78.5b 70.3f
1.0 70.2e 78.5c
2.0 65.5f 78.5c
Data represent s mean with common letters within each column are not significantly
different at p ≤ 0.05 according to DMRT.
Table 4. Germination percentage of somatic embryo on MS medium supplemented with various
concentrations of cytokinines with 2.6 mg/l ABA.
Somatic embryo germination (%)
PGR (mg/l) Hypocotyl Cotyledon
BAP
0.5 50.2d 64.5a
1.0 50.5d 54.6e
2.0 50.3d 45.3g
Kinetin
0.5 50.2d 50.8f
1.0 45.5e 50.3f
2.0 40.6f 40.7h
TDZ
0.5 58.2a 60.4b
1.0 55.6b 58.5c
2.0 55.7b 55.2d
2ip
0.5 52.3c 50.2f
1.0 40.3f 50.3f
2.0 35.2g 45.1g
Data represent s mean with common letters within each column are not significantly different at
p ≤ 0.05 according to DMRT.
(v/v) solution of sodium hypochlorite (Himedia lab, India).
Thereafter, these seeds were rinsed three times for 10 minutes
with sterilized distilled water, immersed in 0.1% mercuric
chloride (Himedia lab, India) for 1 minutes followed by 3–4
washings with sterilized distilled water in the laminar flow
cabinet. The seeds were inoculated in 25 ml culture tubes
(Borosil, India) containing solidified half-strength MS
(Murashige and Skoog, 1962) medium with 1.5% sucrose,
pH 5.8. Culture tubes were kept in the dark for 2 days at 25 ±
2˚C , and later kept in illuminated room with white
fluorescent tube lights ((100 l mol m
–2
s
–1
PFD) from cool-
white fluorescent lamps (F40 T12/CW/EG, Phillips, New
Delhi, India) in a 16 h photoperiod. All the chemicals used in
this study were of analytical grade and of pure quality.
Medium composition
The MS medium contained macro and micro salts, vitamins,
3% sucrose and 0.8% agar-agar. The growth regulators
(BAP, 2, 4-D, Kin, TDZ, 2ip and ABA) were filter-sterilized
and added to the sterilized culture medium.
Callus induction
Hypocotyl and cotyledon explants were taken from five days
old in vitro raised seedlings, and cultured on basal MS
medium supplemented with various concentrations of 2,4-D
(1-5mg/l); and sucrose (0.5-3%). The embryogenic calli,
obtained from hypocotyl and cotyledon, were referred as
HEC (hypocotyl-derived embryogenic calli) and CEC
(cotyledon-derived embryogenic calli), respectively. Ten
explants each, in triplicate were cultured on callus induction
medium.
Embryo development
After twenty days of inoculation of explants on callus
induction medium, the embryogenic calli were cultured on
1788
MS, amended individually with various concentrations of
BAP, Kin, TDZ and 2ip (0.5, 1.0 and 2.0 mg/l). In induction
medium, the embryogenic calli differentiated into somatic
embryos (SEs).
Embryo maturation and germination
After two weeks of culture, these SEs developed further and
showed maturation when early staged embryos were sub-
cultured on embryo development medium. The composition
of germination medium was the same as maturation medium
but it was additionally supplied with various concentrations
of ABA (0, 1.3, 2.6, 3.9, 5.2 mg/l). The pH of both media
was adjusted to pH 5.8. All cultures were incubated in an
automated culture room at 25 ± 2˚C and illuminated with
white fluorescent tube lights (100 l mol m
–2
s
–1
PFD) from
cool-white fluorescent lamps (F40 T12/CW/EG, Phillips,
New Delhi, India) in a 16 h photoperiod.
Transmission Electron microscopy (TEM)
The study of ultra structure of embryogenic calli was done
using TEM. Embryo regenerating tissue samples were fixed
with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4.
They were postfixed in 1% OsO
4
in the same buffer solution.
Embedding was made in Epon–Araldite resin in flat molds
with proper orientation to obtain cross sections of embryos.
Resin blocks were cut on an UC6 Leica ultramicrotome with
a diamond knife and 60-nm sections were stained with uranyl
acetate followed by lead citrate. Stained sections were
examined with a Philips MORGAGNI 268 transmission
electron microscope. The microscope was operated with an
electron beam at 70 kV.
