Content uploaded by Preethi mp
Author content
All content in this area was uploaded by Preethi mp on Jul 24, 2015
Content may be subject to copyright.
BEPLS Vol 4 [7] June 2015 122 | P a g e ©2015 AELS, INDIA
Bulletin of Environment, Pharmacology and Life Sciences
Bull. Env.Pharmacol. Life Sci., Vol 4 [7] June 2015: 122-131
©2014 Academy for Environment and Life Sciences, India
Online ISSN 2277-1808
Journal’s URL:http://www.bepls.com
CODEN: BEPLAD
Global Impact Factor 0.533
Universal Impact Factor 0.9804
ORIGINAL ARTICLE
In vitro propagation and enhancement of phytoconstituents in
Withania coagulans, a rare medicinal plant
Preethi M. Purushotham1, Archana Thottukara Madam1, Pradeepa Duraisamy1, Kalaiselvi
Senthil1,*
1Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home
Science and Higher Education for Women University, Coimbatore 641043, Tamil Nadu, India.
*Corresponding author’s E-mail: aubio.ptc.kalai@gmail.com
ABSTRACT
Withania coagulans, belonging to family Solanaceae, is an important medicinal plant with restricted geographic
distribution. Propagation of Withania coagulans is highly demanded in recent times due to its rich bioactive
withanolides contentand its extinction from natural habitats. Shoot multiplication protocol of Withania coagulans was
standardised by supplementing basal Murashige and Skoog’s media with different concentrations of Benzylaminopurine
(BAP), Kinetin and Thidiazuron (TDZ). Explants were transferred to fresh media with similar hormone constituents every
three weeks. 1 mg/L BAP in combination with 0.5 mg/L Kin induced maximum shoots in this herb under in vitro
conditions. Also among the 25 different combination of auxins (IAA and IBA) tested, MS media supplemented with 1
mg/L IAA and 4 mg/L IBA was found to be the best medium for in vitro adventitious root induction in Withania
coagulans. The developed roots were then transferred for suspension cultures and mass cultivated in bioreactors
successfully. These roots resulted in increased biomass and accumulations of active compounds. Culturing in vitro
adventitious roots in bioreactors could also be used as a fast and efficient method of generating roots that would offer
unique opportunities for producing root drugs without having to depend on field cultivation. Following this, the in vitro
propagated Withania coagulans tissues were subjected to phytochemical analysis and were found to accumulate the
constituents in high amount when compared to field grown roots. Also withanolides quantification using HPTLC proved
high withaferin A and withanone accumulations when propagated under in vitro conditions.
Keywords : Withania coagulans; in vitro; bioreactor; HPTLC.
Received 02.02.2015 Revised 12.03.2015 Accepted 03.05. 2015
INTRODUCTION
In vitro techniques have been found to be useful in the propagation of a large number of threatened and
endangered plants [1]. The technology bears many advantages over conventional agricultural methods
such as, production is independent of variation in crop quality or failure, yield of target compounds would
be constant and geared to demand, there is no difficulty in applying good manufacturing practice to the
early stages of production, production would be possible anywhere under strictly controlled conditions,
independency of environmental problems, free from risk of contamination with pesticides, herbicides,
agrochemicals or fertilizers and new methods of production could be patented [2].
Production of secondary metabolites in tissue cultures is usually higher when plant cells are organized
into tissues/organs. The expression of secondary metabolic pathways in organized cultures is not
surprising because it mimics exactly what the plant does. Root cultures are typical examples that can be
used for production of phytochemicals. Root cultures have been used as a standard experimental system
in studies of inorganic nutrition, nitrogen metabolism, plant growth regulation, and root development [3].
Among the twenty-three known species of Withania, only two (Withania somnifera (L.) Dunal and
Withania coagulans Dunal) are economically significant and widely cultivated [4]. Withania coagulans
Dunal belonging to the family Solanaceae, is a small bush which is spread across South Asia and is
commercially important for its milk coagulating properties [5]. Withania coagulans is well known in the
indigenous system of medicine for the treatment of ulcers, dyspepsia, rheumatism, drowsy, and
consumption debility [6]. It received much attention in recent years due to the presence of a large number
of steroidal alkaloids and lactones known as withanolides. Withanolides, chemically nomenclatured as
BEPLS Vol 4 [7] June 2015 123 | P a g e ©2015 AELS, INDIA
22- hydroxy ergostane-26-oic acid 26, 22-d-lactones, are C28-steroidal lactones based on an intact or
rearranged ergostane frame with appropriate oxidations at C-22 and C-26 to form a d-lactone ring. Major
Withanolides, like Withaferin A, Withanolide A and withanone of the plant have been demonstrated to
possess significant therapeutic actions [7].
