J. Plant Biochemistry & Biotechnology Vol.

18(2), 249-252, July 2009

Short Communication

Micropropagation of Withania coagulans (Stocks) Dunal:

A Critically Endangered Medicinal Herb

Rohit Jain1, Arunima Sinha1, Sumita Kachhwaha1, 2 and S L Kothari1, 2*

1
Department of Botany, University of Rajasthan, Jaipur 302 004, India
2
Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302 004, India

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and
an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken
from adult plant, and cultured on MS medium enriched with BA (0.5 mg l-1), Kn (0.5 mg l-1) and PG (0.5 mg l-1). Nodal segments
and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary
bud multiplication. Addition of PG (0.5 mg l-1) in the regeneration medium significantly improved induction and elongation
of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l-1) and PG (0.5 mg
l-1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l-1)
+ PAA (0.5 mg l-1) + CC (2 mg l-1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4
subcultures of 2 weeks each.

Key words: Withania coagulans, micropropagation, phloroglucinol, choline chloride.

Withania species (Solanaceae) are the natural source of
withanolides (steroidal lactones) which have potential
antitumor, antimicrobial and immunomodulatory properties
(1). Fruits of W. coagulans are also used for milk coagulation
(2). The extract of the plant exhibits free radical scavenging
(3) and hypolipidemic activity (4). Coagulin-H (1), isolated
from W. coagulans (5) has been identified as
immunosuppressive drug (6).
The natural propagation of W. coagulans occurs
through seeds but chances of seed setting get limited due
to unisexual nature of flowers. Overexploitation and the
reproductive failure have rendered the species highly
vulnerable to complete extinction. To date, there have not
been any reports of ex situ conservation of this plant
through tissue culture. We now report an efficient and
reproducible protocol for micropropagation of W.
coagulans.
Only two plants of Withania coagulans were spotted
in the wild in Ajmer district and the explants were taken
from one of these plants. MS (7) basal medium
supplemented with 3% sucrose, pH adjusted to 5.8 before
*Corresponding author. E-mail: kothari-sl@uniraj.ernet.in
Abbreviations: BA - 6-benzylaminopurine, CC - choline chloride,
IAA - indole-3-acetic acid, IBA - indole-3-butyric acid, Kn - kinetin,
NAA - α-naphthaleneacetic acid, PAA - phenylacetic acid, PG -
phloroglucinol, RAPD - random amplified polymorphic DNA
autoclaving at 1.06 kg cm-2 (121°C) for 20 min was used in
all the experiments. The cultures were incubated at 25 ±
1 ° C under a 16-h photoperiod with 25µmol m -2 s -1
photosynthetic photon flux density (PPFD) provided by cool
white fluorescent tubes (40 W; Philips, India). Nodal
segments from field grown plant were thoroughly washed
in 5% (v/v) Teepol, surface sterilized with 70% (v/v) ethanol
for 30s, followed by an aqueous solution of 0.1% (w/v)
freshly prepared HgCl2 solution for 3 min. Finally, the
explants were thoroughly washed with sterile distilled water
and inoculated onto MS medium supplemented with BA or
Kn at 0.5, 1, 2, 3 and 5 mg l-1 either alone or in combination.
Various concentrations (0.5, 1, 2, 5 and 10 mg l-1) of PG
(Sigma, USA) and CC (Sigma, USA) were also tested with
optimal cytokinin concentration. Shoot buds induced in
primary cultures were sectored in clumps of 3-4 and
cultured on fresh medium for further multiplication of shoot
buds.
The in vitro-raised microshoots (2–3 cm in length)
were harvested for rooting. Two step rooting procedure
was followed. Step one involved the pulse treatment of
individual shoots with PG or CC (0.5, 1, 2, 5 and 10 mg l-1)
either alone or in combination with IBA and PAA at 10, 50
and 100 mg l-1 for 7 days on MS liquid medium using a filter
paper bridge. In step two, the pre treated microshoots were
transferred onto ½ or ¼ MS, agar-gelled semisolid medium
250 J Plant Biochem Biotech

