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ORIGINAL PAPER
Elicitor-induced rosmarinic acid accumulation and secondarymetabolism enzyme activities in Solenostemon scutellarioides
Ranabir Sahu • Moumita Gangopadhyay •
Saikat Dewanjee
Received: 6 October 2012 / Revised: 5 December 2012 / Accepted: 11 December 2012 / Published online: 25 December 2012
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012
Abstract This study aimed to improve rosmarinic acid
(RA) production in the whole plant culture of Solenoste-
mon scutellarioides through elicitation. Amongst selected
elicitors methyl jasmonate (MJ), salicylic acid (SA), and
yeast extract (YE) caused significant elevation in RA
accumulation. Elicitation with MJ (50 lM) and SA
(50 lM) caused almost 1.7 and 1.4-fold increase in RA
accumulation, respectively, within day 1. While YE
(100 lg ml-1) elicitation showed highest RA content
(*1.5-fold) in day 3. Preceding the elicitor-induced RA
accumulation, there was a notable alteration in the specific
activities of RA biosynthetic enzymes viz. phenylalanine
ammonia lyase, tyrosine aminotransferase, hydroxyl-phe-
nylpyruvate reductase and rosmarinic acid synthase up on
MJ (50 lM), SA (50 lM) and YE (100 mg ml-1) elicita-
tion. Based on differential responses of aforementioned
enzymes, RA synthesis was further scaled up through
combination of elicitors in pre-optimized doses. In synergy
study, at a time exposure with MJ ? SA ? YE and
MJ ? SA followed by YE after 24 h has been found to
produce significant elevation of RA (2.0 and 1.9-fold,
respectively) within 24 h while later maintained a steady
state increased level (*1.7 ± 0.2-fold) over control up to
day 7.
Keywords Rosmarinic acid � Solenostemon
scutellarioides � Elicitors � Enzyme activity � Biosynthetic
pathway
Abbreviations
BAP 6-benzylaminopurine
GA3 Gibberelic acid
HPR Hydroxyl-phenylpyruvate reductase
IBA Indole-3-butyric acid
MJ Methyl jasmonate
MS Murashige and Skoog
PAL Phenylalanine ammonia lyase
RA Rosmarinic acid
RAS Rosmarinic acid synthase
SA Salicylic acid
TAT Tyrosine aminotransferase
YE Yeast extract
Introduction
Rosmarinic acid (RA), a-O-caffeoyl-30, 40-dihydroxyphe-
nyllactic acid, is a well-known hydroxycinnamic acid ester
that occurs as a principle chemical marker in Solenostemon
scutellarioides (L.) Codd. (Trute and Nahrstedt 1996;
Petersen et al. 1993). RA is a natural polyphenol, which
exhibits various pharmacological activities including anti-
oxidant, antibacterial, antiviral, anti-inflammatory and anti-
allergic action (Yang and Shetty 1998; Hras et al. 2000;
Sanbongi et al. 2004). The antioxidant activity of RA has
been reported to be stronger than even that of vitamin E
Communicated by J. V. Jorrin-Novo.
R. Sahu (&) � M. Gangopadhyay � S. Dewanjee
Advanced Pharmacognosy Research Laboratory,
Department of Pharmaceutical Technology, Jadavpur University,
Raja S C Mullik Road, Kolkata 700032,
West Bengal, India
e-mail: [email protected]
Present Address:M. Gangopadhyay
Genecology Group, Faculty of Science, Health,
Education and Engineering, University of the Sunshine Coast,
Maroochydore, Queensland, DC 4558, Australia
123
Acta Physiol Plant (2013) 35:1473–1481
DOI 10.1007/s11738-012-1188-3
(Park et al. 2008). The antioxidant activity of RA plays
important roles in adsorbing and neutralizing free radicals,
quenching singlet and triplet oxygen or decomposing per-
oxides (Furtado et al. 2008), and thereby reducing the risk
for cancer, atherosclerosis and other diseases associated
with augmented oxidative stress (Park et al. 2008). Due to
wide range of pharmacological attributes, RA is considered
as a commercially important product for the pharmaceutical
and cosmetic industries (Georgiev et al. 2007; Sanchez-
Medina et al. 2007). Because of the aforementioned phar-
macological activities, RA is assumed to participate in the
plant defense mechanism against different stress factors
(Petersen and Simmonds 2003). Over last few decades, the
applications of plant biotechnology have contributed a
renewable source of various important plant secondary
products (Zhou and Wu 2006). It offers alternatives to
classical approach for the production of plant-based
metabolites independent to chemical polymorphism (Pere-
ira et al. 2003) caused by geo-climatic variations. In addi-
tion, tissue culture technology provides many exclusive
advantages such as the optimization of the synthesis and
accumulation of metabolites through manipulation of cul-
ture conditions and elicitation (Gangopadhyay et al. 2011a;
Pitta-Alvarez et al. 2000; Komaraiah et al. 2002; Gango-
padhyay et al. 2011b). Elicitors are signal compounds
which induce or enhance the biosynthesis of metabolites by
activating the pathways in response to exogenous stresses
(Kim et al. 2004; Kuzmaa et al. 2009; Chong et al. 2005).
