Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-011-0039-z
ORIGINAL PAPER
Indoleamines and calcium enhance somatic embryogenesis
in Coffea canephora P ex Fr
A. Ramakrishna • P. Giridhar • M. Jobin •
C. S. Paulose • G. A. Ravishankar
Received: 30 December 2010 / Accepted: 5 August 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Melatonin (MEL) and serotonin (SER) are
important indoleamines that are involved in neural trans-
mission in mammalian cells. They are also known to be
present in various genera of plants. The role (s) of these
indoleamines in plants are not well known. In this study,
the effects of SER, MEL, calcium, and calcium ionophore
(A23187), a calcium channel activator, on somatic
embryogenesis in Coffea canephora have been investi-
gated. Adding 100 lM of either SER or MEL to ‘ strength
Murashige and Skoog (MS) medium and 0.93 lM kinetin
(KN) has resulted in enhanced induction of somatic
embryogenesis, 85 ± 3 and 62 ± 6 embryos/callus,
respectively. In the presence of either 5 mM calcium or
100 lM calcium ionophore A23187, number of somatic
embryos/callus is also increased, with 56 ± 4 and
118 ± 10 somatic embryos/callus, respectively, compared
to 25 ± 3 embryos/callus for control. The presence of
5 mM calcium chloride along with either 100 lM SER or
A. Ramakrishna  P. Giridhar  M. Jobin 
C. S. Paulose  G. A. Ravishankar (&)
Plant Cell Biotechnology Department, Central Food
Technological Research Institute, Constituent Laboratory
of Council of Scientific and Industrial Research,
Mysore 570 0 20, India
e-mail: pcbt@cftri.res.in
M. Jobin
e-mail: jobin.mathew@yahoo.co.in
C. S. Paulose
e-mail: cspaulose@gmail.com
M. Jobin  C. S. Paulose
Molecular Neurobiology and Cell Biology Unit, Centre
for Neuroscience, Department of Biotechnology, Cochin
University of Science and Technology, Cochin,
Kerala 682 022, India
100 lM MEL, respectively, have also promoted somatic
embryogenesis with induction of 105 ± 6 and 78 ± 2
somatic embryos/callus. While, addition of calcium iono-
phore A23187 along with either 100 lM SER or 100 lM
MEL have produced 155 ± 12 or 135 ± 8 embryos/callus,
respectively. In contrast, addition of such indoleamine
inhibitors as 40 lM p-chlorophenylalanine (p-CPA),
20 lM fluoexitine hydrochloride (prozac), 1 mM verapa-
mil hydrochloride (calcium channel blocker), and 1 mM
ethylene glycol-bis (b-amino ethylether)-N, N, N0 , N0 -tetra
acetic acid (EGTA) (a calcium chelator) individually, has
inhibited induction of somatic embryos while reducing
levels of endogenous pools of SER, MEL and indole-3-
acetic acid (IAA) levels. Calcium imaging by laser scan-
ning confocal microscopy (LSCM) has revealed high
fluorescence intensity in callus treated with calcium and
calcium ionophore A23187. Immunolocalization of SER in
different tissues of C. canephora has revealed that it is
localized in vascular tissues of stems, roots, and somatic
embryos, as well as in endocarps (husks) of immature
fruits.
Keywords Somatic embryogenesis  Serotonin 
Melatonin  Indole-3-acetic acid  Calcium  Tissue culture
Abbreviations
EGTA (Ethylene glycol-bis (b-amino ethylether)-N, N,
N0 , N0 -tetra acetic acid)
IAA Indole-3-acetic acid
KN Kinetin
MEL Melatonin
MS Murashige and Skoog
p-CPA p- chlorophenylalanine
SER Serotonin
TBST Tris buffer saline Tween 20
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Introduction
Indoleamines, such as melatonin (N-acetyl-5-methoxy-
tryptamine; MEL) and serotonin (5-hydroxytryptamine;
SER), which are pineal hormones in mammals, are also
reported in various genera of plants, particularly in the
edible portions (Huang and Mazza 2011; Paredes et al. 2009;
Manchester et al. 2000). The specific role of MEL and SER
in higher plants, remains in the dark (Van Tassel and Oneill
2001). MEL, first identified in edible plants (Dubbels et al.
1995) has been subsequently found in many other plants
(Murch et al. 2000; Manchester et al. 2000; Ramakrishna
et al. 2009). Inordinately high levels of MEL have been
reported in several herbs including St. John’s wort (Hyper-
icum perforatum) (Murch et al. 1997). Similarly, fruits and
seeds are the major tissues in which SER and MEL occur
abundantly. SER has been reported in more than 42 species
of plants (Roshchina 2001), since its first report in the
legume Mucuna pruriens (Bowden et al. 1954). There are
only few reports on their physiological role as growth pro-
moting molecules in plants (Hernandez-Ruiz et al. 2005;
Murch and Saxena 2002; Chen et al. 2009; Ramakrishna
