SUPPLEMENTARY MATERIAL
Enhanced bacoside production in shoot cultures of Bacopa monnieri under the influence
of abiotic elicitors
Munish Sharmaa, Ashok Ahujab, Rajinder Gupta b & Sharada Mallubhotlaa*
a School
of Biotechnology, Shri Mata Vaishno Devi University, Katra-182320, India; b Indian
Institute of Integrative Medicine (CSIR), Jammu-180001, India.
a*
Corresponding author: Email: sharda.p@smvdu.ac.in
Abstract
The effect of different abiotic elicitors [Jasmonic Acid (JA), Copper Sulphate (CuSO4),
Salicyclic Acid (SA)] at varying concentrations on the stimulation of biomass and bacoside
production in in vitro Bacopa monnieri shoot culture was studied. A systematic study
conducted over a period of 35 days indicated that the maximum bacoside production (6.74
mg g-1 DW) was obtained after a lag of 7 days and thereafter, the content decreased gradually
to again increase at 28 days (5.91 mg g-1 DW). Therefore, elicitation experiments were
carried out over a period of 3, 6 and 9 days. The shoot cultures treated with 45 mg L-1 of
CuSO4 exhibited the highest bacoside content of 8.73 mg g-1 DW (~1.42 fold higher) than in
control cultures (6.14 mg g-1 DW). The present study indicated effectiveness of abiotic
elicitation on bacoside production in in vitro shoot cultures of this medicinally important herb
known for its memory enhancing properties.
Key words: Brahmi; elicitation; HPLC; shoot biomass; secondary metabolite production
1. Experimental
1.1 Collection and establishment of source plant
Plant material of Bacopa monnieri was collected from Indian Institute of Integrative
Medicine - CSIR, Jammu (formerly Regional Research Laboratory) in the form of stem
cuttings and established under net house conditions at the Herbal Garden, Shri Mata Vaishno
Devi University, Kakryal, Katra (J&K) India (Latitude = 28°66'67" North, Longitude =
77°21'67" East), in the month of May, 2009. The identification of the plant was confirmed
with the specimen voucher deposited in Janaki Ammal Herbarium, IIIM, Jammu bearing
Accession No: 18554.
1.2 Explant preparation and culture initiation
In vitro multiple shoot cultures of B. monnieri were initiated from of 60 day old net house
grown plants. Nodal segments (~0.5 cm) collected were washed thoroughly under running tap
water and subsequently with a 1 % solution of Tween-20. They were then aseptically surface
sterilized with 0.1% (w/v) mercuric chloride for 5 min followed by rinsing 3 to 4 times with
sterile double distilled water and cultured on MS medium (Murashige and Skoog, 1962) as
earlier reported (Ahuja et al., 2005). The pH of the medium was adjusted to 5.8 before
autoclaving at 121 0C for 20 min. All aseptic cultures were incubated in the culture room at
24 ± 2°C under cool, white fluorescent light (Philips, India) with a total irradiance of 3000
lux under a 16 h photoperiod.
1.3 Kinetics of bacoside production
Growth pattern as a measure of biomass and bacoside accumulation in agar solidified cultures
was studied for a period of 35 days (7 day intervals) for determining the optimum culture age
to be used for elicitation experimentations.
1.4 Elicitor treatment
On the basis of the results of the above study, elicitation was carried out using different
abiotic elicitors at varying concentrations of 0.1, 0.5, 1.0 mg L-1 jasmonic acid (JA), 15, 30,
45, 60 mg L-1 copper sulphate (CuSO4) and 50, 100, 200 μM salicyclic acid (SA) and for 3, 6
and 9 days in all culture treatments. Stock solutions of CuSO4 and SA [1.0 mg mL-1 (w/v)]
were prepared in sterilized distilled water while JA was prepared in sterilized ethanol at a
concentration [1.0 ml mL-1 (v/v)]. Appropriate volumes of stock solutions were filter
sterilized using a 0.22 µm PTFE syringe filter and added to autoclaved molten medium. The
control for the JA treatments contained similar concentrations of absolute ethanol whereas
medium without elicitor served as control for CuSO4 and SA treatments.
1.5 Growth measurements
Growth in terms of growth index (GI) on fresh weight (FW) and dry weight (DW) basis was
calculated for all the treatments to assess increase in biomass.
1.6 Extraction and analysis of bacosides
Cultures were harvested periodically for each treatment, air dried, finely powdered using a
mortar and pestle and extracted in a soxhlet apparatus with methanol for 5 h at a temperature
of not more than 60°C. The methanolic extract was filtered with a 0.45 μm PVDF syringe
filter, air dried and further dissolved in HPLC grade methanol and subjected to HPLC
analysis to estimate the total bacosides (A3 and A2). Quantification of bacosides was carried
out on Agilent- 1100 series HPLC system equipped with dual channel interface 35900E,
quaternary pump, degasser, auto sampler, column oven and ultraviolet (UV) detector.