The defensive functions of RA within plants against natural
stresses encouraged investigating the influence of several
exogenous elicitors on its production in in vitro cultures of
S. scutellarioides. However, the effect of elicitors on the
accumulation of RA has not been described for whole plant
cultures of S. scutellarioides despite, the whole plant pro-
duces RA. The synthetic capacity of dedifferentiated tissue
often differs from fully differentiated tissues due to the
organ-specific expression of biosynthetic genes which
regulates enzyme activities for product formation. In
addition, genetic instability and heterogeneity in suspen-
sion culture is greatly reduced through tissue culture based
on clonal propagation which offers as an efficient, alter-
native to raise more genetically stable cultures essential for
maintaining quality and quantity of phytochemicals. The
present study was undertaken to enhance RA production in
in vitro derived S. scutellarioides under the influence of
different elicitors. Since the biosynthetic capacity of
metabolites is dependent on specific expression regulating
enzymes’ activities in biosynthetic pathways, it was further
aimed to find out their role of elicitors on the activities of
regulating enzymes in the biosynthesis of RA. Based on the
activities of enzymes upon elicitation, it was further aimed
to scale up RA production employing synergy between
elicitors.
Experimental
Plant material
In vitro regenerated S. scutellarioides established in
Advanced Pharmacognosy Research Laboratory, Depart-
ment of Pharmaceutical Technology, Jadavpur University,
India, was used as source material for this experiment. The
nodal segments were excised from well maintained in vitro
grown S. scutellarioides (Fig. 1a, clone S 21) and cultured
as per the pre-established protocol (Sahu and Dewanjee
2012). Briefly, the explants showing axillary buds (Fig. 1b)
after 1 week in a hormone-free solid MS (Murashige and
Skoog 1962) media were cultured in MS media containing
GA3 ? BAP (0.5 mg dm-3, each) for shoot development
(Fig. 1c). After sufficient healthy shoot development the
in vitro regenerated shoots were cultured with IBA
(1.0 mg dm-3) for rooting (Fig. 1d). The plants were
allowed to grow up to 8 weeks at 22 ± 2 �C and 16 h
photoperiod (light intensity 100 lmol m-2 s-1 provided
by cool white fluorescent tubes, Philips, India). Eight-
week-old, in vitro grown whole plants were subjected to
elicitation studies.
Preparation of elicitors
MJ and SA procured from Sigma-Aldrich, USA. MJ and
SA solution was prepared by dissolving in 70 % ethanol
and filtered through a syringe filter (25 lm, Gelman sci-
ences, USA) and then added to autoclaved media asepti-
cally in desired concentrations (Wiktorowska et al. 2010).
YE, the polysaccharide fraction isolated from Saccharo-
myces cerevisiae, was prepared by ethanol precipitation
method (Chena et al. 2001) with little modification. Briefly,
50 g of the yeast extract was dissolved in 250 ml double
distilled water. Ethanol was added to 80 % (v/v). After
incubation at 6 �C for 4 days, the precipitate was collected.
The process was repeated thrice and the total precipitate
was dissolved in 200 ml double distilled water, yielding the
crude preparation which was lyophilized to powdered yeast
carbohydrate fraction. Chloride salts of Ag, Cu, Ca, Mn,
and Zn were used for the elicitation. Stock solutions were
prepared separately by dissolving in autoclaved distilled
water. The pH of individual salt solution was adjusted to
5.6 before autoclaving. The solutions were autoclaved for
15 min at 120 �C and 15 psi.