et al. 2009). Both MEL and SER, structurally related to
Indole-3-acetic acid (IAA), probably have a possible role in
plant morphogenesis (Murch et al. 2001). Exogenously
applied, MEL induces lateral leaflet regeneration and inhi-
bition of flowering (Kolar et al. 2003), stimulates coleoptiles
elongation in barley (Hernandez-Ruiz and Arnao 2008),
induces shoot and root multiplication (Murch et al. 2001;
Ramakrishna et al. 2009) and improves seed germination in
cucumber (Posmyk et al. 2009). MEL also exerts specific
physiological influences such as facilitating fruit ripening
(Van Tassel and Oneill 2001), inhibiting apoptosis during
cold treatment (Lei et al. 2004), and delaying of senescence
(Arnao and Hernandez-Ruiz 2009). Similarly, SER also
shows physiological effect on growth, flowering, xylem sap
exudation, and ion permeability (Kang et al. 2009; Rama-
krishna et al. 2011a). Recently, we have reported the mor-
phogenetic potential of indoleamines with reference to in
vitro shoot multiplication in Mimosa pudica L (Rama-
krishna et al. 2009). The endogenous profiles of both SER
and MEL by HPLC and LCMS in coffee beans (Rama-
krishna et al. 2011b) and different tissues of C. canephora
have also been reported by us (Ramakrishna et al. 2011c).
Attempts have been made to identify the changes in
endogenous indoleamine pools under the influence of a
growth regulator- thidiazuron (TDZ) in tissues of Echinacea
purpurea L (Jones et al. 2007) and similar effects of MEL in
Brassica juncea (Chen et al. 2009). Similarly light/dark
cycle has profound influence on indoleamine profile in
Dunaliella bardawil (Ramakrishna et al. 2011d). Partial
characterization of biosynthetic pathway of MEL and SER
from tryptophan in cultured cells of Hypericum perforatum
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Plant Cell Tiss Organ Cult
L. (Murch et al. 2000) and rice plants (Kang et al. 2007) have
been reported.
Calcium is crucial for plant growth and development
(White 2000), particularly in the initiation of wide array of
signal transduction processes in cells of higher plants (Sudha
and Ravishankar 2002, 2003) including bud formation, polar
growth and hormone-regulated growth as well as develop-
ment (White 2000). Moreover, exogenously administered
calcium and calcium ionophore A23187 enhance in vitro
shoot multiplication and root induction in cultures of
M. pudica (Ramakrishna et al. 2009). In Echinacea purpu-
rea L, enhanced levels of MEL, SER and IAA have been
observed upon activation of calcium channels (Jones et al.
2007). Chemical fluorescent dyes are widely used for Ca2?
imaging in plants in vivo (Walczysko et al. 2000). Fluo-
rescence intensity increases upon the binding of the dye to
intracellular calcium (Zottini and Zannoni 1993). There are
no reports on the influence of SER and MEL on somatic
embryogenesis. Further, no direct link has been established
between the Ca2? and SER/MEL mediated somatic
embryogenesis, in plants. Therefore, the correlation of cal-
cium and MEL/SER mediated somatic embryogenic
response in C. canephora is taken up for investigation.
Materials and methods
Sterilization of coffee seeds, callus induction
and somatic embryogenesis
Freshly harvested seeds of Coffea canephora P ex Fr CxR
variety were procured from Central Coffee Research Sta-
tion, Balehonnur, Chikmagalur District, Karnataka, India.
The seeds were washed and surface sterilized (Kumar et al.
2007). Subsequently seeds were blot dried and inoculated in
200 ml glass jars containing 40 ml solid MS basal medium
(Murashige and Skoog 1962) with 3% (w/v) sucrose and
0.8% agar (w/v). The jars were closed with polypropylene
caps. The pH was adjusted to 5.7 using a pH meter (Cyber
Scan 510, Oakton, USA) prior to autoclaving at 121°C and
1.2 kg cm-2 pressure for 20 min. The cultures were main-
tained at 25 ± 2°C in the dark for 120 days. Leaf explants
(45 days old, 10 mm diameter disks) were cultured with
their abaxial surface in contact with the culture medium i.e.
callus induction medium comprising MS salts and B5 vita-
mins, along with 9.8 lM isopentenyladenine (2-iP), 2.2 lM
2,4-dichloroacetic acid (2, 4-D) (Van Boxtel and Berthouhly
1996). The cultures were maintined in petri dishes, each
containing 20 ml of the callus induction medium. After
75 days, the weight of the culture was *50 mg/culture. The
whole callus was maintained on hormone free medium
(‘ strength MS media) for 30 days. The cultures were
maintained at 25 ± 2°C in the dark for 45 days and the
Plant Cell Tiss Organ Cult

