RESEARCH NOTE

Adventitious shoot regeneration and in vitro biosynthesisof steroidal lactones in Withania coagulans (Stocks) Dunal

Rohit Jain • Arunima Sinha • Devendra Jain •

Sumita Kachhwaha • S. L. Kothari

Received: 5 June 2010 / Accepted: 30 August 2010

� Springer Science+Business Media B.V. 2010

Abstract A micropropagation system through leaf explant

culture has been developed for Withania coagulans. Shoot

bud proliferation occurred through both adventitious and de

novo routes depending on the hormonal regime of the culture

medium. Green compact nodular organogenic callus devel-

oped on Murashige and Skoog (MS) medium supplemented

with 2.3 lM kinetin (Kn) and lower levels of 6–benzylade-

nine (BA) (13.3 lM) while multiple adventitious shoot bud

differentiation occurred on medium fortified with 2.3 lM

kinetin (Kn) and higher levels of BA (22.2 lM). Shoot buds

were transferred to proliferation medium containing 2.2 lM

BA, 2.3 lM Kn, and 3.9 lM phloroglucinol (PG) for further

growth and development of shoot system. Elongated shoots

were rooted using a two-step procedure involving pulse

treatment of 7 days in a medium containing 71.6 lM choline

chloride (CC) and 3.9 lM PG and then transferred to rooting

medium containing � MS, 1.2 lM IBA, 3.6 lM PAA, and

14.3 lM CC for 3 weeks. Well-rooted plants were trans-

ferred to a greenhouse for hardening and further growth.

Random amplification of polymorphic DNA (RAPD)

showed monomorphic bands in all the plants thereby con-

firming clonality of the regenerants. Thin layer chromatog-

raphy (TLC) showed the presence of withanolides in the

regenerated plants. Quantification through reverse-phase

HPLC revealed increased concentration of withanolides in

the regenerated plants compared to the field-grown mother

plant. Accumulation of withaferin A and withanolide A

increased up to twofold and that of withanone up to tenfold.

Direct regeneration via leaf explants will be useful for

Agrobacterium-mediated genetic transformation, and will

facilitate pathway manipulation using metabolic engineering

for bioactive withanolides.

Keywords Micropropagation � HPLC � TLC � RAPD �Withania coagulans � Withanolides

Abbreviations

BA 6–benzyladenine

CC Choline chloride

DAD Diode array detector

IAA Indole–3–acetic acid

IBA Indole–3–butyric acid

Kn Kinetin

MS Murashige and Skoog

NAA a–naphthaleneacetic acid

PAA Phenylacetic acid

PG Phloroglucinol

RAPD Random amplification of polymorphic DNA

TLC Thin layer chromatography

Introduction

Withania coagulans (fam. Solanaceae) is commercially

important for its ability to coagulate milk, in the treatment

of ulcers, rheumatism, dropsy, consumption and sensile

debility (Bhandari 1995). Antimicrobial, anti-inflamma-

tory, antitumor, hepatoprotective, antihyperglycemic, car-

diovascular, immunosuppressive, free radical scavenging

and central nervous system depressant activities of the

R. Jain � A. Sinha � D. Jain � S. Kachhwaha � S. L. Kothari

Department of Botany, University of Rajasthan,

Jaipur 302004, India

S. Kachhwaha � S. L. Kothari (&)

Centre for Converging Technologies (CCT),

University of Rajasthan, Jaipur 302004, India

e-mail: slkothari@lycos.com

123

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-010-9840-3

plant have also been demonstrated (Maurya and Akanksha

2010). Pharmacological investigations have elucidated

association of these activities with the specific steroidal

lactones known as withanolides present in Withania (Atta-

ur-Rahman et al. 1998). Withaferin A, withanolide A and

withanone are the major withanolides present in W. som-

nifera and W. coagulans. Overexploitation and the repro-

ductive failures forced the species W. coagulans towards

the verge of extinction (Jain et al. 2009b). The in vitro

shoot cultures could provide an alternative to field plant

harvesting for the production of therapeutically valuable

compounds (Sangwan et al. 2007). There are no reports of

in vitro plant regeneration in W. coagulans except our

earlier report using nodal and shoot tip explant cultures

(Jain et al. 2009b). Here, we report regeneration from leaf

explants and production of withanolides from the regen-

erated plants for the first time.