with 3% sucrose supplemented with IBA /IAA/ NAA/ PAA Table 1. Shoot bud formation from nodal segments of W. coagulans
cultured on MS medium supplemented with BA and Kn
(0.25 – 1 mg l-1) either alone or in combination. Cultures
were evaluated after 4 weeks. Histological preparations BAP Kn Percent response Mean No. of
(mg l-1) (mg l-1) (%) Buds/Explant ± S.E.
were made as described (8).
0.5 0 57 3.8a ± 0.4
Plantlets were then removed from the vessels, washed 1 0 68 6.4b ± 0.4
gently with water and transferred to pots containing 1:1 2 0 74 7.0c ± 0.4
mixture of garden soil and organic manure. 3 0 79 9.0d ± 0.4
5 0 83 11.2e ± 0.5
DNA was extracted from the leaves of 19 randomly
0 0.5 43 2.4f ± 0.1
selected regenerated plants and from the leaves of mother
0 1 55 3.0c ± 0.4
plant (WM). The sample was powdered in liquid nitrogen (-
0 2 55 3.7d ± 0.2
196°C) and stored at -20°C until use for DNA extraction by
0 3 66 5.0x ± 0.3
CTAB method (9). Twelve RAPD primers were taken to
0 5 73 3.2g ± 0.3
assess the clonal fidelity of the regenerated shoots. The 0.5 0.5 83 18.6 h ± 0.5
PCR amplification conditions were, an initial denaturation S.E. – Standard error
at 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, Means in a column followed by different letters are significantly
50 ° C for 45 seconds and 72°C for 1 min, and a final different from each other

extension at 72°C for 5 min.

The data on shoot formation and rooting were collected
after 4 weeks. Each treatment consisted of twenty
replicates. Three explants were cultured per conical flask
and single explant was cultured per test tube. All
experiments were repeated twice. The data was analyzed
statistically using one –way analysis of variance (ANOVA)
by Fischer’s least significant difference (P = 0.05; 10).
obtained after 3 weeks (Table 1; Fig. 1a). Proliferating shoot
cultures were established by subculturing the shoots on
MS medium with BAP (0.5 mg l-1) + Kn (0.5 mg l-1) in clumps
of 3-4 buds. Nodal segments and shoot tips were also
used from regenerated shoots after 4 weeks of shoot bud
initiation. Each explant formed up to 21 shoot buds but
these were too short (0.3-0.5 cm), and not suitable for
micropropagation.

The explants inoculated on MS medium responded
differently on BA and Kn (Table 1). BA gave better response
than Kn in terms of induction of shoot buds. BA (0.5 mg l-1)
in combination with Kn (0.5 mg l-1) proved best for induction
of multiple shoots. An average of 19 shoots (1cm) could be
PG is a phenolic compound that stimulates shoot and
root growth in shoot cultures (11). The addition of PG (0.5
mg l-1) along with BA (0.5 mg l-1) and Kn (0.5 mg l-1) in MS
medium improved the establishment of nodal explant
cultures (Table 2, Fig. 1b). The use of PG during

Table 2. Shoot bud formation from nodal segments and shoot-tips of W. coagulans (excised from in vitro raised shoots) cultured on MS
medium supplemented with BA (0.5 mg l -1) + Kn (0.5 mg l -1) and different concentrations of PG or CC
PG CC Nodal segments Shoot-tips
-1 -1
(mg l ) (mg l ) Mean No. of Mean length of Mean No. of Mean length of
Buds/Explant ± S.E. Shoots (cm) ± S.E. Buds/Explant ± S. E. Shoots (cm) ± S. E.
0 0 20.9a ± 0.3 0.5a ± 0.1 22.3a ± 0.4 0.3a ± 0.1
0.5 0 23.4b ± 0.2 4.3b ± 0.2 24.6b ± 0.3 4.7b ± 0.2
1 0 21.1c ± 0.3 4.1b ± 0.1 23.3c ± 0.1 4.4bc ± 0.1
2 0 19.2d ± 0.3 3.5c ± 0.2 20.4d ± 0.3 4.2c ± 0.2
3 0 18.3e ± 0.2 3.1d ± 0.1 19.3e ± 0.3 3.9d ± 0.2
5 0 15.5f ± 0.5 3.0d ± 0.1 17.5f ± 0.2 3.6d ± 0.1
0 0.5 21.0c ± 0.4 3.8e ± 0.2 23.9g ± 0.3 4.6be ± 0.2
0 1 17.7g ± 0.5 3.3f ± 0.1 22.4a ± 0.3 4.3e ± 0.1
0 2 14.3h ± 0.3 2.8d ± 0.2 21.5h ± 0.5 3.7d ± 0.2
0 3 13.5i ± 0.2 2.4g ± 0.1 19.1i ± 0.6 3.4d ± 0.0
0 5 13.1j ± 0.3 2.2g ± 0.1 14.5j ± 0.3 2.9e ± 0.1
S.E. – Standard error
Means in a column followed by different letters are significantly different at P = 0.05 from each other
 Short Communication 251