Table 1 Influence of indoleamines and their inhibitors on somatic Table 2 Influence of indoleamines, calcium, calcium ionophore
embryogenesis in C. canephora A23187, verapamil and EGTA on somatic embryogenesis in C.
canephora
Treatment No. of somatic
embryos (mean ± SE) Treatment No. of
somatic
Control (EG medium) 25 ± 3cd embryos
SER* (100 lM) 84 ± 2a (mean ± SE)
MEL* (100 lM) 62 ± 6b
Control (EG medium) 25 ± 3d
SER* (100 lM) ? MEL (100 lM) 75 ± 5ab
SER* 100 lM 85 ± 3bc
MEL* (100 lM) ? p-CPA (40 lM) 32 ± 2c
MEL* 100 lM 62 ± 6c
MEL* (100 lM) ? Prozac (20 lM) 23 ± 4c
Calcium 5 mM 56 ± 4c
SER* (100 lM) ? p-CPA (40 lM) 45 ± 3bc
Calcium ionophore (A23187) 100 lM 118 ± 10b
SER* (100 lM) ? Prozac (20 lM) 40 ± 3bc
MEL* 100 lM ? Calcium 5 mM 78 ± 2bc
p-CPA* (40 lM) 05 ± 1d
MEL* 100 lM ? Calcium ionophore (A23187) 135 ± 8ab
Prozac* (20 lM) 08 ± 1d 100 lM
Values are mean of three determinants SER* 100 lM ? Calcium 5 mM 105 ± 6b
a, b, c, d
values indicate mean values with different alphabets are SER* 100 lM ? Calcium ionophore (A23187) 155 ± 15a
significantly differ at P \ 0.05 100 lM
Control EG medium (MS, B5 vitamins, 11.4 lM IAA and 0.93 lM MEL* 100 lM ? Verapamil 1 mM 34 ± 4cd
KN) SER* 100 lM ? Verapamil 1 mM 40 ± 4cd
* EG medium without IAA Verapamil 1 mM 04 ± 1d
EGTA 100 lM 08 ± 1d
Values are mean of three determinants
resulting calli were yellow and friable. These callus cultures a, b, c, d
values indicate mean values with different alphabets are
were used as inoculum (*150 mg/culture) to study the significantly differ at P \ 0.05
interplay of indoleamines, calcium and IAA. EG (embryo- Control EG medium- (MS, B5 vitamins, 11.4 lM IAA and 0.93 lM
genic) medium comprising MS salts, B5 vitamins (Gamborg KN)
et al. 1968), 11.4 lM IAA and 0.93 lM Kinetin (KN) was * EG medium without IAA
employed. Callus cultures of C. canephora were also treated
with indoleamines, indoleamine inhibitors (p- chlorophe- of the MS culture medium. The cultures were incubated at
nylalanine (p-CPA), fluoexitine hydrochloride (prozac) and 25 ± 2°C in the light 16 h photoperiod 25 lmol m-2 s-1
calcium channel blocker (verapamil hydrochloride) were light intensity provided by white fluorescent tubes (Philips,
added individually. All chemicals were procured from India) and at 55–60% relative humidity. The similar set of
Sigma–Aldrich, Bangalore, India. Treatment details were cultures were also maintained under darkness at 25 ± 2°C.
provided in Tables 1 and 2. The number of somatic embryos After 75 days, the cultures were evaluated to record the
formed in each callus mass and endogenous indoleamines number of embryos per explant and compute the percentage
and IAA levels were recorded. One set of respective cultures of explants with somatic embryos.
were incubated under dark, another set of the treated cultures
were incubated at 25 ± 2°C in the light (16 h photoperiod, Regeneration of plants through somatic embryos
25 lmol m-2 s-1). After 75 days, the number of somatic
embryos produced from each callus was recorded. The conversion of somatic embryos to plantlets was carried
out in medium containing half strength MS salts, 10 mg l-1
Culture media and experimental conditions thiamine hydrochloride, 3.2 mg l-1 pyridoxine hydrochlo-
ride, 0.88 lM BA, 2% (w/v) sucrose, 0.8% agar. The cul-
The media were solidified with 0.8% (w/v) tissue culture tures were incubated for 60 days. The rooting of regenerated
grade agar powder (Himedia Mumbai, India) before adding plantlets was done by culturing the plantlets in a medium
3% sucrose (Himedia Mumbai, India) as carbon source. The containing half strength MS salts, 10 mg l-1 thiamine HCl,
pH of the media was adjusted to 5.7 before autoclaving. The 3.2 mg l-1 pyridoxine HCl, 2% (w/v) sucrose, and 100 lM
media were autoclaved at 121°C, 1.2 kg cm2 pressure for calcium ionophore. The cultures were incubated at
20 min. Each treatment consisted of eight plates containing 25 ± 2°C in the light 16 h photoperiod 25 lmol m-2 s-1
five explants each. The experiment was repeated twice. for 30 days. The rooted plantlets, were transferred to micro-
Callus cultures (*150 mg) were cultured in contact with the pots containing sand & compost mixture (1:2) and main-
culture medium in petri dishes (Torson) each containg 20 ml tained in a greenhouse (80% relative humidity).

