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*1Department of Botany, University of Rajasthan, Jaipur 302 004, India2Centre 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 andan important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments takenfrom 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 segmentsand shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillarybud multiplication. Addition of PG (0.5 mg l-1) in the regeneration medium significantly improved induction and elongationof shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l-1) and PG (0.5 mgl-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 4subcultures 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

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-2s-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 waterand inoculated onto MS medium supplemented with BA orKn 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 withoptimal cytokinin concentration. Shoot buds induced inprimary cultures were sectored in clumps of 3-4 andcultured on fresh medium for further multiplication of shootbuds.

The in vitro-raised microshoots (2–3 cm in length)were harvested for rooting. Two step rooting procedurewas followed. Step one involved the pulse treatment ofindividual 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

*Corresponding author. E-mail: kothari-sl@uniraj.ernet.inAbbreviations: 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

250 J Plant Biochem Biotech

with 3% sucrose supplemented with IBA /IAA/ NAA/ PAA

(0.25 – 1 mg l-1) either alone or in combination. Cultures

were evaluated after 4 weeks. Histological preparations

were made as described (8).

Plantlets were then removed from the vessels, washed

gently with water and transferred to pots containing 1:1

mixture of garden soil and organic manure.

DNA was extracted from the leaves of 19 randomly

selected regenerated plants and from the leaves of mother

plant (WM). The sample was powdered in liquid nitrogen (-

196°C) and stored at -20°C until use for DNA extraction by

CTAB method (9). Twelve RAPD primers were taken to

assess the clonal fidelity of the regenerated shoots. The

PCR amplification conditions were, an initial denaturation

at 94°C for 5 min followed by 35 cycles of 94°C for 30 sec,

50°C for 45 seconds and 72°C for 1 min, and a final

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).

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

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.

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 1. Shoot bud formation from nodal segments of W. coagulanscultured on MS medium supplemented with BA and Kn

BAP Kn Percent response Mean No. of(mg l-1) (mg l-1) (%) Buds/Explant ± S.E.

0.5 0 57 3.8a ± 0.4

1 0 68 6.4b ± 0.4

2 0 74 7.0c ± 0.4

3 0 79 9.0d ± 0.4

5 0 83 11.2e ± 0.5

0 0.5 43 2.4f ± 0.1

0 1 55 3.0c ± 0.4

0 2 55 3.7d ± 0.2

0 3 66 5.0x ± 0.3

0 5 73 3.2g ± 0.30.5 0.5 83 18.6 h ± 0.5

S.E. – Standard errorMeans in a column followed by different letters are significantlydifferent from each other

Table 2. Shoot bud formation from nodal segments and shoot-tips of W. coagulans (excised from in vitro raised shoots) cultured on MSmedium 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

(mg l-1) (mg l-1) Mean No. of Mean length of Mean No. of Mean length ofBuds/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 ± 0.10.5 0 23.4b ± 0.2 4.3b ± 0.2 24.6b ± 0.3 4.7b ± 0.21 0 21.1c ± 0.3 4.1b ± 0.1 23.3c ± 0.1 4.4bc ± 0.12 0 19.2d ± 0.3 3.5c ± 0.2 20.4d ± 0.3 4.2c ± 0.23 0 18.3e ± 0.2 3.1d ± 0.1 19.3e ± 0.3 3.9d ± 0.25 0 15.5f ± 0.5 3.0d ± 0.1 17.5f ± 0.2 3.6d ± 0.10 0.5 21.0c ± 0.4 3.8e ± 0.2 23.9g ± 0.3 4.6be ± 0.20 1 17.7g ± 0.5 3.3f ± 0.1 22.4a ± 0.3 4.3e ± 0.10 2 14.3h ± 0.3 2.8d ± 0.2 21.5h ± 0.5 3.7d ± 0.20 3 13.5i ± 0.2 2.4g ± 0.1 19.1i ± 0.6 3.4d ± 0.00 5 13.1j ± 0.3 2.2g ± 0.1 14.5j ± 0.3 2.9e ± 0.1

S.E. – Standard errorMeans 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 withBA (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 (0.5mg 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 MSmedium 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. coagulansshowing 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 eachleaf, a distinct meristematic zone of small densely stainedcells was present over a differentiated zone. A ring ofmultiple shoot primordia could be observed arising directlyfrom base of cultured shoot tip (Fig. 1c). In the culturednodes, at a later stage of development, vertical andsideways 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 inMS medium containing 10 mg l-1 CC and 0.5 mg l-1 PGfollowed by their transfer to ½ strength MS medium withIBA (0.25 mg l-1), PAA (0.5 mg l-1) and CC (2 mg l-1) after 7days (Fig. 1e). Two-step procedure for rooting has beenused to advantage in several woody species (14). Theincorporation of CC at different concentrations enhancedthe response of rooting of shoots significantly. CC and PGhave enhanced rooting in Bambusa tulda (15). Thesecompounds are reported to enhance rooting by acting asauxin protectors to increase the free endogenous IAA levelsduring the inductive phase of rooting (16).

The plantlets were successfully hardened inside theculture room under diffused light on MS medium for 2weeks, followed by their establishment in pots containing(1:1) soil and manure in greenhouse. About 75% of themicropropagated plants survived after transfer to soil andorganic manure (1:1). All the established plants wereapparently uniform and did not show any detectablevariation.

Clonal fidelity of the regenerated shoots was checkedthrough RAPD. Of 12 random primers, 8 generated distinct,reproducible products. A total of 580 amplification productswere detected. The primers OPA-5 and OPA-19 (Fig. 1f)gave highly reproducible banding pattern. Fingerprintingprofiles of regenerants were monomorphic and there wasno variation amongst mother and tissue culture raisedplants. There are number of reports demonstrating thesuitability of enhanced axillary branching for raising trueto type plants (17). Similar results have been obtained inpresent 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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