Plant Cell Reports (1998) 17: 742 – 746
V. B. Mhaske · K. Chengalrayan · S. Hazra
Influence of osmotica and abscisic acid on triglyceride accumulation
in peanut somatic embryos
Received: 4 April 1997 / Revision received: 15 February 1998 / Accepted: 2 March 1998
Abstract Attempts were made to determine the influence
of sucrose, mannitol, sorbitol and abscisic acid on accu-
mulation of triglycerides in peanut (Arachis hypogaea L.)
somatic embryos. The results revealed that 0.584 M su-
crose in the medium produced increased triglycerides in
the embryos compared to the control. At 0.730 M sucrose,
embryos were necrotic although the triglyceride content
was high. Sorbitol at 0.6 M or abscisic acid at 20 µM were
effective in increasing triglycerides in the embryos. The
increase in triglycerides on a fresh-weight basis observed
with increasing concentration of osmoticium was not ap-
parent when determined in terms of dry weight. However,
an increase in triglyceride as percent fresh weight observed
in the presence of 20 µM abscisic acid remained unaltered
when determined in terms of percent dry weight. An in-
crease in storage lipid did not improve conversion of pea-
nut somatic embryos.
Key words Sucrose · Mannitol · Sorbitol · Osmolality ·
Abscisic acid
Abbreviations ABA Abscisic acid · DW Dry weight ·
EDM Embryo development medium · EIM Embryo induc-
tion medium · TG Triglyceride · FW Fresh weight
Introduction
Somatic embryos of oilseed crops show promise for pro-
duction in vitro of lipids normally produced in cotyledons
(Avjioglu and Knox 1989; Janick 1991; Pence et al. 1981).
The presence of storage lipids (triglycerides; TGs) dem-
onstrated in cocoa (Pence et al. 1981), rapeseed (Avjioglu
Communicated by G. Phillips
V. B. Mhaske · K. Chengalrayan · S. Hazra
Plant Tissue Culture Division, National Chemical Laboratory,
Pune 411008, India
Fax no: +91-212-349038
E-mail: shazra@dalton.ncl.res.in
© Springer-Verlag 1998
and Knox 1989) and peanut (Mhaske and Hazra 1994) so-
matic embryos has further strengthened this possibility.
Moreover, it is presumed that maturation of somatic em-
bryos might increase the accumulation of storage sub-
stances and result in improved conversion. The factors of-
ten associated with maturation and accumulation of stor-
age reserves in somatic embryos are the medium osmolal-
ity (Dutta and Appelqvist 1989; Etienne et al. 1993), con-
centration of abscisic acid (ABA) (Dunstan et al. 1991; Fu-
jii et al. 1990; Maquoi et al. 1993) and concentration of su-
crose (Avjioglu and Knox 1989; Janick 1991; Pence et al.
1981). However, there is no report on the influence of these
factors on TG accumulation in peanut somatic embryos.
Information generated from such studies will be needed for
manipulation of metabolite production in vitro and for ge-
netic transformation of peanut for altered TG biosynthe-
sis. In the present investigation, attempts were made to
study the influence of these factors on peanut somatic em-
bryos. The status of the somatic embryos was assessed from
the morphology, TG content, dry weight (DW) as well as
their ability to germinate and form plantlets in medium
without growth regulators.
Materials and methods
Embryogenesis
Pods of Arachis hypogaea L. cv. JL 24 were collected from Mahat-
ma Phule Agricultural University, Pune, India. The pods were shelled
and somatic embryogenesis was induced in immature leaflets fol-
lowing the method described by Chengalrayan et al. (1994) (Fig. 1,
steps 1, 2). These embryos were subjected to fresh embryo develop-
ment medium (EDM) with various supplements (Fig. 1, step 3). Af-
ter 4 weeks of incubation, the morphology, TG content and DW of
embryos were assessed (Fig. 1). The embryos cultured in EDM de-
void of growth regulators served as a control.
Embryogenesis media and culture conditions
Embryo induction medium (EIM) comprised Murashige and Skoog’s
basal medium (Murashige and Skoog 1962) supplemented with
 743
Replicates of four to five such groups of embryos were taken for each
Immature leaflets treatment and the %DW was determined from the means of these
replicates. The data were analysed statistically using the F-test and
subsequently Student’s t-test. The values of TG %DW were deter-
mined from TG %FW and %DW.
STEP 1 4 weeks embryo induction medium

Embryogenic Mass Germination

STEP2 4 weeks embryo development The embryos cultured in various media for 4 weeks were transferred
to germination medium (Fig. 1, step 4) composed of half-strength
medium (EDM)
basal medium with 2% sucrose and 0.25% charcoal (Chengalrayan
et al. 1994). After incubation for 4 weeks in this medium, root and
Embryo shoot development responses were recorded.