Withania plants were abundant until a few decades ago, but ruthless collection for medicinal purposes,
habitat destruction and climate changes makes the species to become endangered in their natural
habitats. Jain et al. (2009) reported that overexploitation and reproductive failures budge the species W.
coagulans towards verge of extinction [8]. Therefore propagation and conservation of these plants is
important to meet up with future demands. The conventional propagation of this species is performed
through seeds and cuttings of stem since root is too slow and laborious to grow. In vitro propagation
technique may be the best solution for its rapid multiplication and re-establishment in nature [9]. The in
vitro cultures could provide as an alternative tool to field harvesting of this plant in order to produce
therapeutically valuable compounds [10] and is reported that the withanolide contents of the in vitro
hairy root cultures of W. coagulans were higher than in the root of the field grown plant [4]. Therefore,
there is a massive need to develop an efficient protocol for the induction of in vitro adventitious roots and
the shoot multiplication of W. coagulans.
Thus the present study was formulated to optimise the concentrations of growth hormones for root
induction and shoot multiplication in Withania coagulans under in vitro conditions and was found that
major phytochemicals and therapeutic withanolides excessively accumulated in in vitro roots of W.
coagulans compared to the field grown roots.
MATERIALS AND METHODS
Plant material: Withania coagulans seeds were obtained from Banaras Hindu University, Varanasi. The
seeds were germinated in vitro after surface sterilization with Tween20 and 1% HgCl2. The seedlings
were maintained on MS basal medium with regular sub culturing. The nodes from two month old aseptic
plantlets were used as explants for shoot multiplication and leaves excised were applied as explants for
root induction.
Media and explants used: MS basal medium was prepared essentially based on the procedure described
by Murashigae and Skoog [11]. The hormones and chemical were purchased from HiMedia Laboratories
Ltd, Mumbai, India and Elix-3 water was used for the entire study. For root induction, MS basal media
supplemented with varying concentrations and combinations of indole butyric acid (IBA) and indole
acetic acid (IAA) and 3% sucrose were used. The leaves were trimmed into pieces of about 1cm2 and
inoculated on to the respective media. In vitro induced roots were maintained in suspensions consisting
of liquid basal MS media supplemented with respective hormone combinations. It was then transferred to
air-lift bioreactor provided with liquid basal MS media for mass cultivation of roots. For shoot
multiplication varying concentrations of hormones 6-Benzylaminopurine (BAP), Kinetin and Thidiazuron
(TDZ) were used. The internodes from MS media grown plants were excised, trimmed at both the ends
and were applied as explants. The inoculated explants were maintained in controlled temperature of 25ºC
and observed regularly for their morphological growth. A photoperiod of 16 hrs was maintained using
fluorescence lights (Philips India Ltd., Mumbai) followed by 8 hrs of dark period and a relative humidity
of 60-70%.
Establishment of multiple shoots: All cultures were observed periodically and the shoots produced in
various combinations were recorded on the basis of visual observation. The combination with increased
shoot multiples was identified as the best. Subculturing was carried out at an interval of 3 wks and the
experiment was repeated three times. The effect of different treatments was measured on the basis of
number of shoots produced and the height achieved by the shoots.
Establishment of roots and mass cultivation: After a period of 30 days, the induced roots in various
hormone combinations were analysed and the combination with more number of induced roots was
identified as the best. The root of that particular combination was then transferred to suspension cultures
with liquid MS basal media supplemented with and without the respective hormones. Thirty root tips or
branch (1.5 gm) measuring in length 2-3 mm were cut under sterile conditions and transferred to conical
flasks containing 30 mL liquid MS with the respective hormone combination. This study was taken to find
out the growth pattern of roots in suspension culture under the influence of hormones. Fresh media were
replaced regularly at 15 days intervals in culture bottles. After 30 days root growth and stabilization in
suspension culture, a part of the well grown roots were transferred to air-lift bioreactor for their mass
propagation. The bioreactor was provided with proper aeration and temperature was maintained at
22°C. The roots were harvested and their wet and dry weight was noted down. Increase in the root mass
was calculated.
Purushotham et al
BEPLS Vol 4 [7] June 2015 124 | P a g e ©2015 AELS, INDIA
Preparation of extracts from roots: The in vitro grown plant parts and field grown roots of W. coagulans
were ground thoroughly and one gram of powdered samples was extracted using 200ml methanol by
repeated sonication and shaking at 104rpm at 22ºC. The extracts were then filtered using Whatmann no:
1 filter paper and was concentrated using flash evaporator maintained at 45ºC and 150 rpm. After
complete solvent evaporation, the residue was dissolved using HPLC grade methanol for their estimations
and quantifications [12].
Phytochemical estimations and withanolides quantification: The plant extracts were subjected to
quantitative phytochemical screening of total flavonoids, steroids, alkaloids, phenols and saponins [13].