Scanning electron microscopy (SEM)
SEs on embryogenic calli were cleaned with 0.1 M phosphate
buffer (pH 7.4) and fixed for 18 hour at 4ºC in modified
Karnovsky’s fluid made in 0.1 M Phosphate buffer (pH 7.4).
The specimens were dehydrated in a graded acetone solution.
Critical Point Drying was done with liquid CO
2
using
Polaron Jumbo Critical Point Dryer, and Gold Sputter
Coating was carried out under reduced pressure in an inert
argon gas atmosphere (Agar Sputer Coater P 7340). After
sputter coating, the tissues were examined under Scanning
Electron Microscope (Leo 435VP) operated at 15 kV (David
et al., 1973).
Histological studies
Embryogenic calli with developing embryos were fixed in
FAA (v/v, formaldehyde/100% ethanol/acetic acid, 95:5:5),
dehydrated in an alcohol series (30-100% ethanol), and then
embedded in pure paraffin wax. Paraffin blocks containing
the embedded samples were sectioned to 10 µm thickness
with a microtome. The sections were deparaffinized in xylol,
stained with 1% (w/v) toludine blue for 2 min, viewed under
a compound microscope (Olympus CX41RF) and
photographed.
Statistical analysis
The data on the effects of growth regulators on different
stages of embryogenesis were analyzed by one-way analysis
of variance (ANOVA). Values are means of five replicates,
and the presented mean values were separated using
Duncan’s Multiple Range Test (DMRT) at p ≤ 0.05.
Acknowledgements
The first author is thankful to Council of Scientific and
Industrial Research (CSIR) for providing the research
fellowship for carrying out this research. We are thankful to
Dr. R.K Katiyar, IARI, New Delhi for providing seeds of
Brassica juncea cv. Pusa Jai kisan, used in this study.
References
Agnihotri A, Prem D, Gupta K (2004) Biotechnology in
quality improvement of oilseed Brassicas In: Srivastava PS,
Narula A, Srivastava S (eds) Plant Biotechnology and
Molecular Marker Anamaya Publishers New Delhi, pp, 10.
Angoshtari R Afshari RT, Kalantari S, Omidi M (2009)
Effects of abscisic acid on somatic embryogenesis and
induction of desiccation tolerance in Brassica napus. Asian
J Plant Sci 8:276-284.
Bewley JD, Black M (1985) Seeds: germination, structure
and composition, in physiology of developmental and
germination, Bewley JD and Black M, Eds. Plenum Press,
New York, 1.
Bhattacharya NM, Sen SK (1980) Production of plantlet
through somatic embryogenesis in Brassica campestris, Z.
pflanzenphysiol 99:357-365.
Das B, Goswami L, Ray S, Ghosh S, Bhattacharyya S, Das S
Majumder AL (2006) Agrobacterium-mediated
transformation of Brassica juncea with a cyanobacterial
(Synechocystis PCC6803) delta-6 desaturase gene leads to
production of gamma linolenic acid. Plant Cell Tiss Org
Cult 86:219–231.
David GFX, Herbert J, Wright CDS (1973) The ultra
structure of the pineal ganglion in the ferret. J Anat 115:79–
97.
Eapen S Abraham V Gerdemann M Schieder O (1989) Direct
somatic embryogenesis, plant regeneration and evaluation
of plants obtained from mesophyll protoplasts of Brassica
juncea. Ann Bot 63:369-372.
Gaj MD (2004) Factors influencing somatic embryogenesis
induction and plant regeneration with particular reference to
Arabidopsis thaliana (L.) Heynh. Plant Growth Reg 43:27-
47.
Imin N, De Jong F, Mathesius U, Noorden G, Saeed NA,
Wang XD, Rose RJ, Rolfe BG (2004) Proteome reference
maps of Medicago truncatula embryogenic cell cultures
generated from single protoplasts. Proteomics 4:1883–1896.
Imin N, Nizamidin M, Daniher D, Nolan KE, Rose RJ, Rolfe
BG (2005) Proteomic analysis of somatic embryogenesis in
Medicago truncatula explant cultures grown under 6-
benzylaminopurine and 1-naphthaleneacetic acid
treatments. Plant Physiol 137:1250-1260.