STEP 3 4 weeks EDM with and without

supplements
Osmolalities recorded
Results and discussion
(for sucrose & osmotica )

Somatic embryos developed in EDM for 4 weeks showed

Embryo
an increase in the accumulation of lipids from this stage
onwards (Mhaske and Hazra 1994). Therefore this point
a b c
was chosen to test the influence of sucrose, osmotica and
ABA on TG content, %DW and on the germination beha-
Morphology Triglyceride Dry weight viour of these embryos.

STEP 4 4 weeks Germination

medium Osmotica

Rooted embryos
Fig. 1 Flowchart of various steps involved in the experiment and
the different parameters studied
20 mg/l 2,4-dichlorophenoxyacetic acid and 0.175 M sucrose. EDM
was the same as EIM except that it contained 3 mg/l 2,4-dichloro-
phenoxyacetic acid. All media were solidified using 0.55% agar (Hi
Media, India). Prior to autoclaving, the pH of the media was adjust-
ed to 5.8. The osmolalities of the media with sucrose, mannitol or
sorbitol were measured by determining the dew point depression us-
ing a Wescor vapour pressure osmometer (Model 5500). Cultures
were incubated in a 16-h photoperiod at 25±2°C, under white fluo-
rescent light, intensity 32 µE/m2 per second.
Determination of TGs
The chemical determination of TG was based on a colorimetric meth-
od used for conifer calli (Feirer et al. 1989) and peanut tissues
(Mhaske and Hazra 1994). The amount of TG was expressed as per-
cent fresh tissue weight (%FW). The data were analysed using
Student’s t-test. Prior to this, the data sets were subjected to an
F-test. Where the F-test showed a significant difference between var-
iances, a modified t-test was performed (Snedecor and Cochran
1967). The experiments using various media supplements were car-
ried out in batches. Each treatment was repeated three times and data
derived from these were pooled for statistical analysis.
Dry weights
Embryos cultured in various media were blotted free of adherent me-
dium, wrapped in preweighed aluminium foil and weighed. Each foil
contained two to five embryos. These embryos were then dried in an
oven adjusted to 60°C until constant weights were attained (48–72 h).
Sucrose
Although it is generally incorporated in the culture me-
dium at a concentration of 2–6% as the primary carbon
source, sucrose also acts as an osmoticum (Pence et al.
1981). Incorporation of increasing amount of sucrose in
the EDM elevated the osmolality from 283 mOsm/kg H2O
in 0.175 M to 974 mOsm/kg H2O in 0.730 M sucrose
(Table 1). Precocious germination of the somatic embryos
observed on transfer to fresh medium of the same compo-
sition with 0.175 M sucrose noted in our previous study
(Mhaske and Hazra 1994) was suppressed in the presence
of high sucrose concentrations. This effect was partial in
embryos cultured in 0.292 M and 0.438 M sucrose, which
only showed the emergence of root primordia. In 0.584 M
and 0.730 M sucrose, emergence of root primordia was sup-
pressed completely. Compared to the embryos in 0.175 M
sucrose, TG %FW was higher in embryos grown at higher
concentrations of sucrose (Table 1). The TG concentration
in peanut somatic embryos cultured in 0.175 M sucrose was
8.07%FW whereas it was 12.64%FW and 12.74%FW in
0.584 M and 0.730 M sucrose, respectively. FW has been
employed earlier to study the accumulation of storage re-
serves in somatic embryos (Burns and Wetzstein 1994;
Feirer et al 1989).
Percent germination of the embryos decreased from
89.5% in 0.175 M sucrose to 77.3 and 57.8% in 0.584 M
and 0.730 M sucrose, respectively. This decrease in the
frequency of germination could be due to toxic or detri-
mental effects of using high concentrations of a permeat-
ing osmoticum like sucrose. Similar toxic or detrimen-
tal effects can possibly be avoided by using a non-per-
744
Table 1 Effect of varying su-
crose concentrations on TG Concentration Osmolality TG TG Percentage
content and germination of pea- (M) (mOsm/kg (% fresh (% dry rooting in
nut somatic embryos. TG val- H2O) weight ± SE) weight ± SE) germination
ues with different superscripts medium
are significantly different at the
5% level using Student’s t-test 0.175 283 8.07 a ± 1.2 30.34 a * ± 4.3 89.5
(* indicates a modified t-test) (control)
0.292 393 9.82 a ± 0.8 29.16 a * ± 2.3 86.8
0.438 582 10.28 a ± 1.0 20.46 b * ± 2.0 79.5
0.584 792 12.64 b* ± 0.5 24.08 a * ± 1.0 77.3
0.730 974 12.74 b ± 1.4 24.33 a * ± 0.6 57.8