The withanolides, withaferin A and withanone was quantified using High Performance Thin Layer
Chromatography (HPTLC). The sample extracts were applied on 20x10cm pre-coated silica gel aluminium
60F254 plates (E.MERCK, Germany) as 8mm band, by means of Semiautomatic CAMAG Linomat 5 device
(Camag, Muttenz, Switzerland) fitted with 100 μL Hamilton syringe. Linear ascending development to a
distance of 8 cm, was carried out in 20x10cm twin trough chamber saturated with 20 mL Toluene : Ethyl
acetate: formic acid (5:5:1) for 30 mins at room temperature. Densitometric scanning was performed and
withanolides were quantified using Camag winCATS software (1.4.4.6337).
Statistical analysis: In all the experiments, the experiment was repeated twice with 4 replicates and three
explants in each. The mean ± SE of the results were determined. Data were statistically analysed by
ANOVA and significant differences between means were assessed by Duncan’s multiple range test using
SAS.
RESULTS
Shoot multiplication of Withania coagulans
The nodal explants developed different responses in different media combinations. Shoots remained
fresh and green but failed to induce multiple shoots in growth regulator free basal MS media. All nodal
explants cultured on growth regulator supplemented MS media grew length wise and began to proliferate
multiple shoots in the 2nd week. Combinations of media consisting of BAP, Kin and TDZ were formed. Fig.
1 shows the formation of multiple shoots from nodal explants of in vitro Withania coagulans with
different concentrations of hormone regulators. BAP and Kin alone discretely did not induce remarkable
shoot formations and the explants cultured on combination media of BAP+Kin induced more axillary
shoot proliferation when compared to the TDZ. The mean number and length of shoot is represented in
Table 1. Maximal shoot generation was achieved in MS media containing 1BAP+0.5 Kin followed by
1BAP+0.1Kin and 0.5BAP+0.5Kin. It was also evident that among these three combinations, 1BAP+0.5 Kin
resulted in the highest number of shoots and mean shoot length. Abnormal callusing was observed in the
explants incorporated even in the lowest concentration of TZD. Complete shoot inhibition and excessive
callusing was observed in the media supplemented with 0.8TDZ.
In vitro rhizogenesis in Withania coagulans
The results of the present investigation showed that increasing concentration of IBA resulted in positive
effect on root induction in W. coagulans. The results are presented in Fig.2 where different combinations
of IAA and IBA gave different responses. The results exhibited that the presence of auxin plays an
important role in inducing roots as there was no sign of roots in MS media without auxin
supplementation. 20 days of culture in media supplemented with auxins produced maximum induced
roots and upon increasing the culture period to 30 days, no significant result was observed. Increasing
concentrations of IAA has also shown to have a positive effect on root induction but a concentration
higher than optimal displayed a negative impact.
The present finding suggest a maximum root induction in MS media supplemented with 1.0 mg/L IAA and
4.0 mg/L IBA with 100% root induction response as shown in Table 2. Followed by MS media
supplemented with IBA alone i.e. 4.0 mg/L showed 94.4% root induction. A combination of 6.0mg/L IBA
with 0.5, 1.0, 2.0, 4.0 mg/L of IAA were also carried out in the present study and was observed that a
higher percentage of roots were induced on increasing medium concentration of IBA to 6.0mg/L
compared to that supplemented with 4.0 mg/L IBA (data not shown). But the roots mainly aroused from
the high amount of callus induced in response to the increased concentration of IBA which was found to
be insignificant and thus excluded.
Mass propagation of roots in suspension and bioreactor
The mass proliferation of the induced roots were further carried out using suspension cultures and
bioreactors (Fig. 3). For the establishment of root cultures in suspension, the roots obtained from MS
media supplemented with 4.0 mg/L IBA and 1.0 mg/L IAA with 3% sucrose were considered, which was
selected as the best auxin combination for root induction. After the culture period, the fresh weight of the
root was noted and finally growth index was calculated (Wu et al. 2008) (Table 3). Media change was
given every 15 days in order to supply adequate nutrients for the culture growth. At the end of 45th day,
Purushotham et al
BEPLS Vol 4 [7] June 2015 125 | P a g e ©2015 AELS, INDIA
the growth index was found to be 69.0, indicating an increase in root mass. The roots appeared healthy,
thin and white in colour.
The stabilised roots were then mass cultivated in bioreactors consisting liquid MS media without auxin
supplements. The results showed an increase in root mass by 41 fold after a period of 30 days.
Quantitative analysis of essential phytoconstituents
The quantitative estimation of important phytoconstituents such as flavonoids, saponins, steroids,
alkaloids and phenols in the methanolic extracts of W. coagulans are presented in Table. 4. The
phytoconstituents present in the sample is expressed as mg/g. All the phytoconstituents were quantified
highest in in vitro extracts of W. coagulans than field grown root extract.
