International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 4(1) pp. 19-24, January 2013
Available online http://www.interesjournals.org/IRJPS
Copyright © 2013 International Research Journals
Full Length Research Paper
Somatic embryogenesis from in vitro cultured
internode explants in grass pea (Lathyrus sativus L.)
1
1
1
2
2
*Swapan K. Tripathy , Rajesh Ranjan , Devraj Lenka , Bhumika Ray Mohapatra and Shovina Pal
1
Sinha Molecular Breeding Laboratory, Department of Plant Breeding and Genetics, College of Agriculture, OUAT,
Bhubaneswar-3 (Odisha), India
2
MITS school of Biotechnology, Bhubaneswar, India
Abstract
Internode explants of grass pea (Lathyrus sativus L.) pre-cultured in B5 with 2, 4-D (2mg/l) + BAP
(0.5mg/l) when transferred to BM medium containing 0.5mg/l NAA + 2mg/l BAP; produced
conspicuous, glossy, dark green and well developed somatic embryos. These were found to be ideal
for whole plantlet differentiation in a suitable germination medium (hormone-free MS). The
embryogenic cultures were maintained in BM + 0.5mg/l NAA + 2mg/l BAP for about two years with
continuous induction of globular to torpedo SEs in addition to proliferation of greenish organogenic
callus. Multiple shoots occasionally grew from such green calli on transfer to a shoot regenerating
medium, B5 + 0.5mg/l IAA + 1mg/BAP. Dhenkanal local yielded the highest somatic embryogenic
regeneration response (19.8 + 1.32%) followed by Nirmal (12.5 + 1.66%), while P24 did not respond at
all. The somatic embryo- derived regenerated plantlets were readily converted into sexually mature
plants.
Keywords: Lathyrus sativus L., Somatic embryogenesis, internode explants, 2,4-D (2, 4-Dichloro-phenoxy
acetic acid), NAA (Naphthalene acetic acid), BAP(6-benzyl aminopurine) and Kn (Kinetin).
INTRODUCTION
Grass pea (Lathyrus sativus L.) is one of the important
food legumes in countries like India, Pakistan,
Bangladesh and Ethiopia. It can thrive well in adverse
climatic conditions (drought, salinity and water-logging)
and has substantially high protein content (25-30%) than
other food legumes. The largest collections of grass pea
genetic resources are in India (Raipur, 2659 genotypes)
followed by Syria (ICARDA, 1560 genotypes) and
France (University of Pau, 1807 genotypes (Campbell,
1997), but unfortunately it harbors neurotoxins
particularly β-N-oxylaminoalanine (BOAA) that cause
neurolathyrism (an upper motor neuron disease in the
cerebral cortex) upon prolonged consumption more than
three months. However, In vitro selection of low
neurotoxin plants and gene transfer may offer genetic
rectification in grass pea and make it suitable for
consumption. An efficient in vitro culture system is a pre-
*Corresponding
Author
swapankumartripathy@gmail.com
Email:
requisite for success in isolation of somaclonal variants,
or somatic mutants and also genetic transformation. In
this context, somatic embryogenesis is gaining
importance as the regenerants are derived from single
somatic cells. But, legumes in general, are recalcitrant to
regeneration in vitro
(Mroginski and Kartha,
1981).Tripathy and Cole (2001) reported that
Organogenesis and somatic embryogenesis are under
separate genetic control and therefore, genotypes of the
same crop species are most likely to respond
differentially with regard to morphogenetic potential even
in an optimum regeneration medium. Bencheikh and
Gallais (1996) studied inheritance of the ability to form
somatic embryos using a diallel cross among six
different pure lines of pea (Pisum sativum). About 80%
of the observed genotypic variation was due to additive
effects. Analysis of the distribution of F3 family means
from crosses between divergent lines indicated presence
of a few major genes to control somatic embryogenesis
in pea. Effect of genotype or genotype x medium
interaction on callus induction and regeneration potential
has been well documented in number of crop plants
20 Int. Res. J. Plant Sci.
including grass pea (Gharywal and Maheswari, 1983;
Suresh Kumar et al., 1983; McKently et al., 1989; OziasAkins et al., 1992). Moreover, successful callus induction
does not necessarily correlate with induction of somatic
embryogenesis and regeneration ability of a genotype.