Application of elicitors
Eight-week-old in vitro regenerated whole plants were
collected from the flasks and aseptically transferred to hor-
mone-free solid MS medium gelled with 0.8 % Bacto agar
(Hi-media, India) supplemented different concentrations of
1474 Acta Physiol Plant (2013) 35:1473–1481
123
selected elicitors viz. MJ (10, 25, 50, and 100 lM), YE (10,
25, 50, and 100 lg ml-1), SA (10, 25, 50, and 100 lM), and
inorganic salts (10, 25, 50 and 100 lM). A set of culture
without elicitor were served as control. Intracellular RA
content was estimated at 0 h followed by day 1, 3, and 7.
Extraction and quantification of RA
The plants were collected from culture flask and the root
portion was washed properly with autoclaved distilled
water for removing adhering agar. The plants were soaked
on blotting paper and then immersed in liquid nitrogen and
pulverized thoroughly in a mortar and pestle pre-cooled
with liquid nitrogen. The powdered plant tissues were
extracted with methanol in an ultrasonic bath at 70 �C for
20 min. The cellular residues were settled at 300 g for
10 min. The supernatant was separated and dried under
vacuum. The quantification of RA content (mg 100 g-1
FW) was done in Dionex Ultimate 3000 HPLC system
(Dionex, Germany), using a reverse phase C-18 column
(250 9 4.6 mm, particle size 5 l) by Acclaim 120,
Germany and UV detector. The standard stock solution of
RA (Sigma-Aldrich, USA) was prepared at 1 mg ml-1 in
HPLC grade methanol. The test solution was prepared by
dissolving dried crude extract in HPLC grade methanol
to give 1 mg ml-1 stock solution. All solutions were fil-
tered through cellulose nylon membrane filter (0.45 lm)
(PALL, Life Sciences), and eluted with isocratic solvent
mixture comprising methanol: water: o-phosphoric acid
(49.5:50.0:0.5, v/v/v) with the flow rate at 1 ml min-1 and
detected at 325 nm. All of the above experiments were
replicated three times each.
The elicitors which resulted significant elevation of
intracellular RA accumulation were subjected further to
find out their roles at optimized concentrations on enzymes
involved in biosynthesis of RA with respect to intracellular
RA accumulation in hour basis up to 7 days i.e., 168 h.
Protein extraction
Fresh plant tissues were homogenized three times for 30 s
in a homogenizer with 1 ml 0.1 M potassium phosphate
buffer pH 7.0 with 1 mM dithiothreitol. The homogenate
was centrifuged at 3,000g for 20 min at 4 �C. The super-
natant was used to determine enzyme activities. Protein
concentration was determined according to Bradford (1976)
with bovine serum albumin (1 mg ml-1) as a standard.
Determination of enzyme activities
The activities of enzymes involved in the biosynthesis of
RA (Fig. 2) viz. PAL, TAT, HPR, and RAS were deter-
mined as described by Petersen et al. (1993), De-Eknamkul
and Ellis (1987); Hausler et al. (1991); Petersen (1991),
respectively. Values are expressed as specific activities
(lkat kg-1 protein).
Determination of synergistic effect of elicitors
To study the synergistic effect of elicitors, in vitro regen-
erated S. scutellarioides were elicited with selected elicitors
(based on the specific activities of enzymes involved in
RA biosynthesis) in combination of application, namely
MJ (50 lM) ? SA (50 lM) ? YE (100 lg ml-1), MJ
(50 lM) ? YE (100 lg ml-1) followed by SA (50 lM)
after 24 h, and MJ (50 lM) ? SA (50 lM) followed by and
YE (100 lg ml-1) after 24 h. Intracellular RA accumulation
was measured in hour basis up to 7 days. This study was
conducted in semisolid media (0.4 % Bacto agar) for easy
imbibitions of elicitors applied after 24 h of initial elicitation.
Fig. 1 Different stages of micropropagation of S. scutellarioides(a–d): a In vitro regenerated S. scutellarioides as source material for
this study, b sprouting of shoot buds excised from in vitro mother
plant clone S-21, c initiation of shoot regeneration, d well established
in vitro culture for elicitation studies
Acta Physiol Plant (2013) 35:1473–1481 1475
123
Data analysis
Three replicates were made for each experimental set.
