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: [email protected]: 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.
References
1 Agarwal R, Diwanay S, Patki P & Patwardhan B, JEthnopharmacol, 67 (1999) 27.
2 Bhandari MM, Flora of the Indian desert, MPS Repros,Jodhpur, India (1995) pp 246.
3 Hemalatha S, Wahi AK, Singh PN &Chansouria JPN, JEthnopharmacol, 93 (2004) 261.
4 Maurya R, Jayendra A, Singh AB & Srivastava AK, BioorgMed Chemis Lett, 18 (2008) 6534.
5 Atta-ur-Rahman, Yousaf M, Gul W, Qureshi S,Choudhary MI, Voelter W, Hoff A, Jens F & Naz A,Heterocycles, 48 (1998) 1801.
6 Mesaik MA, Zaheer-ul-Haq, Murad S, Ismail Z, AbdullahNR, Gill HK, Atta-ur-Rahman, Yousaf M Siddiqui RA,Ahmad A & Choudhary MI, Mol Immun, 43 (2006)1855.
7 Murashige T & Skoog T, Physiol Plant, 15 (1962) 473.
8 Johansen DA, Plant microtechnique, Mc Graw-Hill BookCompany, Inc., New York, USA (1940).
9 Doyle IJ & Doyle JL, Focus, 12 (1990) 13.
10 Gomez KA & Gomez AA, Statistical procedures foragricultural research, John Wiley and Sons, New York (1984)
11 Sarkar D & Naik PS, Plant Cell Tiss Org Cult, 60 (2000) 139.
12 Ibanez MR & Amo-Marc JB, Plant Growth Reg, 26 (1998)49.
13 Rastogi S, Rizvi SMH, Singh RP & Dwivedi UN, Biol Plant,52 (2008) 743.
14 Husain MK & Anis M, In Biotechnology for a better future(L D’Souza, M Anuradha, S Nivas, S Hegde, K Rajendra,Editors). SAC, Mangalore (2004) p 294.
15 Mishra Y, Patel PK, Yadev S, Shirin F & Ansari SA, SciHort, 115 (2008) 315.
16 Faivre-Rampant O, Kevers C & Gaspar T, Plant Sci, 153(2004)73.
17 Rani V & Raina SN, In Vitro Cell Dev Biol-Plant, 36 (2000)319.