Therefore, the present investigation was attempted to
optimize a protocol for efficient regeneration through
somatic embryogenesis taking four genotypes of grass
pea (Lathyrus sativus L.).
MATERIALS AND METHODS
Four genetically pure lines of grass pea (Lathyrus
sativus L.) including P24, Nirmal, Dhenkanal local and
Nayagarh local were tested for somatic embryogenic
response at Sinha Molecular Breeding Laboratory,
Department of Plant Breeding and Genetics, College of
Agriculture, OUAT, Bhubaneswar-3(India) during 200912. Internode explants from aseptically grown seedlings
were initially cultured in B5 (Gamborg et al., 1968)
supplemented with 2,4-D (2mg/l) + BAP (0.5mg/l) or
with NAA (2mg/l) + BAP (0.5mg/l) for primary callus
induction. Calli from both the sources were sub-cultured
in B5 and BM (Blaydes 1966) medium supplemented
with four auxins including 2,4-D,
NAA, IAA and
IBA(0.25, o.5, o.75 and 1.0mg/l each) in combination
with two cytokinins, BAP and Kn (1.0, 2.0, 3.0 and
4.0mg/l each) to assess induction of somatic
embryogenesis. In fact, internode explants pre-cultured
on B5 with 2, 4-D (2mg/l) + BAP (0.5mg/l) produced
somatic embryos on transfer to BM medium containing
0.25mg/l 2,4-D + 1.0mg/l BAP or 0.5mg/l NAA + 2mg/l
BAP. A follow up step was also undertaken to arrive
further suitable combination and optimum concentration
of phytohormones in the regeneration medium for
somatic embryogenic response in Lathyrus (to be
discussed later). Finally, the well developed somatic
embryos were germinated on basal MS medium.
For plant establishment, primary hardening of
regenerated plantlets was done initially in 1/10 basal MS
liquid medium under complete shading. For secondary
hardening, plants were transferred to sterilized garden
soil (Soil: FYM= 1:1) in pots and nurtured for 15 days by
adding 1/10 basal MS liquid medium in the morning and
tap water in the afternoon in the green house. After
transfer to the field, plants were shaded with card boards
and irrigated twice a day for first 10 days, during which
shading was gradually reduced by one hour each day.
The whole in vitro culture experiment comprising
four varieties of grass pea was carried out in six lots,
each lot being considered as single replication. The
observations recorded in terms of % - response of
internode explants for somatic embryo induction and no.
of SEs/ responsive internode explant were analysed
following standard RBD design (Panse and Sukhatme,
1985).
RESULTS AND DISCUSSION
An attempt was made to obtain suitable hormone
recipe(s) for successful somatic embryo (SE) induction
using internode primary callus (from B5 + 2mg/l 2,4D +
0.5mg/l BAP and B5 + 2mg/l NAA + 0.5mg/l BAP ) as
explants in B5 and BM medium supplemented with four
auxins including 2,4-D, NAA, IAA and IBA(0.25, 0.50,
0.75 and 1.0mg/l) in combination with two cytokinins,
BAP and Kn(1.0, 2.0,3.0 and 4.0mg/l). Induction of SEs
were realized only when primary internode cultures
grown on B5 + 2mg/l 2,4-D + 0.5mg/l BAP transferred to
BM with 2, 4-D(0.25mg/l) + BAP(1.0mg/l) and BM with
NAA(0.5mg/l) + BAP(2mg/l). Internode explants precultured on B5 + 2mg/l NAA + 0.5mg/l BAP never
developed SEs. Thus, development of somatic embryos
is pre-conditioned by presence of 2,4-D in the initial
callus induction media (Barna and Mehta,1995). Jiangbo
et al. (2000) used hypocotyle explants of Lathyrus
maritimus and reported that optimum concentration of 2,
4-D in the regeneration medium should be 0.5mg/l
beyond which the embryo development was inhibited
and could not pass even beyond heart shaped stage.