Table 2 Effect of mannitol

and sorbitol on TG content and Concentration Osmolality TG TG Percentage
germination of peanut somatic (M) (mOsm/kg (% fresh (% dry rooting in
embryos. TG values with dif- H2O) weight ±SE) weight ±SE) germination
ferent superscripts are signifi- medium
cantly different at the 5% level
using Student’s t-test Control 283 6.10 a ± 0.8 22.93 a ± 2.9 89.5
(* indicates a modified t-test) 0.2 mannitol 440 7.35 a * ± 1.3 22.18 a * ± 3.8 94.2
0.6 mannitol 961 8.28 a ± 0.8 20.6a * ± 1.9 76.9
0.2 sorbitol 474 5.78 a* ± 1.2 21.7a * ± 4.7 94.9
0.6 sorbitol 959 8.39 b ± 0.7 20.11 b ± 1.8 70.3

meating osmoticum such as polyethylene glycol (Attree
et al. 1992).
In our earlier experiments (Mhaske and Hazra 1994),
the TG level in peanut somatic embryos was 7.9%FW af-
ter 4 weeks of incubation in EDM containing 6% sucrose,
and the germination frequency was 100% (Chengalrayan
et al. 1994). In the present experiments, the embryos in the
set of controls with 0.175 M sucrose were in EDM for 8 in-
stead of 4 weeks. Although there was no alteration in TG
content or morphological appearance, the germination fre-
quency was reduced to 89.5%. This suggests that a longer
exposure of embryos even in 0.175 M sucrose restricted ra-
dicle emergence. This inhibitory effect of sucrose was pre-
dominant in embryos cultured at higher concentrations.
The restriction of radicle emergence was associated with
an increase in TG %FW. Sucrose osmolalities above
792 mOsm/kg H2O were deleterious, causing browning
and necrosis of peanut somatic embryos.
Mannitol and sorbitol
There are very few reports on the accumulation of storage
lipids in somatic embryos under the influence of osmotica
(Avjioglu and Knox 1989; Pence et al. 1981). An increase
in TG in somatic embryos of carrot (Dutta and Appelqvist
1989) and pecan (Burns and Wetzstein 1994) was noted in
the presence of high levels of osmoticum. In the current
study, concentrations of 0.2 M and 0.6 M sorbitol and man-
nitol were chosen to obtain an osmolality within
400–1000 mOsm/kg H2O. This is approximately the range
obtained with sucrose at different concentrations. The os-
molalities attained with 0.2 M and 0.6 M mannitol and sor-
bitol are included in Table 2. Morphological observations
of the cultures did not reveal any marked difference in the
embryos grown in 0.2 M sorbitol or mannitol compared to
the control. Moreover, the precocious germination was
not restricted. Although similar in osmolality to 0.730 M
sucrose, 0.6 M mannitol and sorbitol were detrimental to
embryos. The embryos appeared brown, stunted and ne-
crotic in all the media with osmolalities higher than
792 mOsm/kg H2O. Compared to the control, the increase
in TG determined in the presence of 0.2 M mannitol or sor-
bitol was not significant. In 0.6 M sorbitol, the increase in
TG %FW was significant at the 5% level whereas with
mannitol it was not.
Rooting frequency in germination medium remained
similar to the control (94%) in embryos grown in 0.2 M
mannitol and sorbitol whereas it was reduced to 76.9%
and 70% for 0.6 M mannitol and sorbitol, respectively
(Table 2). None of the embryos from 0.2 M or 0.6 M of
either osmoticum developed shoots. Instead, callusing was
observed around the middle region of the embryos cultured
in 0.6 M mannitol or sorbitol. At 0.6 M, with osmolalities
similar to that of medium with 0.730 M sucrose, there was
a reduction in germination frequencies and dedifferentia-
tion of the embryo, suggesting that increased storage lip-
ids in the embryos did not improve the conversion fre-
quency of peanut somatic embryos.
ABA
Both ABA and osmotica are known to promote synthesis
of storage products in developing embryos (Skriver and
Mundy 1990; Xu and Bewley 1995). Compared to storage
proteins, information on the accumulation of storage lip-
ids (TG) in response to ABA is limited to a few studies
(Attree et al. 1992; Feirer et al. 1989). It is known that ABA
influences the process of embryo maturation by restricting
precocious germination and by inducing desiccation toler-
ance (Compton and Gray 1993; Emons et al. 1993; Ooms
et al. 1994). The present experiments with ABA were de-
signed to determine the concentration at which TG accu-
 745
Table 3 Effect of ABA on TG content and germination of peanut %DW increased significantly with an increase in sucrose
