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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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