Besides, they opined that mild concentration of NaCl in
the medium is important to enhance SE induction and
improve maintenance of embryogenic calli. This
suggested further that biochemical and physiological
events occurring within cells during first two weeks of
culture in the regeneration medium are very important
and could throw light on our understanding of the
mechanisms of differentiation and organ development in
vitro. In general, as cells underwent embryogenesis they
enlarged with glossy surface and the surrounding walls
become thinner with the onset of embryogenesis and the
cell wall diminished further as embryos matured. Thus,
kinetics of cell wall thickness and cell surface are
reported to be early indicators of the competence of cells
to undergo somatic embryogenesis (Ochatt et al., 2008).
Somatic embryos were induced on BM alone. This
+
might be due to optimum level of NH4 (1000mg/l) and
NO3 (1000mg/l) in BM as compared to B5 medium which
+
has high NO3 (2500mg/l) and no NH4 . Meijer and Brown
+
(1987) reported an absolute requirement of NH4 in MS
medium for SEs induction and differentiation in
Medicago sativa. SEs could be readily produced in
+
media containing high amount of NH4 and NO3 , but
never on medium with low amount of NO3 (Mascarenhas
1981). Halperin and Wetherell (1965) observed that
+
NH4 and casein hydrolysate at low concentration were
strongly stimulatory to somatic embryogenesis in
comparison to NO3 .
Further, it is worthwhile to mention that Kn with any
of the test auxins could not induce SEs in either B5 or
BM medium. Hence, it is reasonable to assume that BAP
is essential for induction of SEs. Patel et al. (1991) in
mungbean and Malik and Saxena(1992) in Phaseolus
spp. Stressed the importance of BAP for somatic
Tripathy et al. 21
Table 1. Effect of 2,4-D and NAA in combination with BAP in BM medium for somatic embryo induction in Lathyrus sativus
(Var. Nayagarh local, Explant : Internodes pre-cultured on B5 + 2mg/l 2,4-D + 0.5mg/l BAP).
Hormone
recipe(mg/l)
2,4-D + BAP
0.1 + 1.0
0.2 + 1.0
0.3 + 1.0
0.4 + 1.0
0.5 + 1.0
0.0 + 0.5
0.0 + 1.0
0.0 + 1.5
0.0 + 2.0
% response
No. of SE/responsive
internode explant
Remarks
2.9 + 0.09*
1.3 + 0.09
0.0
0.0
0.0
0.0
3.8 + 0.23
3.5 + 0.35
5.7 + 0.47
5.3 + 0.31*
2.8 + 0.26
0.0
0.0
0.0
0.0
0.1 + 0.53
9.0 + 0.48
10.5 + 1.08
0.0 + 2.5
0.0 + 3.0
NAA + BAP
0.25 + 2.0
0.50 + 2.0
0.75 + 2.0
1.0
+ 2.0
0.5 + 1.0
0.5 + 2.0
0.5 + 3.0
0.5 + 4.0
5.8 + 0.39
7.8 + 0.61
11.2 + 1.23
12.3 + 1.13
Less viable SEs
Rudimentary SEs
No response, but callusing
-do-doNo response
Few viable SEs
Less viable SEs
SEs not viable and failed to develop beyond
globular stage
-do-do-
3.8 + 0.25
4.7 + 0.30
4.1 + 0.26
2.1 + 0.18
0.0
4.8 + 0.35
6.9 + 0.59
7.2 + 0.47
7.1 + 0.50
12.5 + 1.23
9.5 + 1.00
3.9 + 0.32
0.0
11.9 + 1.29
7.3 + 0.68
3.5 + 0.41
More or less normal green glossy SEs
Conspicuous dark green glossy SEs
Green and more or less normal SEs
SEs induced in late and less viable
No response
Dark green, glossy and well developed SEs
SEs bit reduced in size and less viable
SEs small in size and not viable
*Values are mean + SE.
embryogenesis. Besides, Mroginski and Kartha (1981)
demonstrated more effectiveness of BAP than both Kn
and 2- ip for plant regeneration in pea.