HPLC analysis was conducted using Acetonitrile:Water (48:52, v/v) as the mobile phase with
a flow rate of 0.4 ml min-1 at wavelength of 210 nm on a column RP-18 (EMerck, 5 μm, 4.0 ×
250 mm). The column temperature was maintained at 30° C for maximum peak efficiency
with injection volume of 10 μL and a run time of 30 min for each separation. Data acquisition
was performed by Chemstation software. All solvents used for the HPLC analysis were of
HPLC grade (Acetonitrile, Milli Q distilled water, Methanol). A 1.0 mg mL-1 stock solution
of standard bacoside (mixture of A3 and A2) was prepared in methanol and 1.0 mL of this
solution was diluted to 10 ml with methanol to get a 100 μg mL-1 working solution. Standard
calibration curves were established by plotting the area of peaks against different
concentrations of 2 μl, 4 μl, 6 μl, 8 μl and 10 μl. Quantification of bacosides in the samples
was determined using regression equation of calibration curves. On the basis of the standard
curves obtained, the quantity of bacoside A3 and A2 were determined in samples of shoot
cultures.
1.7 Statistical analysis
Each experiment was repeated thrice with 6 replicates each and the results are represented
graphically as mean ± SE. Statistical analysis was performed with SPSS (version 20) using
one-way ANOVA (analysis of variance). Significance differences among means were
analyzed using Duncan’s multiple range test (DMRT) at P ≤ 0.05.
References
Ahuja A, Gupta KK, Khajuria RK, Sharma A, Kumar A, Sharada M, Kaul MK. 2005.
Production of Bacosides by multiple shoot cultures and in vitro regenerated plantlets of
selected cultivar of Bacopa monnieri (L.) Wettst. In: Kumar A, Roy S (eds.) Plant
Biotechnology and its application in Plant Tissue Culture. New Delhi: IK Publishers; p.
155-162.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant. 15: 473-497.
Table S1
Time course studies on biomass and bacoside production on agar solidified medium. Mean
within a column followed by common letters are not significantly different at P< 0.05,
according to DMRT. Data show means ± SE from triplicate experiments.
* Growth index (GI) = Final weight–Initial weight / Initial weight
Culture period (days)
Shoot No.
Shoot Length (cm)
Leaves No.
Nodes No.
*GI (FW)
*GI (DW)
Bacoside A3
( mg g-1 DW)
Bacoside A2
( mg g-1 DW)
Total bacoside
( mg g-1 DW)
7
14
21
28
35
0.61±0.12a
0.72±0.14 a
1.39±0.16 b
1.94±0.33 b
0.56±0.14 a
0.75±0.17 ab
0.8±0.11 ab
1.12±0.14 b
2.5±0.58 a
0.89±0.25 a
1.88±0.31 a
0.89±0.25 a
3.79±0.43 b
2.83±0.65 ab
1±0.29 ab
2.41±0.29 a
1.2±0.28 ab
3.8±0.06 b
3.53±0.4 abc
1.03±0.17 ab
2.57±0.3 a
1.55±0.26 ab
2.07±0.65 a
4.42±0.71 bc
1.64±0.31 bc
5.41±0.77 b
3.62±1.4 bc
2.45±0.25 ab
3.61±0.23 c
1.02±0.09 b
5.05±0.36 c
1.8±0.16 c
6.83±0.32 c
5.04±0.79 c
2.18±0.61 a
2.95±0.24 ab 1.75±0.69 ab 0.97±0.97 a
3.46±0.61 b
2.6±0.79 ab
6.74±0.44 a
5.91±0.83 a
4.78±1.4 a
5.55±0.63 a
3.04±1.62 a
Table S2
Effect of Jasmonic acid (JA) on growth and bacoside production in selected culture line on
agar solidified medium.