123

Immuno localisation of SER in different tissues
of C. canephora
The study of localization of SER in different tissues of
C. canephora using anti-SER antibody was attempted.
Localisation of SER was performed according to the
method of Perrot-Rechenmann and Gadal (1986). C. cane-
phora tissues, including somatic embryos (6 weeks) in
vitro stem (8 weeks), in vitro root (3 weeks) and in vivo
immature fruit (12 weeks) (without feeding of SER/MEL),
were fixed with 2% formaldehyde and 0.5% glutaraldehyde
in 0.025 M phosphate buffer (pH 7.0). Tissue sections were
cut manually and dehydrated by alcohol (15, 30, 50, 70,
90% ethanol and absolute alcohol) and followed by alco-
hol:xylene series (2:1), (1:1), (1:2). Tissue sections were
incubated with primary antibodies—anti-SER antibody (5-
HT) developed in rabbit was procured from Sigma Aldrich,
USA), 1:10000 in TBST (Tris buffer saline Tween 20)
buffer for 1 h at room temperature. The sections washed
thoroughly with TBST buffer and incubated with second-
ary antibody (Goat anti rabbit IgG- Flouroscene Isothio
cyanate conjugated, Bangalore Genei, India) (1:8000 in
TBST) for 1 h were washed thoroughly with TBST buffer.
Sections, covered with cover slip were observed under
fluorescent microscope (Olympus, CKX41).
Quantification of endogenous levels of MEL and SER
Extraction and sample preparation
Extraction of MEL and SER was performed according to
the method of Murch et al. (2000). One gram fresh weight
callus cultures was ground in 1 ml of Tris buffer (1 M
Tris–HCl, pH 8.4) and homogenized in 500 ll of buffer
(0.4 M perchloric acid, 0.05% sodium meta bisulphate,
0.1% ethylenediaminetetraacetic acid (EDTA). Particulate
matter was removed by centrifugation at 12,000 rpm for
15 min. The resulting supernatant was dried with nitrogen
gas and resuspended in 500 ll methanol for HPLC.
HPLC conditions
MEL and SER were separated using C-18 Column
(3.9 9 150 mm, particle size 3 lm) (Ultralab Products,
Bangalore, India). Isocratic buffer (0.1 M sodium acetate,
0.1 M citric acid, 0.5 mM sodium octane sulfonate, 0.15 M
EDTA, adjusted to pH 3.7, 5% methanol) was used as a
mobile phase for the separation of indoleamines. Samples
were injected (20 lL) for HPLC analysis with an isocratic
condition maintained at a flow rate of 1 ml/min. Indole-
amines were monitored using fluorescence detector (FD)
at, kEm 348/E 9 280, (Shimadzu, Japan). The peak iden-
tities of these compounds were confirmed by their retention
123
Plant Cell Tiss Organ Cult
times and characteristic spectra of standard chromato-
grams, recorded with a Shimadzu model LC-10Avp series,
equipped with SPD-10AVP detector. MEL and SER were
quantified from their peak areas in relation to the respective
reference standards. Indoleamine contents were expressed
in lg/g fresh weight. SER and MEL were also analyzed in
embryogenic callus of C. canephora by LC–MS-ESI for
authentication of their presence using the method described
by Cao et al. (2006). The molecular mass of each peak in
LC–MS-ESI was obtained by monitoring the parent
molecular ion 233 and 176 peak of MEL and SER,
respectively coupled with H?, 2H? ions. MEL was
detected in the positive mode as the parent compound with
a molecular mass of 233. SER was detected in the ESI
positive mode with mass of parent compound of 176 (Data
not shown).
Extraction and estimation of endogenous auxin
(Indole 3- acetic acid)
Endogenous IAA was extracted by following a modified
method of Stossel and Venis (1970). One gram of plant
tissue, homogenized completely using liquid nitrogen, was
suspended in 10 ml methanol and filtered through a sin-
tered glass filter (G2) under suction. The residue on the
filtration was reextracted twice each with 10 ml of meth-
anol the total filtrate was evaporated to an aqueous residue
on a rotary evaporator (BUCHI Rotavapor R-205). The
aqueous residue was made alkaline (pH 8.5) by adding
10 ml of cold 0.5 M K2HPO4. The contents were trans-
ferred to a separating funnel and shaken with 10 ml diethyl
ether. After discarding the lipid fraction the pH of the
aqueous layer was adjusted to 3.0 by adding suitable
quantities of phosphoric acid (2.8 M). IAA was finally
extracted 10 ml diethyl ether. The ether was evaporated to
dryness under reduced pressure and the residue was dis-
solved in 2 ml cold distilled methanol. To a known volume
of the IAA extract, dried using nitrogen gas in a clean test
tube, about 0.2 ml of ice-cold trifluoroacetic acid (TFAA)
and acetic anhydride (1:1) reagent mixture was added and
mixed thoroughly. The tubes were incubated in ice for
15 min. IAA content, estimated by recording fluorescence
(excitation at 440 nm and emmision 490 nm) using a
spectro-fluorimeter (Spectro-fluorophotometer, Shimadzu,
RF 5301 PC), was expressed in ng/g f.wt.
Calcium imaging in (in vitro) coffee tissues by Laser
Scanning Confocal Microscope (LSCM)
Coffee (in vitro) somatic embryos were used for calcium
imaging studies. Single cells were isolated according to
Aziz et al. (2006). Coffee (in vitro) grown somatic embryos
(6 weeks) (*200 mg) were macerated with 0.5%
Plant Cell Tiss Organ Cult