As a follow- up step, several combinations of 2,4-D
and NAA with BAP at different concentrations were tried
to further optimize the hormone recipe in BM
regeneration medium(Table 1). Increased concentration
of 2, 4-D with constant BAP at 1mg/l led to induction of
rudimentary SEs or even no response, while BAP
without 2,4-D in BM exhibited linear increase in number
of SEs /explants and general increase in % response
with increase in its concentration(Table -1). This may be
due to the fact that somatic embryogenesis is generally
suppressed by addition of Kn and 2, 4-D which can be
stimulated by latter’s withdrawal from the regeneration
medium in a sequential manner (Steward et al., 1963;
Bachs-Husemann and Reinert, 1970; Rao et al., 1973;
Sengupta and Raghavan, 1980). At higher concentration
of BAP (2.0-4.0mg/l) alone, SEs developed were not
viable and failed to develop SEs beyond globular stage.
In Phaseolus spp., Malik and Saxena (1992) reported no
response of BAP for somatic embryogenesis at lower
concentration, while beyond optimum (90-120µM), SEs
were highly vitrified and showed stunted growth or even
die. Production of SEs in BM without 2,4-D may be due
to carry over- effects of auxin (2,4-D) from primary
inoculation to subculture as also reported by Reinert and
Tazawa (1969). They further reported cytokinins in initial
medium to be responsible for partial or complete
inhibition of SE induction. This might be one of the
causes for lower recovery of SEs in the present study,
since the initial media were supplemented with BAP
(0.5mg/l).
Lower concentration of NAA (0.25-0.75mg/l) with
2mg/l BAP elicited more number of SEs/responsive
internode explants (Table 1), but higher concentration of
NAA (1mg/l) in combination with 2mg/l BAP delayed SE
induction and SEs were less viable. Induction of SEs
increased with the number of subculture. The
embryogenic calli at lower conc. of NAA + 2mg/l BAP
turned white to glossy green. However, conspicuous,
glossy, dark green and well developed globular SEs
induced in 0.5mg/l NAA + 2mg/l BAP were found to be
ideal (Plate 1) for whole plantlet (with defined root and
shoot) differentiation (Plate 2) in a suitable germination
medium (hormone-free MS), but those induced in 2,4-D
+ BAP were less viable with poor regenerating potential
and often produced abnormal cotyledon like structures
without any shoot initial (Plate 3). Embryogenic cultures
were maintained in BM + 0.5mg/l NAA + 2mg/l BAP for
about two years with continuous induction of globular
SEs in addition to proliferation of greenish organogenic
callus. Multiple shoots occasionally grew from
such green organogenic calli on transfer to a standard
22 Int. Res. J. Plant Sci.
Plate 1. Induction of large glossy green globular somatic embryos
from pre-cultured (B5 + 2mg/l 2, 4-D + 0.5mg/l BAP) internode
explants up on transfer to BM + 0.5mg/l NAA + 2.0mg/l BAP in
Lathyrus sativus L., cv. Nayagarh local.
Plate 2. Regeneration of plantlet (with defined root and
shoot) from somatic embryo derived from BM + 0.5mg/l
NAA + 2.0mg/l BAP up on transfer to hormone- free MS
medium in Lathyrus sativus L. ,cv. Nayagarh local.
Tripathy et al. 23
Plate 3. Formation of abnormal cotyledon like structures without
any shoot initial from somatic embryos derived from BM + 0.2mg/l
2, 4-D + 1.0mg/l BAP up on transfer to hormone-free MS
medium in Lathyrus sativus L. ,cv. Nayagarh local.
Table 2. Response of internode explants (pre-cultured in B5 + 2mg/l 2,4-D + 0.5mg/l BAP) of grasspea (Lathyrus sativus L)
genotypes to somatic embryogenesis in BM medium supplemented with NAA(2mg/l) and BAP (0.5mg/l).
Genotype
% Response
P 24
Nirmal
Nayagarh local
Dhenkanal local
0.0
12.5 + 1.66
4.9 + 0.45
19.8 + 1.32
No. of SEs/responsive
internode explant
0.0
5.8 + 0.47
11.2 + 1.23
17.3 + 1.21
Total no. of plantlets
produced
0
11
10
28
No. of plants
survived
0
3
5
9
Pry. Callus culture medium : B5 + 2mg/l 2,4-D + 0.5mg/l BAP
regenerating medium, B5 + 0.5mg/l IAA + 1mg/BAP;
while the SEs failed to differentiate.