Variability in data was expressed as the mean ± standard
deviation followed by Dunnett’s t test using computer-
ized GraphPad InStat version 3.05, Graph pad software,
USA. The values were considered significant when
p \ 0.05.
N H 2
C O O HH O
C O O H
H O O
C O O H
O HH O
C O O H
N H 2
C O O H
C O O H
H O
H O
O
O
C O O H
O H
H O
O
O
C O O H
H O
O
O
C O O H
O H
H O
H O
O H
H O
O
O
C O O H
O H
H O
H O
T A T
P A L
H P R
C 4 H
R A S
3 'H ?3 H (C Y P 8 9 8 A 6 )
3 'H 3 H (C Y P 9 8 A 6 ? )
C O S C o A
H O
4 C L
R A
T y r
4 -O H -p h e n y lp y ru v ic a cid
4 -O H -p h en y l-la ctic a c id
P h e
C in n a m ic a c id
4 -O H -cin n a m ic a c id
C H P L
C a H P L C o D P L
4 -O H -cin n a m in y l-C o A
Fig. 2 The metabolic pathway
leading to RA (Ogata et al.
2004). Tyr tyrosine, Phephenylalanine, TAT tyrosine
aminotransferase, PALphenylalanine ammonia lyase,
HPR 4-hydroxyl-
phenylpyruvate reductase, C4Hcinnamic acid 4-hydroxylase,
4CL 4-coumaric acid:CoA
ligase, RAS rosmarinic acid
synthase, 3H 4-coumaroyl-40-hydroxyphenyllactic acid
3-hydroxylase, 30H caffeoyl-
40-hydroxyphenyllactic acid
30-hydroxylase, CHPL4-coumaroyl-
40-hydroxyphenyllactic acid,
CaHPL caffeoyl-
40-hydroxyphenyllactic acid
1476 Acta Physiol Plant (2013) 35:1473–1481
123
Results and discussion
Variation in elicitors and concentrations
on production of RA
The quantification of RA was performed by HPLC anal-
ysis and the values were expressed as (mg 100 g-1 FW).
Figure 3 depicted the HPLC chromatogram of standard
RA and extract. Elicitation with MJ, SA, and YE has been
shown to exert positive effect in intracellular RA accu-
mulation in in vitro cultures of S. scutellarioides
(Table 1). However, the effects of aforementioned elicitors
were different in terms of concentration and exposure time
of elicitation to achieve maximum accumulation of RA. A
significant increase in RA accumulation (*1.2–1.7-fold)
was noticed between 25 and 100 lM of MJ. Accumulation
of RA has been found to be highest in day 1 after MJ
elicitation in all variants. The RA content gradually
decreased with time, but remains significantly higher than
control up to 7 days after elicitation. Elicitation with
50 lM of MJ found to be most effective and intracellular
RA content reached almost 1.7-fold higher in day 1 as
compared to that of control culture. YE was found to be
significantly effective between 50 and 100 lg ml-1 and
RA content remained nearly constant between day 1 and 3.
Maximum RA concentration was achieved at the dose of
100 lg ml-1 in day 3, which was nearly 1.5-fold than
control. RA content within tissues was reduced in day 7.
SA exhibited significant intracellular RA accumulation
between 50 and 100 lM concentrations. Maximum RA
concentration was found within 24 h after SA elicitation
and remained almost similar up to day 3. A maximum
accumulation of RA (*1.4-fold) was obtained at 50 lM
of SA in day 1. RA content in all variants was found to be
reduced after day 3 of elicitation. Elicitation with inor-
ganic salts did not show significant alteration in intracel-
lular RA accumulation in S. scutellarioides cultures
(Table 2) except AgCl, which caused a significant increase
in RA production in day 7 at the dose of 100 lM. Chlo-
ride salts of Cu and Zn exhibited negative response to RA
production.