macerozyme R10 w/v, 1.5% cellulose Onozuka R-10, 16%
mannitol at pH 5.7. The isolated single cells of C. cane-
phora were loaded with 5 lM Fura-2AM as reported by
Anil and Rao (2000). Single cells were incubated with
buffer (MS ? 2% v/v mannitol ? Ca2? fluorescent dye,
5 lM Fura-2AM (dissolved in DMSO) for 1 h at room
temperature. Fluorescent intensity was analysed using the
quantitation mode in LAS-AF software from Leica
Microsystems, Mannheim, Germany. A region of interest
(ROI) was drawn within a field of view. For each ROI, the
pixel intensity (fluorescence intensity) was calculated to
analyze the intracellular Ca2? in the single cell.
Statistical analysis
Each experiment consisted of eight plates containing five
explants each. The experiment was conducted thrice. The
data were analyzed by one-way analysis of variance
(ANOVA) using Microsoft Excel XP (Microsoft Corpora-
tion, Seattle WA.USA), and the mean separations were
performed by Duncan’s Multiple Range Test (DMRT) at
P \ 0.05 (Harter 1960).
Results
Immunolocalisation of SER in different tissues
of C. canephora
Results of the present study indicated that the localisation
of SER in different tissues of Coffea could be confirmed by
fluorescence immunolocalization technique. SER was
found in the vascular tissues of stem, root, somatic
embryos and endocarp region (husk) of the immature fruits.
SER was strongly stained in vascular tissues of stem and
root compared to somatic embryos (Fig. 1). SER

Fig. 1 Immunolocalization of
serotonin in different tissues of
C. canephora using anti-
serotonin antibody. a control
(stem), b control (root),
c Localization of SER in
vascular tissues of the C.
canephora in in vitro stem
(Magnification 409), d In vitro
root (409), e in vitro somatic
embryos (1009), f Endocarp
region (husk) of immature fruit
(1009)

123

localization studies could be useful to elucidate cellular
signalling mechanism of SER in plants.
Influence of indoleamines and their inhibitors
on somatic embryogenesis
Yellow friable callus cultures, incubated in the dark on EG
(embryogenic) medium (MS medium with 11.4 lM IAA,
0.93 lM K and 100 mg/l meso- inositol, 3% sucrose (w/v),
produced only 25 ± 3 embryos (Table 1). Out of the var-
ious concentrations of MEL or SER (each 50, 100,
Fig. 2 Influence of SER, MEL and their inhibitors (p-CPA and
prozac) on somatic embryo induction in C. canephora under dark and
light conditions. a Formation of yellow friable callus cultures of
C. canephora from hypocotyls and leaf explants (Bar 10 mm) under
dark, b Induction of somatic embryos by 100 lM SER under dark
123
Plant Cell Tiss Organ Cult
250 lM) tried in preliminary experiments, it was found
that 100 lM of MEL or SER is optimal for induction of
somatic embryogenesis in C. canephora. EG medium
(without IAA) supplemented with 100 lM SER, induced
84 ± 3 embryos from each callus (Table 1; Fig. 2b) with
concomitant enhancement of endogenous pools of SER
(65.5 lg/gf. wt.) and IAA (42 ng/gf.wt.) increased the
induction of embryos 1.6 and 2.6 folds, respectively
(Fig. 3a,b). Similarly, incorporation of MEL into EG
medium produced 62 ± 6 embryos (Table 1; Fig. 3c).
MEL fed callus cultures exhibited enhanced endogenous
(Bar 5 mm), c Somatic embryos induction by 100 lM MEL under
dark (Bar 10 mm), d Suppression of somatic embryo induction by
40 lM p-CPA (Bar 10 mm), e Induction of somatic embryos by
100 lM SER under light, f Formation of somatic embryos by 100 lM
MEL under light
Plant Cell Tiss Organ Cult