Kirti PB, Chopra VL (1989) A simple method of inducing
somatic embryogenesis in hypocotyl explant culture of
mustard, Brassica juncea (L.) Czern & Coss. Plant Breed
102:73-78
Kirti PB, Chopra VL (1990) Rapid plant regeneration
through organogenesis and somatic embryogenesis from
cultured protoplasts of Brassica juncea. Plant Cell Tiss Org
Cult 20:65-67.
Kumari A, Cheema GS, Munshi SK (1995) Induction, growth
and development of somatic embryos from hypocotyl
explants in mustard (Brasica juncea L. Czern & Coss).
Cruciferae Newslett 17:30–31.
Kumari A, Cheema GS, Munshi SK (2000) A hypocotyl-
derived somatic embryogenic system in Brassica juncea
Czern & Coss and its manipulation for enhanced storage
lipid accumulation. Plant Cell Tiss Org Cult 63:109–120.
1789
Maheswaran G, Williams EG (1984) Direct somatic
embryoid formation on immature embryos of Trifolium
repens, T. pratense and Medicago saliva and rapid clonal
propagation of T. repens. Ann Bot 54:201−211.
Mujib A, Samaj J (2006) Somatic embryogenesis. Springer-
Verlag, Berlin, Heidelberg, New York.
Murashige T, Skoog F (1962) A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol
Plant 15:473– 497.
Narasimhulu SB, Kirti PB, Prakash S, Chopra VL (1992)
Somatic embryogenesis in Brassica nigra (Koch) J Exp Bot
43:1203-1207.
Nolan KE, Irwanto RR, Rose RJ (2003) Auxin up-regulates
MtSERK1 expression in both Medicago truncatula root-
forming and embryogenic cultures. Plant Physiol 133:218–
230.
Pareekh LK, Chandra N (1978) Somatic embryogenesis in
leaf callus from cauliflower (Brassica oleracea). Plant Sci
Lett 11:311–316.
Potts DA, Rakow GW, Males DR (1999) Canola-quality
Brassica juncea, a new oilseed crop for the Canadian
prairies. In: Proc. 10th International Rapeseed Congress,
Canberra, Australia, September, pp, 23-29.
Raemakers K, Pereira I, Putten HK, Visser R (2005) Indirect
somatic embryogenesis in cassava for genetic modification
purposes. Methods Mol Biol 318:101-109.
Raghavan V (2004) Role of 2,4-D in somatic embryogenesis
on cultured zygotic embryo of Arabidopsis: Cell expantion,
cell cycling and morphogenesis during continuous exposure
of embryo to 2,4-D. Am J Bot 91(11):1743-1756.
Senaratna T, Laima K, Beversdorf WD Mc Kersie BD (1991)
Desiccation of microspore derived embryos of oilseed rape
(Brassica napus L.). Plant Cell Rep 10:342-344.
Sharma KK, Bhojwani SS, Thorpe TA (1991) The role of
cotyledonary tissue in the differentiation of shoots and roots
from cotyledon explants of Brassica juncea (L.) Czern.
Plant Cell Tiss Org Cult 24:55-59.
Stoutjesdijk PA, Hurlstone C, Singh SP, Green AG (1999)
Genetic manipulation for altered oil quality in Brassicas. In:
Proc. 10th International Rapeseed Congress, Canberra,
Australia, September, pp, 23-29.
Sugiyama M (1999) Organogenesis in vitro. Curr. Opin.
Plant Biol 2:61–64.
Sung ZR, Okimoto R (1993) Coordinate gene expression
during somatic embryogenesis in carrots. Proc Natl Acad
Sci USA 80:2661-2665.
Thorpe TA (1995) In Vitro embryogenesis in plants. Kluwer
academic publishers, Netherlands. ISBN 0-7923-3149-4
Turgut K, Barghchi M, Scott R (1998) Efficient shoot
regeneration and somatic emhryogenesis from immature
cotyledons of Brassica napus L. Plant Breed 117:503-504.
Yantcheva A, Vlahova M, Antanassor A (1998) Direct
somatic embryogenesis and plant regeneration of carnation
(Dianthus caryophyllus L.). Plant Cell Rep 18:148-153.