There was a general increase in % response for SE
induction (Table 1 ) by increased BAP concentration
(2.0-4.0mg/l ) with constant NAA (0.5mg/l). No. of SEs
per responsive explants, however, decreased abruptly
leading to production of non-viable SEs at NAA (0.5mg/l)
+ BAP (4.0mg/l).
Gharywal and Maheswari(1983) reported somatic
embryogenesis in callus masses of long term subcultures on B5 + IAA(0.5mg/l) + BAP(1mg/l) arising from
shoot meristems and leaf explants in Lathyrus sativus.
Tripathy et al. (1994) also reported somatic
embryogenesis in the same crop using internode
explants. Barna and Mehta (1995) reported direct
somatic embryogenesis from immature leaflets and
internodal segments. The explants were grown on MS
medium supplemented with 2,4-D (1-10mg/l) for one
week and then grown on auxin -free medium for another
four weeks of induction of SEs. Capacity of immature
zygotic embryos or embryo parts, e.g., cotyledon and
embryo axis to undergo somatic embryogenesis have
been reported in other legumes e.g., Finer and
Nagasawa (1988) in soybean and Angelini and Allavena
(1989) in Phaseolus coccineus, Sellars et al. (1990) in
peanut and soybean, Kysely and Jacobsen (1990) in
pea, Ozias-Akins et al. (1992) in peanut and Durham
and Parrott (1992) in peanut.
Genotypic response for somatic embryogenic
regeneration was tested in four varieties of Lathyrus
(Table 2). Dhenkanal local yielded the highest somatic
embryogenic regeneration response (19.8 + 1.32%) as
well as number of SEs/responsive internode explants
(17.3 + 1.21%). Nirmal responded well to somatic
24 Int. Res. J. Plant Sci.
embryogenic regeneration (12.5+1.66), but induced
significantly lower number of SEs/responsive internode
explant. In contrast, Nayagarh local had opposite
response indicating that regeneration via somatic
embryogenesis is genetically controlled. The SE- derived
regenerated plantlets were readily converted into
sexually mature plants although with poor survival
percentage in the field. Somatic embryogenesis has
been reported in Lathyrus sativus (Gharywal and
Maheswari, 1983; Barna and Mehta, 1995), but there are
no available reports for difference in genotypic response.
Kao and Michayluk(1981) could decipher genotypic
differences not only at genetic, species and varietal
levels, but even within different plants of the same
cultivar in Medicago sativa.
REFERENCES
Angelini RR, Allavena A (1989). Plant regeneration from somatic
cotyledon explant cultures of bean (Phaseolus coccineous L.). Plant
Cell Tissue and Organ Culture, 19:167-174.
Bachs-Husemann D, Reinert J (1970). Protoplasma, 70:49.
Barna KS, Mehta SL (1995). Genetic transformation and somatic
embryogenesis in Lathyrus sativus L. J. Plant Biochem. Biotech.;
4:67-71.
Bencheikh M, Gallais A (1996). Somatic embryogenesis in pea (Pisum
sativum L. and Pisum
arvense L.): Diallel analysis and genetic
control. Euphytica, 90(3): 257-264.
Blaydes DF (1966). Interaction of kinetin and various inhibitors in the
growth of soybean tissue. Physiol. Pl.; 19:748-753.
Campbell CG (1997). Grass Pea. Lathyrus sativus L. Promoting the
conservation and use of underutilized and neglected crops. 18.
Institute of Plant Genetics and Crop Plant Res.; Gatersleben
/International Plant Genetic Resources Institute (http://www.
ipgri.org), Rome, Italy: 30.
Durham RE, Parrott WA (1992). Repetitive somatic embryogenesis
from peanut cultures in liquid medium. Plant Cell Reports, 1:122125.
Finer JJ, Nagasawa A (1988). Development of an embryogenic
suspension culture of soybean (Glycine max Mcrrill). Plant Cell
Tissue and Organ Culture, 15:125-136.