a
a b a

ab
bc cd
ab ab c d d
d
c d c d c
d d

c
a
d a
bc ab ab
ab c
c c
cd
cd cd cd d d
d d
de

e a

b f a
bc bc
a a bc b ab
bc
ab ab ab
ab ab c
bc cd bc
cd d d d
cd d

g
a
b
h a
bc ab b
b bc bc ab
ab bc bc bc c c
cd
d d d d cd d
d d d d

Fig. 3 Endogenous indoleamines and IAA content under the exog-
enous feeding of indoleamines (SER and MEL) and their inhibitors
(p-CPA and prozac); calcium, calcium ionophore A23187 and their
inhibitors (Verapamil and EGTA). a Endogenous MEL and SER
content under exogenous feeding of SER and indoleamine inhibitors
(p-CPA and prozac), b Endogenous IAA content under the influence
of exogenous feeding of SER and indoleamine inhibitors, c Endog-
enous levels of indoleamines under the exogenous feeding of MEL
and indoleamine inhibitors d Endogenous IAA content under the
exogenous feeding of MEL and indoleamine inhibitors, e Endogenous
levels of indoleamines under the exogenous feeding of SER and
calcium channel inhibitors, f Endogenous IAA content under the
exogenous feeding of SER and calcium channel inhibitors, g Endog-
enous MEL and SER content under exogenous feeding of MEL and
calcium and calcium channel inhibitors, h Endogenous IAA content
under the influence of exogenous feeding of MEL, calcium and
calcium channel inhibitors. (Values are expressed as mean – SE,
where each treatment possesses eight plates containing five explants
each. The experiment was conducted twice. Values with different
letters are significantly different at P \ 0.05 by DMRT)