Withanolide quantification using HPTLC
The extracts of field grown roots and in vitro propagated tissues of W. coagulans were subjected to HPTLC
profiling for quantification of major withanolides, withaferin A and withanone (Fig. 4a). Multi wavelength
scanning was carried out for withaferin A and withanone to determine their absorption spectra (λmax) as
223nm and 231nm respectively. The detection was then performed using the respective λmax and a
distinct fingerprint profile was developed for withanolide standards and crude samples. The amounts of
these withanolides in the extracts were quantified from the spotted standards and the linear regression
generated.
The quantitative evaluation of the plates was determined in three independent experiments. The
developed plates were then derivatized with 10% sulphuric acid reagent. Characteristic fluorescence for
withanolides were observed under UV light at 366nm which could be applied to identify them among
other separated compounds of the crude extracts. Linear correlation between the applied concentration
and the peak area obtained was found. The Rf values were identified and the quantification of
withanolides were determined with respect to the peak area obtained and is represented in the Fig. 4b
respectively. The concentration withaferin A was found to accumulate in similar manner in W. coagulans
roots with a higher content in the aerial parts when grown in in vitro conditions whereas the withanone
quantity was found to increase profoundly in all the in vitro tissues of W. coagulans when compared to the
commercially utilised field grown roots.
Fig. 1: Influence of growth regulators on in vitro shoot multiplication of W. coagulans. ST14 (1.0
BAP+0.5Kin) was determined to be the best media combination for producing multiple shoots with
appreciable shoot length and leaf counts.
Purushotham et al
BEPLS Vol 4 [7] June 2015 126 | P a g e ©2015 AELS, INDIA
Fig. 2: Influence of Auxins on in vitro root induction of Withania coagulans. Among the 25 different
combinations of root induction media, 1.0 IAA+4.0 IBA induced maximum number of root hairs.
Fig. 3: Mass cultivation of adventitious roots of W. coagulans. (a, b) 0.5 gm of induced adventitious
roots were separated and transferred to suspension media (c, d) roots were grown in suspension culture
of half strength MS media without agar (e, f) 1 gm of suspension grown roots were then transferred to
bioreactor with 1 L of half strength MS media. Complete proliferation was achieved in 30 days (g) the
roots from the bioreactor were harvested
Purushotham et al
BEPLS Vol 4 [7] June 2015 127 | P a g e ©2015 AELS, INDIA
Fig. 4: HPTLC quantification of withanolides. (a) Chromatograms of field root and in vitro tissue
extracts of W. coagulans plotted against the respective withanolides. (b) The Withaferin A and withanone
in all the extracts of W. coagulans was quantified and withaferin A was analysed to accumulate in a similar
pattern in all W. coagulans tissues. The withanone quantity was found to profoundly accumulate in all the
in vitro tissues of W. coagulans when compared to the field grown roots.
Table 1: Effect of growth regulators on shoot multiplication of Withania coagulans under in vitro
conditions
S.No
BAP
mg/L
Kinetin mg/L
TDZ mg/L
Mean number of shoots
Mean shoot length
ST0
0
0
-
1.18±0.1
e
4.05±0.4
a
ST1
0.1
-
-
1.26±0.07
e
4.0±0.08
a
ST2
0.5
-
-
2.18±0.1
c
3.05±0.1
b
ST3
1.0
-
-
2.37±0.3
b
2.81±0.2
b
ST4
-
0.1
-
1.39±0.1
d
4.0±0.2
a
ST5
-
0.5
-
1.5±0.2
d
3.01±0.1
b
ST6
-
1.0
-
1.96±0.3
c
2.64±0.1
c
ST7
0.1
0.1
-
2.2±0.3
c
3.53±0.9
a
ST8
0.1
0.5
-
2.68±0.5
b
1.99±0.1
d
ST9
0.1
1.0
-
2.41±0.6
b
2.78±0.3
c
ST10
0.5
0.1
-
2.14±0.5
c
1.91±0.1
d
ST11
0.5
0.5
-
2.74±0.06
b
1.95±0.1
d
ST12
0.5
1.0
-
3.33±0.5
a
1.70±0.3
d
ST13
1.0
0.1
-
3.16±0.8
a
2.0±0.3
d
ST14
1.0
0.5
-
3.37±0.6
a
2.12±0.2
d
ST15
1.0
1.0
-
2.56±0.1
b
2.43±0.4
c
ST16
-
-
0.2
1.35±0.1
d
3.24±0.9
b
ST17
-
-
0.4
1.15±0.4
e
2.94±0.3
b
ST18
-
-
0.6
1.97±0.6
c
2.79±0.4
c
ST19
-
-
0.8
1.26±0.35
e
0.9±0.8
e
ST20
-
-
1.0
1.89±0.1
c
2.22±0.2
c
*Means followed by the same letters within a column are not significantly different (P≤0.05).