Fig. 1. In vitro regeneration of W. coagulans. (a) Induction of shoot buds from nodal explants of W. coagulans cultured on MS medium with
BA (0.5 mg l-1) + Kn (0.5 mg l-1), (b) Proliferation and elongation of shoot buds on MS medium with BA (0.5 mg l-1) + Kn (0.5 mg l-1) + PG (0.5
mg l-1), (c-d) Histological details of the shoot bud formation from the shoot tip (c) and nodal segments (d), (e) Rooting on half strength MS
medium with IBA (0.25 mg l-1) + PAA (0.5 mg l-1) + CC (2 mg l-1), and (f) Agarose gel electrophoresis of RAPD fragments of W. coagulans
showing banding patterns of 20 plants amplified by the primer OPA-19.
252 J Plant Biochem Biotech
multiplication has improved shoot multiplication in several
species (12). Rastogi et al (13) have also advocated
incorporation of PG in the medium for better growth of
cultures. The ability of shoot multiplication was maintained
up to 12 subcultures, at 2-wk interval, on MS medium
supplemented with BA (0.5 mg l-1) and Kn (0.5 mg l-1).
Histological studies revealed that in the axil of each
leaf, a distinct meristematic zone of small densely stained
cells was present over a differentiated zone. A ring of
multiple shoot primordia could be observed arising directly
from base of cultured shoot tip (Fig. 1c). In the cultured
nodes, at a later stage of development, vertical and
sideways expansion of the meristematic zone occurred
(Fig. 1d).
The maximum frequency of root formation (80%),
highest number (11.5±0.7) of roots and root length
(7.9±0.3cm) were seen after pulse treatment of shoots in
MS medium containing 10 mg l-1 CC and 0.5 mg l-1 PG
followed by their transfer to ½ strength MS medium with
IBA (0.25 mg l-1), PAA (0.5 mg l-1) and CC (2 mg l-1) after 7
days (Fig. 1e). Two-step procedure for rooting has been
used to advantage in several woody species (14). The
incorporation of CC at different concentrations enhanced
the response of rooting of shoots significantly. CC and PG
have enhanced rooting in Bambusa tulda (15). These
compounds are reported to enhance rooting by acting as
auxin protectors to increase the free endogenous IAA levels
during the inductive phase of rooting (16).
The plantlets were successfully hardened inside the
culture room under diffused light on MS medium for 2
weeks, followed by their establishment in pots containing
(1:1) soil and manure in greenhouse. About 75% of the
micropropagated plants survived after transfer to soil and
organic manure (1:1). All the established plants were
apparently uniform and did not show any detectable
variation.
Clonal fidelity of the regenerated shoots was checked
through RAPD. Of 12 random primers, 8 generated distinct,
reproducible products. A total of 580 amplification products
were detected. The primers OPA-5 and OPA-19 (Fig. 1f)
gave highly reproducible banding pattern. Fingerprinting
profiles of regenerants were monomorphic and there was
no variation amongst mother and tissue culture raised
plants. There are number of reports demonstrating the
suitability of enhanced axillary branching for raising true
to type plants (17). Similar results have been obtained in
present investigation.
The protocol offers a potential system for a large-scale
propagation and conservation of this medicinal plant and
would facilitate its improvement programme using genetic
transformation and metabolic engineering techniques.
Acknowledgements
We thank Council of Scientific and Industrial Research
(CSIR), New Delhi for the financial support in the form of a
R&D project: CSIR-38(1178)/EMR-II/07. Rohit Jain and
Arunima Sinha also thank CSIR for the award of Senior
Research Fellowships.
Received 20 January, 2009; accepted 8 July, 2009.
Online published 18 July, 2009.
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