Media*
+ JA
(mg L-1)
Control
0.1
0.5
1.0
Exposure
time (days)
Growth index
FW
DW
Bacoside (mg g-1 DW)
A3
A2
3
6
9
3
6
9
3
6
9
3
6
9
0.58±0.14a
0.7±0.09a
0.82±0.09a
0.53±0.11a
0.84±0.16a
0.88±0.2a
0.54±0.12a
0.82±0.06a
0.9±0.18a
0.54±0.1a
0.68±0.16a
0.81±0.13a
3.45±1.31a
2.45±0.68a
2.46±0.32a
3.77±1.37a
4.98±1.46a
4.42±0.51a
4.45±1.28a
5.3±0.99a
5.53±2.43a
4.48±2.36a
6.7±1.15a
4.87±0.26a
0.41±0.15a
0.57±0.11a
0.67±0.12a
0.33±0.19a
0.67±0.19a
0.73±0.2a
0.37±0.15a
0.75±0.27a
0.74±0.2a
0.27±0.18a
0.51±0.14a
0.69±0.14a
0.51±0.51a
0.29±0.29a
0.63±0.46a
0.2±0.2a
1.26±1.26a
1.22±1.14a
1.77±1.77a
1.46±0.77a
1.01±0.95a
1.11±1.11a
1.76±1.76a
1.86±1.01a
Total
Bacoside (mg
g-1 DW)
3.96±1.58ab
2.74±0.93a
3.09±0.66 ab
3.97±1.39 ab
6.25±1.99 ab
5.64±1.56 ab
6.22±2.16 ab
6.76±0.76 ab
6.55±2.55 ab
5.6±1.89 ab
8.46±2.19 b
6.73±0.75 ab
Table S3
Effect of Copper sulphate (CuSO4) and Salicyclic acid (SA) on growth and bacoside
production in selected culture line on agar solidified medium
Media*
Control
Exposure Growth index
time
FW
DW
(days)
3
6
9
CuSO4 ( mg L-1)
15
3
6
9
30
3
6
9
45
3
6
9
60
3
6
9
SA (µM)
50
3
6
9
100
3
6
9
200
3
6
9
Bacoside ( mg g-1 DW)
A3
A2
Total
Bacoside
(mg g-1 DW)
0.54±0.11a
0.62±0.1 a
0.78±0.13 a
0.35±0.14 a
0.43±0.17 ab
0.68±0.19 abc
4.11±1.87 a
4.49±1.43 a
5.08±1.5 a
0.71±0.71 a
0.37±0.37 a
1.06±0.87 a
4.82±2.24 a
4.86±1.6 a
6.14±1.67 a
0.51±0.07 a
1.09±0.49 ab
1.49±0.58 bc
1.2±0.07 ab
1.65±0.12 bcd
1.06±0.08 ab
1.53±0.1 bc
1.68±0.12 bcd
2.03±0.02 cde
2.24±0.04 de
2.55±0.13 e
2.64±0.13 e
0.37±0.14 a
0.97±0.5 abcd
1.25±0.54 abcde
0.71±0.24 abc
1.35±0.14 bcde
0.93±0.05 abc
1.04±0.22 abcde
1.09±0.16 abcde
1.4±0.5 cde
1.88±0.35 de
1.95±0.04 e
1.97±0.14 e
5.45±0.93 a
5.15±0.56 a
6.21±0.68 a
4.76±2.13 a
6.66±0.74 a
6.37±1.55 a
6.51±0.95 a
6.09±0.37 a
7.77±0.99 a
5.04±1.62 a
4.52±0.8 a
5±1.52 a
1.12±0.71 a
1.29±0.78 a
0.73±0.73 a
1.63±0.91 a
0.49±0.49 a
0.81±0.81 a
0.74±0.74 a
1.25±0.63 a
0.96±0.96 a
0.02±0.02 a
0.43±0.28 a
1.21±0.9 a
6.57±1.57 a
6.44±1.19 a
6.94±1.12 a
6.39±2.95 a
7.15±0.26 a
7.18±1.77 a
7.26±1.08 a
7.34±0.28 a
8.73±0.1 a
5.06±1.6 a
4.95±0.65 a
6.21±1.86 a
0.55±0.07a
1.08±0.1bc
1.34±0.17cd
1.12±0.07bc
1.09±0.09bc
1.36±0.22cd
1.17±0.14bc
1.65±0.11de
1.77±0.11e
0.35±0.05a
0.81±0.2abc
1.12±0.29c
0.46±0.02ab
0.78±0.16abc
1.07±0.21bc
0.64±0.35abc
0.43±0.18ab
0.52±0.13abc
5.47±0.9ab
6.4±1.33ab
7.42±0.53b
2.14±0.59a
4.15±1.82ab
4.78±1.47ab
4.49±0.93ab
7.48±1.99b
6.05±1.41ab
1.12±0.66a
1.56±1.56a
0.72±0.49a
2.74±2.74a
1.03±1.03a
2.31±1.23a
1.25±1.25a
0.12±0.12a
0.87±0.87a
6.58±0.71a
7.96±0.76a
8.14±0.16a
4.88±3.06a
5.18±2.82a
7.09±2.57a
5.74±1.97a
7.6±2.05a
6.92±1.89a
A
B
C
Figure S1. Effect of different concentrations of abiotic elicitors (A) JA, (B) CuSO4, (C) SA
on bacoside production in shoots extracts of B. monnieri . Error bars indicate ± SE
A
B
C
D
D
Figure S2. HPLC chromatogram of bacoside in methanolic extracts of JA, CuSO4 and SA
treated shoots of B. Monnieri (A) standard bacoside (mixture of bacoside A3 and A2), (B) JA
(1.0 mg L-1) treated culture, (C) CuSO4 (45 mg L-1) treated culture, (D) SA (50 µM) treated
culture .