Materials and methods

Plant material and establishment of in vitro cultures

from leaf explants

Leaf explants (0.8–2 cm) were collected from the field-

grown plants spotted in Ajmer (Rajasthan) in 2007. The

species was identified by the Herbarium, Dept. of Botany,

University of Rajasthan, Jaipur. Explants were thoroughly

washed under running tap water for 15 min followed by

treatment with 20% Extran (liquid detergent; Merck, India)

for 5 min. Eventually, the explants were aseptically surface

sterilized with 0.1% (w/v) HgCl2 (Merck, India) solution

for 3 min. Explants were rinsed 4–5 times with sterile

distilled water and cultured on full- and half-strength MS

(Murashige and Skoog 1962) medium supplemented with

3% sucrose (Merck, India) and 0.9% agar (bacteriological

grade; Merck, India). Various concentrations and combi-

nations of different plant growth regulators (Sigma, India)

including 6–benzyladenine (BA; 2.2, 4.4, 8.8, 13.2 and

22.2 lM), kinetin (Kn; 2.3, 4.6, 9.2, 13.9 and 23.2 lM),

indole-3-acetic acid (IAA; 1.1, 1.7 and 2.8 lM), indole-3-

butyric acid (IBA; 0.9, 1.4 and 2.4 lM), phenylacetic acid

(PAA; 1.4, 2.2 and 3.6 lM) and a–naphthaleneacetic acid

(NAA; 1.0, 1.6 and 2.6 lM) were added in the medium to

optimize growth and differentiation. The pH of the medium

was adjusted to 5.8 followed by sterilization at 1.2 kg/cm2

pressure and 121�C temperature for 20 min. Leaf explants

with or without petiolar parts were placed abaxially on the

medium. Cultures were maintained at 26 ± 1�C under 16/

8 h photoperiod with 25 lmol m-2 s-1 photosynthetic

photon flux density provided by white fluorescent tubes

(40 W; Philips, India). Twenty replicates were maintained

for each treatment. The numbers of responding explants

and shoot buds developed per explant were recorded and

shoot buds were subcultured on first stage proliferation

medium (MS, 2.2 lM BA, and 2.3 lM Kn) containing

3.9 lM phloroglucinol (PG) to further enhance growth and

development of shoot buds. Regenerated shoots of appro-

priate length ([3 cm) were subjected to a two-step rooting

procedure involving pulse treatment of 7 days on � MS,

71.6 lM choline chloride (CC) and 3.9 lM PG and then

transferred to rooting medium containing � MS, 1.2 lM

IBA, 3.6 lM PAA, and 14.3 lM CC prior to hardening as

described previously (Jain et al. 2009b). The data on shoot

bud formation and rooting were collected after 4 weeks.

Three explants per flask and single explant per test tube

was cultured. All experiments were repeated twice.

RAPD analysis

DNA was extracted from the leaves of 17 randomly selected

regenerated plants and from the leaves of mother plant

(WM). The leaf samples were powdered in liquid nitrogen

and stored at -20�C until used for DNA extraction by CTAB

method (Doyle and Doyle 1990). The PCR amplification

conditions were: an initial denaturation at 94�C for 4 min

followed by 40 cycles of 94�C for 45 s, 37�C for 45 s and

72�C for 2 min, and a final extension at 72�C for 10 min. The

amplicons were separated through 1.2% agarose (Himedia,

India) gel electrophoresis and photographed using Gel

Documentation System (Bio-Rad, Germany).

Extraction of withanolides

All the analytical and HPLC grade solvents, reagents and

precoated silica gel TLC plates were purchased from

Merck. Isolation of withanolides from various tissues was

performed using the method described by Sangwan et al.