Time course of RA accumulation and activities
of enzymes involved in RA biosynthesis
Amongst all of selected elicitors, MJ, SA, and YE at the
doses 50 lM, 50 lM, and 100 lg ml-1 have been found to
produce significant improvement in intracellular accumu-
lation of RA in S. scutellarioides. RA biosynthesis in plants
has been suggested to involve both the phenylpropanoid
pathway (for the 4-OH-cinnamic acid moiety) and a tyro-
sine-derived pathway (for the 4-OH-phenyl-lactic acid
moiety) as depicted by Fig. 2. Eight enzymes involved in
RA biosynthesis have been identified and characterized and
amongst them PAL, TAT, HPR, and RAS are categorized
as principle enzymes involved and regulate RA biosyn-
thesis in suspension culture of S. scutellarioides (Petersen
and Metzger 1993; Szabo et al. 1999). PAL is a key
enzyme at the entrance of the phenylpropanoid pathway
(Chevolot et al. 1998); TAT is the first enzyme in the
tyrosine-derived pathway (Petersen and Metzger 1993),
HPR catalyzes the conversion of 4-OH-phenyl-pyruvic
acid to 4-OH-phenyl-lactic acid (Ogata et al. 2004), and
RAS catalyzes the adduct formation between 4-OH-phe-
nyl-lactic acid and 4-OH-cinnamyl-CoA in RA biosyn-
thesis (Berger et al. 2006; Yan et al. 2006). The activities
of these enzymes are coordinately regulated parallel to the
accumulation of RA in the cultures. In this study, the
specific activities of PAL, TAT, HPR, and RAS were
measured and the effect was compared with respect to
intracellular RA accumulation after elicitation with MJ,
SA, and YE (Figs. 4, 5, 6, 7, 8).
After addition of 50 lM MJ to the whole plant culture of
S. scutellarioides, RA accumulation was enhanced. Higher
RA levels could be observed from 6 h after elicitation, and
it stayed higher than the control levels until day 7 after
elicitations (when measurements were discontinued). This
showed that RA levels were increased for several days after
addition of MJ. Highest RA contents (*1.7-fold) were
measured 24 h after elicitation in comparison to the con-
trol. In MJ-treated cultures, the specific activity of PAL
was quickly increased within 12 h compared to control
levels. After 24 h, PAL activity decreased rapidly and
reached near to the control status in day 7. The activity of
TAT was constantly higher at levels over control in
MJ-treated but no discrete activity peak was observed.
HPR activity showed a transient increase with a maximal
activity at 24 h after elicitation with MJ. After 48 h of MJ
elicitation, the activity gradually decreased and remained
comparable to that of control culture. The specific activity
of RAS was not increased significantly by the MJ
treatment.
Six hours after elicitation with YE (100 lg ml-1), the
RA content of the S. scutellarioides culture started to rise
and the maximum RA accumulation was achieved at 24 h
and it remained almost constant (*1.4 ± 0.1-fold) up to
72 h after elicitation. RA content slowly reduced after 72 h
of YE elicitation but remained higher up to 7 days over
control. The PAL activity was suppressed by YE elicita-
tion. However, specific activity of TAT started to rise
within 6 h and after elicitation with a maximum activity at
24 h. The maximum retained almost similar up to 72 h
after elicitation. The activity of TAT was sharply reduced
after 72 h and remained almost comparable to control after
5 days. There is no significant change in HPR activity after
YE elicitation. RAS activity started to rise within 6 h
Acta Physiol Plant (2013) 35:1473–1481 1477
123
achieved maximum activity at 24 h. After attaining a peak
of highest activity (24 h), it reduced sharply even below
control 48 h onward.
SA (50 lM) caused significant elevation of RA content.
The intracellular RA content reached to its maximum value
(*1.4-fold) at 24 h and then slowly reduced with time.
The stimulating effect on specific activity of PAL was
observed in SA elicitation. The PAL activity sharply
increased within 6 h after elicitation, but no specific pattern
of PAL activity was recorded throughout the course of
study. The specific activity of TAT was transiently
increased up to 12 h to reach to maximum level and then
decreased slowly. The specific activities of HPR and RAS
were not significantly increased by the SA treatment.