Gamborg OL, Miller RA, Ojima K (1968). Nutrient requirements of
suspension cultures of soybean root cells. Expt. Cell Res.; 50:151158.
Gharywal PK, Maheswari SC (1983). Genetic and physiological
influences on differentiation in tissue cultures of a legume Lathyrus
sativus. Theor. Appl. Genet., 66:123-126.
Halperin W, Wetherell WF (1965). Ammonium requirement for
embryogenesis in vitro. Nature, 205:519-520.
Jiangbo W, Yumei W, Jingfen J (2000). Embryogenic callus induction
and somatic embryo formation from hypocotyl explants of Lathyrus
maritimus. Acta Botanica Boreali- Occidentalia ,Sinica, 20(3):352357.
Kao KM, Michayluk MR (1981). Embryoid formation in alfalfa cell
suspension cultures from different plant parts. In Vitro, 17:645-648.
Kysely W, Jacobsen H (1990). Somatic embryogenesis from pea
embryos and shoot apices. Plant Cell Tissue and Organ Culture,
20:7-14.
Ozias-Akins P, Anderson WF, Holbrook CC (1992). Somatic
embryogenesis in Arachis hypogaea L.: Genotypic comparison.
Plant Sci., 83:103-111.
Malik KA, Saxena PK (1992). Somatic embryogenesis and shoot
regeneration from intact seedlings of Phaseolus acutifolius A., P.
aureus (L.) Wilczek, P. coccineus L. and P. wrightii L. Plant Cell
Reports, 11:163-168.
Mascarenhas AF (1981). Plant Tissue Culture: its role in studies on
organogenesis. Curr. Sci.; 50(19):835-83.
McKently AH, Moore GA, Gardner FP (1989). In vitro plant
regeneration of peanut from seed explants. Crop Sci., 30:192-196.
Meijer Eltjo GM, Brown Daniel CW (1987). Role of exogenous reduced
nitrogen and sucrose in rapid high frequency somatic
embryogenesis in Medicago sativa. Plant Cell Tissue and Organ
Culture, 10: 11-19.
Mroginski LA, Kartha KK (1981). Regeneration of Pea (Pisum sativum
L. cv. Century) plants by in vitro culture of immature leaflets. Plant
Cell Reports, 1:64-66.
Ochatt S, Muilu R, Ribalta F (2008). Cell morphometry and
osmolarity as early indicators of the onset of embryogenesis from
cell suspension cultures of grain legumes and model systems. Plant
Biosystems, 142(3):480-486.
Panse VG, Sukhatme PV (1985). Statistical methods for agriculture
workers. Fourth edition,97-123.
Patel MB, Bhardwaj R, Joshi A (1991). Organogenesis in Vigna radiata
(L.) Wilczek. Indian J. Expt. Biol., 29:619-622.
Rao PS, Handro W, Harada H (1973). Physiol. Plant, 28:458.
Reinert J, Tazawa M (1969). Wirkung von Stickstoffverbindungen und
von Auxin auf die Embryogenese in Gewebekulturen. Planta,
87:239-248.
Sellars RM, Southward GM, Phillips GC (1990). Adventitious somatic
embryogenesis from cultured immature zygotic embryos of peanut
and soybean. Crop Sci., 30: 408-414.
Sengupta C, Raghavan V (1980). Somatic embryogenesis in carrot cell
suspension : II. Synthesis of ribosomal RNA and Poly (A) + RNA. J.
Expt. Bot., 3:259-268.
Steward FC, Blakely L, Kent A, Mapes MO (1963). Brookehaven
Symp. Biol., 16:73.
Suresh KA, Reddy TP, Reddy GM (1983). Plantlet regeneration from
different callus cultures of pigeonpea (Cajanus cajan L.). Plant Sci.
Lett., 32:271-278.
TripathySK, Pattnaik SN, Kole C (1994). Multiple shooting and somatic
embryogenesis in Lathyrus sativus L. In: Second Asia-Pacific
Conference on Agricultural Biotechnology, Madras, India,172.
Tripathy SK, Cole C (2001). Genotypic response to plant regeneration
in Lathyrus sativus L. Plant Sci. Res., 23(1 and 2):17-20.