Purushotham et al
BEPLS Vol 4 [7] June 2015 128 | P a g e ©2015 AELS, INDIA
Table 2: Response of explants to variation in auxins concentration
S.No IBAmg/L IAAmg/L
Mean roots ± SE (30
days)
Percentage of root
response (30 days)
Percentage of root
induction (30 days)
RT0 0 0 0.333±0.21h 33.33 0.58
RT1
0.5
0
10.167±0.79
e
100
17.68
RT2 1.0 0 15.667±0.67d 100 28.99
RT3
2.0
0
18.667±0.49
d
100
32.46
RT4
4.0
0
56.667±1.05
a
100
98.55
RT5
0
0.5
7.167±0.54
f
100
12.46
RT6 0 1.0 3.333±0.21g 100 5.79
RT7 0 2.0 2.167±1.38g 33.33 3.77
RT8
0
4.0
3.833±0.98
g
83.33
6.67
RT9 0.5 0.5 18.0±0.58d 100 31.3
RT10 1.0 0.5 24.333±0.33c 100 42.32
RT11 2.0 0.5 28.333±0.67c 100 49.28
RT12
4.0
0.5
28.833±0.54
c
100
50.14
RT13 0.5 1.0 9.333±0.76e 100 16.23
RT14 1.0 1.0 16.0±0.68d 100 27.83
RT15 2.0 1.0 22.333±4.51d 83.33 38.84
RT16
4.0
1.0
57.5±1.12
a
100
100
RT17
0.5
2.0
2.333±0.56
g
83.33
4.06
RT18
1.0
2.0
10.333±0.76
e
100
17.97
RT19 2.0 2.0 16.333±0.50d 100 28.40
RT20
4.0
2.0
40.5±0.34
b
100
70.43
RT21
0.5
4.0
9.0±2.9
f
66.67
15.65
RT22 1.0 4.0 11.167±0.6e 100 19.42
RT23 2.0 4.0 8.167±2.6f 66.67 14.20
RT24
4.0
4.0
12.833±0.75
e
100
22.32
* Means followed by the same letters within a column are not significantly different (P≤0.05)
Table 3: Growth of W. coagulans adventitious roots in suspension culture and bioreactor
S. No. Culture Incubation
period (days)
Fresh wt of the
inoculum (gm)
Fresh wt of the
harvested
biomass (gm)
Growth
index
Fold
increase
1 Suspension 45 0.5 35 69 70
2
Bioreactor
30
1.0
41
40
41
Table 4: Quantitative phytochemical analysis of field root and in vitro tissue extracts of
W. coagulans
Sample Total
flavonoids
Total
steroids
Total
phenols
Alkaloids Saponins
Wc field root 0.78±0 4.13±0.36 1.10±0 34.55±0 0.78±0
In vitro propagated tissue extracts
Wc root 9.32±0 7.71±0.35 0.93±0 55.03±2.93 26.28±0.08
Wc stem 6.37±0 28.08±0 2.28±0.04 40.40±0 18.47±0
Wc leaf 16.55±0 26.29±0.35 2.19±0.04 116.47±0 17.46±0.07
DISCUSSION
In vitro propagation is crucially recommended when the metabolite of interest is produced only in
specialized plant tissues or glands in the parent plant. The objective of the present study was to induce
high frequency withanolide rich adventitious roots under in vitro conditions by normalising the
supplementation of growth regulator in the culture media. Among phytohormones, auxin plays an
essential role in regulating roots development and it has been shown to be intimately involved in the
process of adventitious rooting. Among auxins IAA was first used for stimulating roots of cuttings [14] and
soon after another auxin which also promoted rooting; IBA was discovered and was considered even more
effective [15]. Wadegaonkar et al. (2006) reported that combinations of IAA and IBA were effective in
inducing adventitious roots from leaf explants of W. somnifera [16]. Thus, different combinations of IAA
Purushotham et al
BEPLS Vol 4 [7] June 2015 129 | P a g e ©2015 AELS, INDIA
and IBA were checked for Withania coagulans in the present study. Differences in the ability to form
adventitious roots have been attributed to differences in auxin metabolism [17].
The result showed 94.4% root induction in MS media supplemented with 4mg/L of IBA. This showsbetter
performance of IBA over IAA. The reason for these differences in root inducing ability may be higher
stability of IBA and slow but continuous release of IAA from IBA [18, 19] and release of IBA through
hydrolysis of conjugates results in better metabolism and transport [17]. But the results suggested that
IBA alone could not bring 100% root induction, since a better result was obtained in the combination of
both IAA and IBA. This may be due to the fact that IBA is a very simple ‘conjugate’ of IAA and must be
converted to IAA by β-oxidation to have an auxin effect as suggested by Muller et al. (2005) and that
either IBA itself is active or that it modulates the activity of IAA [20, 21].