somatic embryos. TG values with different superscripts are signifi- concentration, reaching the optimum at 0.584 M sucrose.
cantly different at the 5% level using Student’s t-test (* indicates a
modified t-test) Raising the sucrose concentration to 0.730 M did not in-
crease the DW further, and morphologically the embryos
Concentration TG TG Percentage were brown and necrotic at this concentration. Similar ob-
(µM) (% fresh (% dry rooting in servations were noted for other osmotica. The %DW of
weight ± SE) weight ± SE) germination
medium
embryos increased significantly in 0.6 M mannitol or sor-
bitol although the increase was not as large as in sucrose.
Control 8.62 a ± 0.9 32.38 a ± 3.2 89.5 At 0.6 M mannitol and sorbitol, the osmolalities were 961
1 7.72 a ± 0.8 32.28 a ± 3.4 72.1 and 959 mOsm/kg H2O. These were similar to the osmo-
10 11.14 a ± 1.2 36.58 a ± 4.0 90.2 lality of 974 mOsm/kg H2O attained with 0.730 M sucrose.
20 13.66 b ± 1.1 48.86 b ± 4.1 48.8
50 9.37 a ± 0.9 27.78 a ± 2.7 63.3 In spite of the similarities in osmolalities, the %DW of so-
100 10.95 a ± 0.9 26.14 a* ± 2.1 60.5 matic embryos cultured in 0.730 M sucrose was signifi-
cantly higher than the %DW attained with 0.6 M mannitol
or sorbitol. This observation further strengthens the hy-
mulation was altered and whether ABA helps in conver- pothesis that sucrose plays a dual role as both an osmoti-
sion of peanut embryos. The results (Table 3) indicated that cum and a carbon source in plant tissue culture (Pence
1 µM or 10 µM ABA did not significantly affect precocious et al. 1981).
germination or the TG content of peanut somatic embryos. In contrast to the response observed with increasing su-
A further increase in ABA to 20 µM restricted precocious crose concentrations, the %DW did not show a definite
germination and increased the TG content. Elevation of trend with the various concentrations of ABA (Table 4). In
ABA concentrations to 50 and 100 µM in the medium not microspore-derived embryos of Brassica (Pomeroy et al.
only restricted germination but also adversely affected TG 1994), exogenously supplied ABA enhanced oil accumu-
accumulation and the morphology of the embryos. Thus, lation on a DW basis although accumulation of dry matter
among the concentrations tested, 20 µM ABA was most ef- per embryo was markedly inhibited. Presumably accumu-
fective in stimulating maximum TG accumulation in pea- lation of seed reserves is subject to multiple regulatory
nut somatic embryos. The increase in TG was significant mechanisms, not all of which are affected by ABA (Fin-
compared to the control at the 5% level. kelstein and Somerville 1989).
On transfer to germination medium, the frequency of root The values of TG %DW were determined from TG
emergence of peanut somatic embryos precultured in the %FW and the %DW of the embryos. This was necessary
presence of 1 µM and 10 µM ABA remained unaffected, because TGs were estimated in fresh tissues using a de-
whereas in embryos cultured at higher concentrations it was structive method (Feirer et al. 1989). The data are presented
suppressed (Table 3). However, none of these embryos de- in Tables 1–3. Analysis of the data in Tables 1–4 indicates
veloped shoots to form plantlets. Even in the presence of that sucrose, osmotica and ABA influenced the accumula-
20 µM ABA, when the increase in TG content was signifi- tion of metabolic products in peanut somatic embryos in
cantly high, the embryos failed to give rise to plantlets. The different ways. In the presence of sucrose, although there
inability of high-TG-containing somatic embryos to convert was an increase in TG %FW (Table 1) and %DW (Table 4),
into plantlets could be attributed to inadequate desiccation. the proportion of TG %DW was reduced at 0.438 M,
As embryo quality improves, embryos require desiccation 0.584 M and 0.730 M concentrations. However, these dif-
to complete maturation and thereby germinate normally. ferences were not statistically significant except at
0.438 M, where TG %DW was significantly lower than in
the control. Therefore, whether the increase in %DW was
Embryo DWs due to accumulation of other metabolic products needs to
be investigated as sucrose is recognized as a metabolic
Comparison of the DW data of the embryos cultured in in- intermediate (Pence et al. 1981). The presence of alterna-
creasing concentrations of sucrose (Table 4) showed that tive osmotica increased the %DW of the peanut embryos