123

MEL (48.5 lg/gf.wt.) (Fig. 3c) and a concomitant increase
of IAA content (38.40 ng/gf.wt.) (Fig. 3d). Incorporation
of SER—MEL conversion inhibitor (40 lM p-CPA) into
EG medium devoid of IAA led to a drastic reduction in
somatic embryo production; only 5 ± 1 somatic embryos
were produced (Table 1). Apart from this reduction in the
endogenous pools of MEL (4.0 lg/gf.wt.) and SER
(9.5 lg/gf.wt.) also reduced by *7, 2.2 folds, respectively
(Fig. 3c), while reducing IAA (10 ng/gf.wt.) content was
by 1.3 folds (Fig. 3d). Similarly, incorporation of 20 lM
prozac (SER reuptake inhibitor) into the medium induced
only 8 embryos, and the endogenous pools of MEL and
SER (Fig. 3c) and the IAA content (Fig. 3d) were reduced
by *2.4, 9 and 1.6 folds, respectively.
Callus cultures, treated with indoleamines, when incu-
bated under light conditions also induced somatic embryos.
Only 50 ± 4 and 25 ± 2 embryos were obtained in pres-
ence of 100 lM SER/MEL on EG medium in presence of
light which is less than that of cultures incubated under
Fig. 4 Effect of indoleamines, calcium and calcium ionophore
(A23187) on somatic embryogenesis in in vitro cultures of C.
canephora under dark conditions a 100 lM MEL 1 5 mM calcium
(Bar 10 mm), b 100 lM calcium ionophore A23187 ? 100 lM
123
Plant Cell Tiss Organ Cult
dark (Fig. 2e, f). Callus cultures treated with indoleamines
and calcium also raised endogenous SER, MEL and IAA
content by 2.5, 2, and 3 folds, respectively; whereas, cul-
tures treated with indoleamine inhibitors and calcium
channel inhibitors, reduced the endogenous pools of MEL,
SER and IAA content by *2, 5 and 1 fold, respectively
(Data not shown).
Influence of calcium and calcium ionophore A23187
on somatic embryogenesis
Supplementation of 5 mM calcium to EG medium pro-
duced 56 ± 4 embryos (Table 2; Fig. 4a) with increase in
endogenous MEL (29 lg/gf.wt.) and SER (38.6 lg/gf.wt.)
(Fig. 3e) without affecting IAA levels; furthermore, addi-
tion of 100 lM calcium ionophore A23187 induced more
number of somatic embryos (* 118 ± 10) (Fig. 4c).
Endogenous SER and MEL levels (Fig. 3e) and IAA levels
were not affected in these embryos (Fig. 3 e,f).
verapamil (Bar 10 mm), c 100 lM SER 1 100 lM calcium iono-
phore A23187 (Bar 5 mm), d In vitro rooting by 100 lM calcium
ionophore A23187 (Bar 30 mm)
Plant Cell Tiss Organ Cult
Influence of calcium channel inhibitors in C. canephora
To satisfy our curiosity to understand if calcium has any
role in somatic embryogesis. EG medium was supple-
mented with calcium channel inhibitors including 1 mM
verapamil hydrochloride or 100 lM EGTA. A significant
reduction in somatic embryo induction was observed
(Fig. 4b). EGTA treatment reduced the endogenous pools
of SER (10.2 lg/gf.wt.), MEL (6.2 lg/gf.wt.) and that of
IAA (14 ng/gf.wt.) (Fig. 3g, h). Similarly, verapamil
hydrochloride treatment also reduced SER (6.2 lg/gf.wt.),
MEL (8.2 lg/gf.wt.) and IAA levels (14.5 ng/gf.wt.).
Influence of calcium and calcium ionophore A23187
on somatic embryogenesis in C. canphora
under the influence of SER and MEL
EG medium, supplemented with 100 lM SER and 5 mM
CaCl2, induced 105 ± 6 embryos from each callus by
increasing endogenous MEL (32.5 lg/gf.wt.), SER
(57.4 lg/gf.wt.) (Table 2; Fig. 3e). Addition of 100 lM
calcium ionophore and 100 lM SER induced 155 ± 15
somatic embryos per culture and augmented the endoge-
nous MEL (35 lg/gf.wt.; Fig. 3e) and IAA (50 ng/gf.wt.)
levels (Fig. 3f). Similarly, 100 lM MEL and 5 mM CaCl2
treatment produced 78 ± 2 embryos from each callus
(Table 2). A concomitant increase of endogenous levels of
SER (57.4 lg/gf.wt.), MEL (32.5 lg/gf.wt.) was also
observed (Fig. 3g). Similarly, incorporation of MEL and
calcium ionophore A23187 produced 135 ± 8 embryos
along with a slight increase in endogenous profiles of MEL
(32.5 lg/gf.wt.) and SER (41.5 lg/gf.wt.; Fig. 3g).
Regeneration of somatic embryos to plantlets
Somatic embryos, subcultured on ‘ strength MS medium
comprising 0.25 mg l-1 BA with 100 lM SER/MEL
(without IAA) developed into plantlets in 4 weeks. The
percentage of conversion from embryos to plantlets was
50–70%. All the regenerated plants appeared to be mor-
phologically normal (Fig. 4d).
For investigating the effect of calcium on somatic
embryogenesis, imaging of calcium was done for in vitro
callus cultures of C. canephora treated with calcium and
calcium ionophore, calcium chelator (EGTA), calcium
channel blocker verapamil hydrochloride, and also in-
doleamines including SER and MEL at 100 lM and indole
inhibitor p-CPA at 40 lM. High fluorescence intensity was
recorded in cultures treated with calcium (1223656), cal-
cium ionophore A23187 (2099579). SER (2074875) and
MEL (1067922) treated cultures also recorded high fluo-
rescence intensity compared to control (897658),while low
fluorescence intensity was recorded in cultures treated with
Table 3 Calcium imaging of C. canephora in vitro tissues treated
with calcium, calcium ionophore, indoleamines and calcium channel
inhibitors
Treatment Fluorescence Relative
intensity fluorescence
intensity
Control (EG medium) 897,658 Nil
Calcium 5 mM 1,22,3656 1.3
Calcium ionophore (A23187) 100 lM 2,099,579 2.3
Melatonin* 100 lM 1,067,922 1.1
Serotonin* 100 lM 2,074,877 2.3
p- Chloro phenyl alanine (p-CPA)* 116,281 0.13
40 lM
Verapamil hydrochloride 1 mM 749,889 0.8
Control: EG medium- (MS, B5 vitamins, 11.4 lM IAA and 0.93 lM
KN)
*EG medium without IAA
either verapamil hydrochloride (749889) or p-CPA
(116281) (Table 3).
Discussion
In the present study, specific fluorescence of SER was
found in the vascular tissues of the stem, root and somatic
embryos as well as in the endocarp region (husk) of
immature coffee fruits. Studies by Kimura (1968) reported,
SER and catecholamines distribution in Musa paradisica L
(banana), Musa basjoo (plantain) and Allium cepa L
(onion) employing fluorescence histochemical studies. The
present study demonstrated this phenomenon by advanced
methodology. SER localization studies could be useful to
elucidate cellular signaling mechanism of SER in plants.
MEL/SER incorporation into the medium resulted in the
formation of somatic embryos along with elevated endog-
enous pools of indoleamines and IAA, indicating that
exogenous SER/MEL alters the endogenous levels of SER,
MEL and IAA levels, thereby inducing somatic embryo-
genesis. MEL was reported to promote the vegetative
growth in lupin (Lupinus albus L.) hypocotyls, coleoptiles
of wheat, barley, canarygrass and oat in a similar manner to
IAA (Hernandez-Ruiz et al. 2004). Chemical structure of
indoleamine strating from tryptophan suggested that, SER
and MEL might mimic IAA like activity (Van Tassel and
Oneill 2001; Hernandez-Ruiz et al. 2005; Ramakrishna
et al. 2009). Incorporation of p-CPA into the culture
medium inhibited not only somatic embryogenesis but also
reduced MEL and SER content. Hence, it was hypothe-
sized that p-CPA inhibitory role in SER to MEL conver-
sion, may cause suppression of somatic embryo induction.
Our earlier report showed that indoleamines (SER and
123