It was observed that there was differences in pattern of root numbers induced per explant i.e. percentage
of root induction in each combination. Though many of the explants gave fast response in presence of
varying proportions of auxin supplemented, the number of roots induced differed. 100% of root induction
was observed in the combination 1.0 IAA and 4.0 IBA, followed by 94.4% in 4.0 IBA and 70.0% in 2.0 IAA
and 4.0 IBA.
From this study it is suggested that adventitious root cultures of W. coagulans are promising for large-
scale biomass production in suspension cultures and that it can be successfully taken to bioreactors for
mass proliferation. Similarly, adventitious root suspension cultures are proved to be efficient for biomass
accumulation in P. notoginseng [22] and Echinacea purpurea [23]. Since the roots contain a number of
therapeutically applicable withanolides, in vitro mass cultivation of roots will be an effective technique for
the large scale production of these secondary metabolites. The development of a fast growing root
culturing system would offer unique opportunities for producing root drugs under laboratory conditions
without having to depend on field cultivation. Therefore, this study proposes that mass cultivation of roots
of in vitro W. coagulans could be successfully preceded and would offer unique opportunities for
producing drugs from roots. Also the withanolide contents of the hairy root cultures of W. coagulans were
higher than in the root of the plant. In the hairy root cultures all the withanolides were accumulated in the
root tissues and withaferin A or withanolide A were not detected in the culture medium samples [4].
The effect of cytokine on shoot multiplication of Withania coagulans was observed and analysed taking the
shoot number and their attained length into consideration. The media free of cytokinen produced only
single shoot from the nodal explants along with rhizogenesis. TDZ resulted in excessive callusing and
retarded the growth and proliferation of shoots from the explants. Similar callusing problems in the nodal
explants with TDZ media have been previously reported in many studies [24, 25, 26, 27]. In the present
study, among different combinations of media investigated, 1BAP+0.5 Kin was more effective and shoot
multiplication was attained at its best levels. The frequency of shoot proliferation dropped with increasing
concentration of Kinetin (1BAP+1Kin). Also excessive Kin concentration in the media resulted early
maturation and yellowing of leaves in the proliferated shoots. Taking the shoot length into consideration,
it was observed that maximum length was attained in the media supplemented with no growth regulator
and it gradually decreased with increase in the hormone supplementation, indicating the shoot
proliferation and the shoot length are conflict to each other.
Suitable growth regulator type and concentration influences the growth of plants and plant tissues and
varies upon different plant species. This study supports the rapid root induction and shoots multiplication
of this important medicinal plant under in vitro conditions which can be further used in the
transformation studies.
Estimation of biochemical attributes is informative for finding the therapeutic potential of a plant/plant
part. Field roots are the sole part of the W. coagulans consumed orally to cure various diseases and
therefore phytochemical estimations of the in vitro cultured W. coagulans were quantified in comparison
to the field grown root extracts. Steroids, alkaloids and saponins being the important constituent of
withnolides, the major bioactive principle of genera Withania is reported to accumulate high in in vitro
cultures of W. coagulans than the field roots. Hence high content of these compounds in the in vitro
cultures exhibits great deal of pharmacological importance.
A rapid, precise and accurate identification and estimations of active marker componds as the qualitative
and quantitative target to measure the authenticity quality is essential [28]. HPTLC is one such efficient,
fast and reliable alternative to determinate and quantify natural single compounds. Using HPTLC, exact
quantification of the compounds can be achieved by scanning the bands with absorbance after separation
is achieved [29, 30].
In the present study, a densitometric HPTLC analysis was performed for the development of characteristic
finger print profile to quantify the therapeutic withanolides, withaferin A and withanone in the methanolic
extracts of W. coagulans. The extracts of W. coagulans showed the equal presence of withaferin A in all the
samples whereas the in vitro tissues of W. coagulans accumulated withanone in extravagant amounts.
Purushotham et al
BEPLS Vol 4 [7] June 2015 130 | P a g e ©2015 AELS, INDIA
Withaferin A and withanone belong to a class of compounds from Withania known as withanolides have
been identified as prominent phytocompounds with active pharmacological properties [31, 32, 33]. This is
the first report of increased withanone accumulations in in vitro cultures of W. coagulans. The production
of these secondary metabolites from plant under cultivation or grown in nature is constantly not
satisfactory and is activated only during a particular growth or developmental stage, stress or nutrient
availability or in specific season. Therefore, plant cell and tissue cultures have been a possible production
method for secondary metabolites [34, 35]. Production of useful compounds independent of soil profile
and climatic changes under controlled conditions, free of insects and microbes, improve productivity due
to automated cell growth control, rational regulation of metabolite productive process and extractable
organic substances from cell cultures [36]. W. coagulans root cultures should be explored as a potential
efficient method for producing useful withanolides owing to their enormous commercial value, the plants
scarcity in the natural environments and costly synthetic process of the secondary metabolites. In this
regard, adventitious root cultures have been used as a more stable and fast growing technique for
withanolides production. Based on the analytical assays, in vitro cultures of W. coagulans was found to be a
valuable reservoir of bioactive compounds with high medicinal values because of its higher
phytoconstituents, especially the steroids, alkaloids and saponins quantitatively compared to field grown
roots.