(2007).

Qualitative and quantitative analysis of withanolides

Qualitative withanolide profiling was done through TLC

while quantification was carried out through HPLC as

described by Sangwan et al. (2007). For TLC, 10 ll sample

was loaded on precoated silica gel G-60 plates, performed in

a solvent system consisting of chloroform:ethyl ace-

tate:methanol:toluene (74:4:8:30, v/v), and development

was done with anisaldehyde reagent (250 ll anisaldehyde in

a mixture of 20 ml acetone, 80 ml water and 10 ml 60%

perchloric acid) followed by heating at 110�C. HPLC anal-

ysis was performed on Agilent (Germany) model 1200 and

separation was achieved by a reverse-phase column (Eclipse

XDB c-18, 4.5 mm 9 150 mm, particle size 1.8 lm; Agi-

lent) using water (A) and methanol (B), each containing

0.1% acetic acid, as solvent and online UV-Diode Array

Plant Cell Tiss Organ Cult

123

Detector (UV-DAD) at 227 nm. The solvent gradient was set

as A:B, 60:40–25:75, 0–45 min; 10:90, 45–60 min at a flow

rate of 0.6 ml min-1. Sample volume of 10 ll was injected

and the column temperature maintained at 27�C during the

run. Authentic withanolides including withaferin A, witha-

none and withanolide A (Chromadex, CA, USA) were used

as markers to ascertain their discrete resolution from each

other under these conditions for both TLC and HPLC.

Computation of withanolide concentration in the samples

was done through a calibration curve of concentration versus

detector response (peak area) using different concentrations

of standard solutions of withaferin A, withanolide A and

withanone in methanol. The data was analyzed statistically

using one-way analysis of variance (ANOVA) by Fischer’s

least significant difference (P = 0.05) (Gomez and Gomez

1984). HPLC data was analyzed with the Chemstation LC–

3D software (Agilent).

Results and discussion

Leaf explants cultured in the absence of growth regulators

senesced without producing callus or adventitious buds,

whereas they responded with enlargement and swelling at

the cut petiolar end followed by callus formation on MS

medium supplemented with Kn (2.3 lM) or BA

(2.2–13.3 lM). Kn alone (Murch et al. 2004) or in com-

bination with auxins (Kachhwaha and Kothari 1996; Reddy

et al. 2004) and BA alone (Kulkarni et al. 2000; Sharma

et al. 2003; Tilkat et al. 2009) or in combination with

auxins (Koroch et al. 2002; Jain et al. 2009a; Kothari et al.

2010; Sinha et al. 2010) have most frequently been

reported to induce in vitro plant regeneration in a wide

range of monocotyledonous and dicotyledonous plants.

Therefore, we also examined the effect of IAA, NAA or

PAA in combination with BA or Kn on organogenesis. The

combination of BA or Kn with auxins was not conducive to

organogenesis. Brown, compact, nodular callus was

observed on medium supplemented with BA (13.3–22.2

lM) and IAA (1.1 lM) or IBA (0.9 lM) or PAA (1.4 lM),

but it could not induce any shoot buds. The amount of

callus increased with increasing concentration of auxins.

Rhizogenesis was observed all along the lamina cultured

on medium with BA (2.2–22.2 lM) with NAA (1.0–

2.6 lM). Kn in combination with auxins initiated forma-

tion of pale and non–morphogenic callus.

The use of 2.3 lM Kn in combination with BA

(2.2–13.3 lM) promoted the initiation and development of

shoot buds along with callus (Fig. 1a). Clusters of adventi-

tious shoots (17.6 ± 0.5) regenerated mostly from petiolar

base of leaf explants or at leaf midrib region on medium

supplemented with 22.2 lM BA and 2.3 lM Kn (Table 1,

Fig. 1b). This clearly demonstrated that the combination of

BA and Kn was the most important factor for shoot regen-

eration from leaf explants of W. coagulans. Combination of

BA with Kn for inducing shoot bud differentiation from the

explants has also been reported in several other plants (Dayal

et al. 2003; Baskaran and Jayabalan 2005; Sreedhar et al.