Table 4 Influence of various

treatments on %DW of peanut Sucrose %DW ± SE Osmoticum %DW ± SE ABA %DW ± SE
somatic embryos. Values with (M) (M) (µM)
different superscripts are signif-
icantly different at the 5% level 0.175 26.6 a ± 0.9 Control 26.6 a ± 0.9 Control 26.6 a ± 0.9
using Student’s t-test (control)
0.292 33.7 b ± 2.2 Mannitol 1 23.9 a ± 2.0
0.438 50.2 b ± 2.2 0.2 33.2 b ± 0.5 10 30.4 b ± 1.1
0.584 52.5 b ± 1.9 0.6 40.1 b ± 0.6 20 28.0 a ± 0.5
0.730 52.4 b ± 1.1 50 33.7 b ± 1.5
Sorbitol 100 41.9 b ± 1.2
0.2 26.6 a ± 0.9
0.6 38.7 b ± 1.5
746
(Table 4) without altering the TG %DW. As indicated ear-
lier, ABA did not show a definite pattern relative to the
%DW, but compared to the control, there was a statisti-
cally significant increase in TG %DW and TG %FW in the
embryos cultured in 20 µM. Beyond this concentration, TG
contents were reduced. In the experiments with ABA, the
sucrose concentration was 0.175 M. Thus, it is evident that
20 µM ABA was effective in enhancing TG accumulation
in the peanut somatic embryos. Desiccation in vivo is a
normal feature in later stages of embryo development along
with accumulation of storage products (Goldberg et al.
1989). However, incorporation of osmotica and ABA in-
fluenced the TG content and the weight differently in pea-
nut somatic emrbyos.
The data generated in these studies revealed that the in-
crease in TG content in the presence of ABA did not over-
come the limitation caused by the morphological anoma-
lies in the somatic embryos of peanut (Chengalrayan et al.
1994, 1997). However, they suggest that the mechanisms
of TG accumulation in peanut somatic embryos (Mhaske
and Hazra 1994) are susceptible to altered ABA concen-
trations.
Acknowledgements We acknowledge C. S. I. R. (India) for grant-
ing fellowships to V. B. M. and K. C. N. Thanks are due to Prof. V.
Sitaramam, Department of Biotechnology, University of Pune, for
allowing us to use the osmometer facility in his laboratory, and Prof.
S. A. Paranjape, Department of Statistics, University of Pune, for her
help in statistical analysis of the data. This is N. C. L. communica-
tion no. 6394.
References
Attree SM, Pomeroy MK, Fowke LC (1992) Manipulation of culture
conditions for culture of somatic embryos of white spruce for im-
proved triacylglycerol biosynthesis and desiccation tolerance.
Planta 187:395–404
Avjioglu A, Knox RB (1989) Storage lipid accumulation by zygot-
ic and somatic embryos in culture. Ann Bot 63:409–420
Burns JA, Wetzstein HY (1994) Storage reserves in pecan somatic
embryos derived from suspension culture. Plant Sci 102:213–219
Chengalrayan K, Sathaye SS, Hazra S (1994) Somatic embryogen-
esis from mature embryo derived leaflets of peanut (Arachis hy-
pogaea L.). Plant Cell Rep 13:578–581
Chengalrayan K, Mhaske VB, Hazra S (1997) High frequency con-
version of abnormal peanut somatic embryos. Plant Cell Rep
16:783–786
Compton ME, Gray DJ (1993) Sucrose, abscisic acid and methyl
glyoxal bis-(guanylhydrazone) affect grape somatic embryogen-