ACKNOWLEDGEMENT
We kindly acknowledge Department of Biotechnology, New Delhi (No. BT/PR11845/GBD/27/168/2009),
for financial support to carry out the present study.
REFERENCES
1. Sarasan, V., Cripps, R., Ramsay, M.M., Atherton, C., Mcmichen, M., Prendergast, G. & Rowntree, J.K. (2006).
Conservation in vitro of threatened plants-Progress in the past decade. In Vitro Cell. Dev. Biol. – Plant 42: 206-
214.
2. AbouZid, S.F., El-Bassuony, A.A., Nasib, A., Khan, S., Qureshi, J. & Choudhary, M.I. (2010). Withaferin A Production
by Root Cultures of Withania coagulans. Int. J. Appl. Res. Nat. Prod., 3: 23-27.
3. Loyola-Vargas, V. & Miranda-Ham, M. (1995). Root culture as a source of secondary metabolites of economic
importance. In Recent Advances in Phytochemistry, Volume 29. Phytochemistry of Medical Plants, Plenum Press,
NJ, pp. 217-248.
4. Mirjalili, H.M., Fakhr-Tabatabaei, S.M., Bonfill, M., Alizadeh, H., Cusido, R.M., Ghassempour, A.R. & Palazon, J.
(2009). Morphology and Withanolide Production of Withania coagulans Hairy Root Cultures. Eng. Life Sci. 9:197-
204.
5. Ali, N., Ahmad, B., Bashir, S., Shah, J., Azam, S. & Ahmad, M. (2009). Calcium Channel Blocking Activities of
Withania coagulans. Afr. J. Pharm. Pharmacol., 3: 439-442.
6. Hemalatha, S., Kumar, R. & Kumar, M. (2008). Withania coagulans Dunal: A Review. Phcog. Rev. 2: 351-358.
7. Kaileh, M., Berghe, W.V., Heyerick, A., Horion, J., Piette, J., Libert, C., Keukeleire, D.D., Essawi, T. & Haegeman, G.
(2007). Withaferin A Strongly Elicits IKKb Hyperphosphorylation, Concomitant with Potent Inhibition of Its
Kinase Activity. J. Biol. Chem. 282: 4253–4264.
8. Jain, R., Sinha, A., Kachhwaha, S. & Kothari, S.L. (2009). Micropropagation of Withania coagulans (Stocks) Dunal: a
critically endangered medicinal herb. J. Plant Biochem. Biotechnol. 18: 249–252.
9. Valizadeh, J. & Valizadeh, M. (2011). Development of Efficient Micropropagation Protocol for Withania coagulans
(Stocks) Dunal. Afr. J. Biotechnol. 10: 7611-7616.
10. Sangwan, R.S., Chaurasiya, N.D., Lal, P., Misra, L., Uniyal, G.C., Tuli, R. & Sangwan, N.S. (2007). Withanolide A
Biogeneration In invitro Shoot Cultures of Ashwagandha (Withania somnifera Dunal), A Main Medicinal Plant In
Ayurveda. Chem. Pharma. Bull. 55: 1371–1375.
11. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiol. Plant 15: 472-497.
12. Patel, J.B., Lahiri, S.K. & Shah, M.B. (2009). Development of a new method for identification and estimation of
Withania somnifera root, and a method for quantitative analysis of withaferin A in young and old roots. J. Planar
Chromatogr. Mod. TLC 22: 283–286.
13. Harbome, J.B. (1998). Phytochemical methods. In a guide to modern techniques of plant analysis, 3rd edition, pp.
40-137.
14. Cooper, W.C. (1935) Hormones in Relation to Root Formation on Stem Cuttings. Plant Physiol., 10: 789-794.
15. Zimmerman, P.W. & Wilcoxon, F. (1935). Several chemical growth substances which cause initiation of roots and
other responses in plants. Contrib. Boyce Thompson Inst. 7: 209–229.
16. Wadegaonkar, P.A., Bhagwat, K.A. & Rai, M.K. (2006). Direct Rhizogenesis and Establishment of fast Growing
Normal Root Organ Culture of Withania somnifera Dunal. Plant Cell Tiss. Org. Cult. 84: 223-225.
17. Epstein, E. & Ludwig-Muller, J. (1993). Indole-3-butyric Acid in Plants: Occurrence, Synthesis, Metabolism and
Transport. Physiol. Plant 88: 382-389.
