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: ranaju4u@yahoo.co.in

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

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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

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(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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