esis. Proc Fla State Hort Soc 106:124–128
Dunstan DI, Bethune TD, Abrams SR (1991) Racemic abscisic
acid and abscisyl alcohol promote maturation of white spruce
(Picea glauca) somatic embryos. Plant Sci 76:219–228
Dutta PC, Appelqvist LA (1989) The effect of different cultural con-
ditions on the accumulation of depot lipids notably petroselinic
acid during somatic embryogenesis in Daucus carota L. Plant Sci
64:167–177
Emons AMC, Samallo-Droppers A, Toorn C van der (1993) The in-
fluence of sucrose, mannitol, L-proline, abscisic acid and gibbe-
rellic acid on the maturation of somatic embryos of Zea mays L.
from suspension cultures. J Plant Physiol 142:597–604
Etienne H, Montorro P, Michaux-Ferriere N, Carrron MP (1993) Ef-
fect of desiccation, medium osmolarity and abscisic acid on the
maturation of Hevea brasilensis somatic embryos. J Exp Bot
44:1613–1619
Feirer RP, Conkey JH, Verhagen SA (1989) Triglycerides in embryo-
genic conifer calli - a comparison with zygotic embryo. Plant Cell
Rep 8:207–209
Finkelstein R, Somerville C (1989) Abscisic acid or osmoticum pro-
mote accumulation of long chain fatty acids in developing em-
bryos of Brassica napus. Plant Sci 61:213–217
Fujii JA, Slade D, Olsen R, Ruzin SE, Redenbaugh K (1990) Alfal-
fa somatic embryo maturation and conversion to plants. Plant Sci
72:93–100
Goldberg RB, Barker SJ, Perez-Grau L (1989) Regulation of gene
expression during plant emrbyogenesis. Cell 56:149–160
Janick J (1991) Production of seed lipids via culture of somatic em-
bryos. In: Rattray J (ed) Biotechnology of plant fats and oils.
American Oil Chemists Society, Champaign, Ill, pp 76–103
Maquoi E, Hanke DE, Deltour R (1993) The effects of abscisic ac-
id on the maturation of Brassica napus somatic embryos: an ultra-
structural study. Protoplasma 174:147–157
Mhaske VB, Hazra S (1994) Appearance of storage lipids (triglyce-
rides) in somatic embryos of peanut (Arachis hypogaea L.). In
Vitro Cell Dev Biol 30P:113–116
Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 15:
473–497
Ooms JJJ, Veen R van der, Karssen CM (1994) Abscisic acid and os-
motic stress or slow drying independently induce desiccation tol-
erance in mutant seeds of Arabidopsis thaliana. Physiol Plant
92:506–510
Pence VC, Hasegawa PM, Janick J (1981) Sucrose mediated regu-
lation of fatty acid composition in asexual embryos of Theobro-
ma cacao. Physiol Plant 53:378–384
Pomeroy K, Brown DCW, Takahata Y (1994) Response of Brassica
napus L. microspore derived embryos to exogenous abscisic ac-
id and desiccation. In Vitro Cell Dev Biol 30P:196–203
Skriver K, Mundy J (1990) Gene expression in response to abscisic
acid and osmotic stress. Plant Cell 2:503–512
Snedecor GW, Cochran WG (1967) Statistical methods 6th edn.
Oxford and IBH, New Delhi
Xu N, Bewley D (1995) The role of abscisic acid in germination,
storage protein synthesis and desiccation tolerance in alfalfa
(Medicago sativa L.) seeds, as shown by inhibition of its synthe-
sis by fluridone during development. J Exp Bot 46:687–694