2008). Presence of petiolar part along with lamina was

essential for morphogenesis as no response was observed

when lamina without petiolar part was cultured. Previous

reports have shown the same impact including petioles for

enhancing shoot regeneration in several other plant species

such as Paulownia tomentosa (Corredoira et al. 2008),

Prunus persica (Gentile et al. 2002; Zhou et al. 2010), and P.

serotina (Liu and Pijut 2008). Shoot buds induced on

explants in the primary cultures were transferred to the

proliferation medium containing 2.2 lM BA and 2.3 lM Kn

for further differentiation of new shoot buds, but the elon-

gation of the shoot buds did not occur (Fig. 1c). A combi-

nation of 2.2 lM BA, 2.3 lM Kn and 3.9 lM PG was

required in the proliferation medium for the elongation of

shoot buds up to 2–3 cm, a length which was required for

rooting (Fig. 1d). PG has similarly been used by other

workers (Sarkar and Naik 2000; Feeney et al. 2007). Elon-

gated shoots ([3 cm) were transferred to � MS medium

containing 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC after

7 days of pulse treatment with 71.6 lM CC and 3.9 lM PG

for rooting. The incorporation of CC and PG enhanced

rooting significantly. These compounds have been reported

to act as auxin protectors and increase the endogenous IAA

levels during the inductive phase of rooting (Faivre-Rampant

et al. 2004). Use of CC and PG in enhancing rooting has also

been reported in Dendrocalamus hamiltonii (Sood et al.

2002) and Bambusa tulda (Mishra et al. 2008). The rooted

plantlets (Fig. 1e) were successfully transferred to the

greenhouse for hardening.

The regenerated plants were subjected to RAPD analysis

to check their clonality. Twenty random primers (OPF

1–10 and OPT 1–10) were used, of which 15 produced

distinct and reproducible bands. A total of 1,197 amplicons

were obtained and primer OPF-3 generated a highly

Table 1 Shoot bud formation from leaf explants of W. coagulanscultured on MS medium supplemented with BA and Kn

BA (lM) Kn (lM) % response Shoot buds

(Mean ± SE)

2.2 2.3 80 4.6 ± 0.5 e

4.4 2.3 86 7.7 ± 0.6 d

8.9 2.3 73 9.3 ± 0.6 c

13.3 2.3 93 12.1 ± 0.2 b

22.2 2.3 80 17.6 ± 0.5 a

SE Standard error

Means in a column followed by different letters are significantly

different from each other at P = 0.05

Plant Cell Tiss Organ Cult

123

reproducible banding pattern (Fig. 2). DNA fingerprinting

profiles of regenerants revealed that there was no variation

amongst mother and tissue culture-raised plants. There are

many reports demonstrating the suitability of enhanced

axillary branching for raising true-to-type plants (Rani and

Raina 2000).

Analysis of withanolide content in in vitro shoot cultures

of W. somnifera has been reported by several workers (Ray

and Jha 2001; Sangwan et al. 2004, 2007), but there are no

such reports for W. coagulans. The study used an analytical

reverse phase HPLC system providing symmetrical and

high resolution peaks of three important withanolides in the

plant. TLC of different extracts revealed that withaferin A,

withanolide A and withanone were biosynthesized in

regenerated plants of W. coagulans (Fig. 3). Withanolide

content was analyzed by HPLC, and standard samples of

withaferin A, withanolide A and withanone were used to

construct a calibrated graph by plotting peak areas versus

the amount of respective withanolide over a range of

50–1,000 ng ll-1. The response was linear over the tested

concentration range. The identification of withanolides was

confirmed on the basis of retention time and absorption

spectra on UV-DAD (32.46 min, 215 nm; 38.38 min,

Fig. 1 Shoot bud induction from leaf explants of W. coagulans.