18. Krieken, W.M., Breteler, H., Visser, H.M. & Mavridou, D. (1993). The role of the conversion of IBA into IAA on root
regeneration in apple: Introduction of a test system. Plant Cell Rep. 12: 203-206.
Purushotham et al
BEPLS Vol 4 [7] June 2015 131 | P a g e ©2015 AELS, INDIA
19. Liu, Z.H., Wang, W.C. & Yen, Y.S. (1998). Effect of hormone treatment on root formation and endogenous indole-3-
acetic acid and polyamine levels of Glycine max cultivated in vitro. Bot. Bull. Acad. Sin. 39: 113-118.
20. Muller, L.J., Vertocnik, A. & Town, C.D. (2005). Analysis of indole-3-butyric acid-induced adventitious root
formation on Arabidopsis stems segments. J. Exp. Bot. 56: 2095-105.
21. van der Krieken, W.M., Breteler, H., Visser, M.H.M. & Mavridou, D. (1993). The role of IBA into IAA on root
regeneration in apple: Introduction of a test system. Plant Cell Rep. 12: 203-206.
22. Gao, X., Zhu, C., JIa, W., Gao, W., Oiu, M. & Xiao, P. (2005). Induction and characterization of adventitious roots
directly from leaf explants of Panax notoginseng. Biotechnol. Lett. 27: 1771-1775.
23. Wu, C.H., Murthy, H.N., Hahn, E.J. & Paek, K.Y. (2008). Establishment of adventitious root co-culture of Ginseng
and Echinacea for the production of secondary metabolites. Acta Physiol. Plant 30: 891-896.
24. Faisal, M. & Anis, M. (2006) Thidiazuron induced high frequency axillary shoot multiplication in Psoralea
corylifolia L. Biol. Plantarum 50: 437-440.
25. Ray, A. & Bhattacharya, S. (2008). An improved micropropagation of Eclipta alba by in vitro priming with
chlorocholine chloride. Plant Cell Tiss. Org. Cult. 92: 315-319.
26. Sivanesan, I., Hwang, S.J. & Jeong, B.R. (2008). Influence of plant growth regulators on axillary shoot multiplication
and iron source on growth of Scrophularia takesimensis Nakai-a rare endemic medicinal plant. Afr. J. Biotechnol.
7: 4484-4490.
27. Sivanesan, I., Song, J.Y., Hwang, S.J. & Jeong, B.R. (2011). Micropropagation of Cotoneaster wilsonii Nakai-a rare
endemic ornamental plant. Plant Cell Tiss. Org. Cult. 105: 55-63.
28. Shrikumar, S. & Ravi, R.K. (2007). Approaches towards development and promotion of herbal drugs. Phcog. Rev.
1: 180-184.
29. Hans-Deinstrop, E. (2000) Applied thin layer chromatography: Best practice and avoidance of mistake, 2nd
edition, Published by willy –VCH verlag GmbH and co, Weinheim.
30. Khoobdel, M., Nematollah, J. & Babak, S. (2007). Quantitative and qualitative determination of dimethyl phthalate
and N,N-diethyl-m-toluamide in repellents commercial formulation by high performance thin layer
chromatography. Pak. J. Biol. Sci. 10: 3678-3682.
31. Priyandoko, D., Ishii, T., Kaul, S.C. & Wadhwa, R. (2011). Ashwagandha leaf derived withanone protects normal
human cells against the toxicity of methoxyacetic acid, a major industrial metabolite. PLoS One 6: e19552.
32. Grover, A., Priyandoko, D., Gao, R., Shandilya, A., Widodo, N., Bisaria, V.S., Kaul, S.C. & Wadhwa, R. (2012).
Withanone binds to mortalin and abrogates mortalin-p53 complex: computational and experimental evidence.
Int. J. Biochem. Cell Biol. 44: 496–504.
33. Grover, A., Shandilya, A., Bisaria, V.S. & Sundar, D. (2010). Probing the anticancer mechanism of prospective
herbal drug withaferin A on mammals: a case study on human and bovine proteasomes. BMC Genomics 4: S4-S15.
34. Rao, S. & Ravishankar, G.A. (2002). Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol.
Adv. 20: 101-153.
35. Verpoorte, R., Contin, A. & Memelink, J. (2002). Biotechnology for the production of plant secondary metabolites.
Phytochem. Rev. 1: 13-25.
36. Vijayasree, N., Udayasri, P., Kumar, A.Y., Babu, R.B., Kumar, P.Y. & Varma, V.M. (2010). Advancements in the
production of secondary metabolites. J. Nat. Prod. 3: 112-123.
CITATION OF THIS ARTICLE
Preethi M. P, Archana T M, Pradeepa D, Kalaiselvi S. In vitro propagation and enhancement of phytoconstituents in
Withania coagulans, a rare medicinal plant. Bull. Env. Pharmacol. Life Sci., Vol 4 [7] June 2015: 122-131
Purushotham et al