Synergistic effect of elicitors
There always exists a possibility in theory to synergisti-
cally improve the secondary metabolite production by
treatment of the cultures with some combined elicitors, if
these elicitors could enhance the stimulating effect each
other by related mechanism (Zhao et al. 2001). Therefore,
based on differential responses in the activities of PAL,
TAT, HPR, and RAS with the exposure of MJ, YE and
Fig. 3 Chromatogram obtained
from standard RA solution (top)
and RA in S. scutellarioidesextract (bottom)
Table 1 Effect of MJ, SA, YE
elicitors on intracellular RA
accumulation in
S. scutellarioides
Values presented as mean ± SDa Values significantly elevated
from control (p \ 0.01)b Values significantly elevated
from control (p \ 0.05)
according to Dunnett’s test
Name of
elicitors
Concentrations of
elicitors (lM)
RA (mg 100 g-1 FW)
0 h Day 1 Day 3 Day 7
Control – 11.32 ± 1.28 11.42 ± 1.41 11.46 ± 1.35 11.92 ± 1.36
MJ 10 lM 11.27 ± 1.33 12.56 ± 1.45 13.10 ± 1.33 12.77 ± 1.26
25 lM 11.45 ± 1.40 14.85 ± 1.67b 14.67 ± 1.54b 14.12 ± 1.42b
50 lM 11.25 ± 0.98 19.12 ± 1.45a 18.76 ± 1.96a 16.01 ± 1.50a
100 lM 11.30 ± 1.55 17.11 ± 1.71a 16.98 ± 1.33a 15.84 ± 1.45b
YE 10 lg ml-1 11.29 ± 1.12 11.98 ± 0.48 12.45 ± 1.73 12.08 ± 1.37
25 lg ml-1 11.25 ± 1.21 12.85 ± 1.27 13.38 ± 0.45 13.42 ± 1.48
50 lg ml-1 11.36 ± 1.33 15.67 ± 0.56b 16.18 ± 1.64a 13.95 ± 1.25
100 lg ml-1 11.18 ± 1.38 16.52 ± 1.42a 16.65 ± 1.30a 14.32 ± 2.05b
SA 10 lM 11.36 ± 1.45 12.98 ± 1.25 13.18 ± 1.47 13.02 ± 1.33
25 lM 11.20 ± 1.11 13.88 ± 1.38 13.44 ± 1.45 13.67 ± 1.28
50 lM 11.42 ± 1.14 15.78 ± 1.36b 15.71 ± 1.50b 14.92 ± 1.42b
100 lM 11.22 ± 1.54 15.58 ± 1.57b 15.62 ± 1.33b 14.96 ± 1.44b
1478 Acta Physiol Plant (2013) 35:1473–1481
123
SA at the optimized doses, it was aimed to find out the
synergy between elicitors to further scale up RA synthesis
within in vitro S. scutellarioides. The activities of most of
enzymes were found maximum between 6 and 24 h with
MJ and SA, while TAT activity was observed maximum
in between 24 and 72 h after YE elicitation. Based on this
study, a steady state higher level of RA content in
S. scutellarioides was aimed. The study of synergy
between elicitors with respect to intracellular RA accu-
mulation was depicted in Fig. 9. Amongst selected
combinations, at a time treatment with MJ (50 lM) ? SA
(50 lM) ? YE (100 lg ml-1) caused a transient peak of
RA content within 24 h (*2-fold) but decrease slowly
with time. While, initial exposure of MJ (50 lM) ? SA
(50 lM) followed by YE (100 lg ml-1) after 24 h has
been found to possess highest accumulation of RA within
24 h (*1.9-fold) and a steady state increased level
(*1.7 ± 0.2-fold) over control was maintained through-
out the course of study. Elicitation with MJ
10
12
14
16
18
20
0 24 48 72 96 120 144 168
RA
(m
g 10
0 g
F
W)
-1
Hours
ControlMJYESA
Fig. 4 Time courses of intracellular RA accumulation in S. scutel-larioides elicited with pre-optimized elicitors viz. MJ (50 lM), YE
(100 lg ml-1), and SA (50 lM) up to 7 days after inoculation
0
10
20
30
40
50
0 24 48 72 96 120 144 168
Spec
ific
act
ivit
y (µ
kat
kg-1
prot
ein)
Hours
PAL Control
PAL MJ
PAL YE
PAL SA
Fig. 5 Time courses of PAL activities in S. scutellarioides after
treatment with pre-optimized elicitors viz. MJ (50 lM), YE