a Indirect induction on MS, 13.3 lM BA and 2.3 lM Kn. b Direct

induction from petiolar end on MS, 22.2 lM BA and 2.3 lM Kn.

c Shoot buds developed on the first stage proliferation medium.

d Proliferation and elongation of shoots on MS, 2.2 lM BA, 2.3 lM

Kn and 3.9 lM PG. e Rooting on � MS, 1.2 lM IBA, 3.6 lM PAA

and 14.3 lM CC

Fig. 2 Agarose gel electrophoresis of RAPD fragments showing

banding pattern amplified by OPF–3 primer. M Molecular marker,

C control

Fig. 3 TLC profile of W. coagulans. Lanes 1 standard withaferin A, 2standard withanolide A, 3 standard withanone, 4 sample extracted

from in vitro shoots, 5 samples extracted from field leaves, 6 samples

extracted from callus, 7 samples extracted from field roots

Plant Cell Tiss Organ Cult

123

230 nm; and 40.90 min, 230 nm for withaferin A (Fig. 4a),

withanolide A (Fig. 4b) and withanone (Fig. 4c), respec-

tively). The accumulation of all the three withanolides was

higher in regenerated plants than in the samples taken from

field-grown plants (Fig. 4d, e). A shift towards organ dif-

ferentiation resulted in improved potential of the cultures to

synthesize withanolides. The quantities of withaferin A and

withanolide A increased up to two-fold while the witha-

none content increased up to ten-fold in the regenerated

plantlets as compared to field-grown plants (Table 2).

Withanolide A accumulates in small amounts in shoots

(Fig. 4e) and more in roots (Fig. 4f) in field-grown plants,

but in the present study the amount of withanolide A was as

good in regenerated shoots as in the roots of field plants

(Table 2, Fig. 4d). Several factors, e.g., the difference in

chemotype utilized as source for initiation of multiple

shoot buds, and culture conditions such as basal media

composition and growth regulator types utilized to estab-

lish cultures might have contributed to withanolide pro-

duction. The positive correlation between withanolide

synthesis and morphological differentiation suggests that

synthesis is regulated in a tissue-specific way and organ-

ogenesis is the key regulatory factor which stimulates

production of withanolides in vitro. The detection of higher

content in differentiated cultures also points out that the

enzymes responsible for biogenesis of withanolides in vitro

might be optimally active in the culture conditions as has

been shown earlier in W. somnifera (Sharada et al. 2007).

Taken as a whole, our results demonstrate that leaves of

W. coagulans have a great organogenic potential for shoot

bud formation; however, the response is highly sensitive

and directly related to the combinations of exogenous

growth regulators in the culture medium. The results also

Fig. 4 DAD–HPLC chromatogram of standards. a Withaferin A, b withanolide A, c withanone. Samples from d in vitro developed shoots,

e field leaves, and f field roots (insets are UV-DAD spectra of the specified withanolide)

Table 2 Withanolide content in different tissues of W. coagulans

Sample Withanolide Content (mg gfw-1) Mean ± SE

Withaferin A Withanolide A Withanone

Field leaves 0.084 ± 0.004 0.059 ± 0.014 0.031 ± 0.001

In vitro leaves 0.192 ± 0.005 0.123 ± 0.009 0.282 ± 0.006

Field roots Nil 0.113 ± 0.009 Nil

Plant Cell Tiss Organ Cult

123

confirm the potential of this plant to biosynthesize the

active principle (withanolides) under in vitro culture con-

ditions. In vitro regeneration of adventitious shoots is an

essential component for most of the genetic transformation

protocols. The system described here will be useful in this

respect and for conservation of elite germplasm of this

important medicinal plant species.

Acknowledgments Financial support from Council of Scientific

and Industrial Research (CSIR) in the form of R&D project: CSIR–

38(1178) EMR–II/2007 is gratefully acknowledged. Rohit Jain,

Arunima Sinha and Devendra Jain thank CSIR for the award of Senior

Research Fellowships.

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