MEL), calcium and calcium ionophore A23187 induce
morphogenesis in in vitro cultures of Mimosa pudica,
whereas, verapamil hydrochloride and EGTA significantly
could suppress shoot multiplication and root induction
(Ramakrishna et al. 2009).
Elevation in calcium concentration is reported to reduce
somatic embryogenesis potential in Hevea cells (Montoro
et al. 1995). Further, studies by Sudha and Ravishankar
(2002) have demonstrated the significance of Ca2? on
secondary metabolites production in Capsicum. Similarly,
the calcium ionophore A23187 elicits anthocyanin in callus
cultures of D. carota (Sudha and Ravishankar 2003).
Calcium ionophore A23187 stimulates cytokinin produc-
tion and is involved in mitosis leading to bud formation in
Funaria hygrometrica by increasing the intracellular cal-
cium levels (Saunders and Helper 1982). In tobacco cells,
the calcium ionophore A23187 is known to enhance
intracellular Ca2? levels (Chandra and Low 1997). In the
present study, incorporation of CaCl2 to the EG medium
along with MEL/SER is found to induce more number of
somatic embryos in C. canephora. Earlier reports have
suggested that, increase in cytosolic calcium triggers
numerous cellular processes by modulation of protein
kinases, ion channels and other cellular proteins (White
2000). Calcium imaging technique is useful to measure
cytoplasmic calcium levels in plant cells using confocal
microscopy (Clarakson et al. 1988). The fluorescence
probes i.e. Fura-2 and indo-1 have been developed for
intracellular calcium measurements in root hairs of Bras-
sica napus (Clarakson et al. 1988). Thus the changes in
calcium level may elicit morphogenetic response in C.
canephora. In this study, calcium, calcium ionophore
A23187 and indoleamines treated cultures have recorded
high fluorescence intensity; whereas, low fluorescence
intensity has been recorded when the cultures are treated
with indoleamine inhibitors and calcium channel inhibitors.
In this study, in vitro grown C. canephora plants, sup-
plemented with 5 mM calcium and 100 lM calcium ion-
ophore A23187, induced rooting, whereas supplementation
of calcium channel blocker effectively reduce root induc-
tion. Similarly, calcium channel blockers have been
administered to study the involvement of calcium during
the MEL and SER mediated somatic embryogenesis. Cal-
cium channel inhibitors are reported to bind to plant
membranes and block Ca2? entry into protoplast (Graziana
et al. 1988) and pollen tubes (Obermeyer and Weisenseel
1991). Results of this study suggest that elevation in the
cytoplasmic calcium levels and endogenous levels of MEL
and SER is led to enhancement of somatic embryogenesis.
Another important finding, in this study is, the massive
production of somatic embryos from yellow friable callus
cultured on EG medium supplemented with 100 lM cal-
cium ionophore A23187. Somatic embryogenesis has been
123
Plant Cell Tiss Organ Cult
achieved under the influence of BA or KN in combination
with IAA in many plants viz, C. arabica (Menendez-Yuffa
et al. 2010), Pulsatilla koreana Nakai (Lin et al. 2011),
Ochna integerrima (Ma et al. 2011), Desmodium motorium
(Chitra Devi and Narmathabai 2011), Crambe abyssinica
(Don Palmer and Keller 2011), Dendrocalamus hamiltoni
(Zhang et al. 2011), Schisandra chinensis (Chen et al.
2010) and Colocasia esculenta (Deo et al. 2010). In the
present study, IAA and KN have been used during initial
stage of embryogenesis. Subsequently, BA is used along
with indoleamines MEL/SER to obtain regeneration on
medium devoid of IAA. This response may be due to auxin
like activity of indoleamines (Hernandez-Ruiz et al. 2004;
Ramakrishna et al. 2009; Murch et al. 2001). In the present
study, we have found that the induction of somatic
embryogenesis is more effective under the influence of
darkness than in presence of light (16 h photoperiod). Light
quality and intensity are found to affect the growth and
development of somatic embryos (Takanori and Cuello
2005). The effect of light quality on somatic embryogen-
esis has also been reported in Agave tequilana Weber var.
Azul (Rodriguez-Sahagum et al. 2010).
In conclusion, exogenous indoleamines and calcium are
found to induce somatic embryogesis in C. canephora. The
synergistic effect of indoleamines and calcium ionophore
A23187 more effectively drives cellular machinery
towards embryogenesis. Further investigation of the role of
these compounds in plant organogenesis are likely to be of
great significance. The data from this study may further
help to realize the multiple roles of the indoleamines in
plant morphogenesis and somatic embryogenesis. In future,
it may be helpful for elucidating MEL and SER dependent
cellular signaling mechanisms in plants.
Acknowledgments The authors are thankful to Department of
Science & Technology, New Delhi, India for financial assistance.
Mr.A.Ramakrishna acknowledges CSIR, New Delhi for awarding
Senior Research Fellowship. Authors thank Mr.P.S.Kulashekar for his
help in manuscript preparation.
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