(100 lg ml-1), and SA (50 lM)
Table 2 Effect of different
inorganic salts elicitors on
intracellular RA accumulation
in S. scutellarioides
Values presented as mean ± SDa Values significantly elevated
from control (p \ 0.05)
according to Dunnett’s test
Name of
elicitors
Concentrations of
elicitors (lM)
RA (mg 100 g-1 FW)
0 h Day 1 Day 3 Day 7
Control – 11.32 ± 1.28 11.42 ± 1.41 11.46 ± 1.35 11.92 ± 1.36
Silver chloride 10 11.30 ± 1.12 11.36 ± 0.33 11.45 ± 1.23 11.37 ± 0.87
25 11.32 ± 0.32 12.25 ± 1.34 13.31 ± 0.24a 14.12 ± 1.33
50 11.54 ± 0.45 12.82 ± 0.44 13.56 ± 0.46 12.67 ± 0.39
100 11.24 ± 0.20 12.24 ± 0.55 13.49 ± 1.07 13.98 ± 1.04a
Copper chloride 10 11.18 ± 1.10 10.88 ± 1.08 11.14 ± 0.27 11.12 ± 1.22
25 11.54 ± 1.25 10.45 ± 0.36 10.91 ± 0.45 10.87 ± 1.41
50 11.42 ± 1.14 9.42 ± 0.46 10.58 ± 1.01 10.17 ± 0.28
100 11.19 ± 1.21 10.38 ± 1.14 10.32 ± 0.85 10.37 ± 0.54
Calcium chloride 10 11.61 ± 1.17 11.67 ± 0.45 11.45 ± 0.15 11.42 ± 1.40
25 11.48 ± 1.09 11.43 ± 1.22 11.38 ± 0.21 11.41 ± 1.15
50 11.13 ± 1.67 11.63 ± 0.64 11.48 ± 0.98 11.52 ± 0.78
100 11.82 ± 1.33 12.02 ± 0.82 11.98 ± 1.42 12.19 ± 1.34
Manganese chloride 10 11.55 ± 0.89 11.35 ± 0.67 11.42 ± 0.45 11.39 ± 1.38
25 11.33 ± 0.38 11.36 ± 1.02 11.38 ± 1.38 11.42 ± 0.56
50 11.21 ± 0.57 11.55 ± 0.25 11.78 ± 1.45 11.82 ± 1.08
100 11.50 ± 1.20 11.41 ± 0.31 11.63 ± 1.48 11.59 ± 0.71
Zinc chloride 10 11.23 ± 0.45 10.47 ± 0.23 10.55 ± 0.27 10.67 ± 0.62
25 11.28 ± 1.33 9.38 ± 0.75 10.11 ± 0.46 10.85 ± 0.42
50 11.20 ± 0.42 9.87 ± 1.08 9.89 ± 0.67 10.07 ± 1.01
100 11.36 ± 0.35 9.86 ± 0.33 9.95 ± 0.92 9.72 ± 0.21
Acta Physiol Plant (2013) 35:1473–1481 1479
123
(50 lM) ? YE (100 lg ml-1) followed by SA (50 lM)
after 24 h also caused significant elevation of RA content
over control, but not so effective as compared with other
two selections.
Conclusion
The present study indicated the potential of these bio-
technology-based methodologies for mass production of
RA. The changes in RA production can be influenced by
manipulation of type of elicitors, elicitation time and also
their concentration in vitro. The results clearly indicated
that incorporation of MJ, SA and YE in culture medium
caused significant elicitation of RA production in the cul-
ture of S. scutellarioides. The effect of aforementioned
elicitors on intracellular RA accumulation was found to be
dependent on specific activity of regulating enzymes in
biosynthetic pathways. The selected elicitors have been
found to possess differential response on specific activities
of PAL, TAT, HPR, and RAS. RA synthesis was further
scaled up through combination of elicitors based on
observed activities of metabolic enzymes. In conclusion,
the experiment presented here would serve for the high
yielding of RA in S. scutellarioides for phytochemical
industry, subject to economic consideration.
Author contribution R.S. performed experiment for
elicitation and phytochemical analysis. M.G. performed
experiment for enzyme estimation and assisted the elici-
tation experiment. S.D. designed the experiments, super-
vised entire work and wrote the manuscript.
Acknowledgments The financial support of the University Grants
Commission, New Delhi, India is gratefully acknowledged.
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