Acad.

Form 10a

KERALA AGRICULTURAL UNIVERSITY

FACULTY OF AGRICULTURE
COLLEGE OF AGRICULTURE, VELLAYANI

PROGRAMME OF RESEARCH WORK FOR THESIS FOR MASTER’S DEGREE

1. Title of thesis 7. Practical/Scientific utility

Stimulation of withanolide accumulation in the Withania somnifera (L) Dunal (ashwagandha)

multiple shoot cultures of ashwagandha has been used for over 3,000 years in the
ayurvedic medicinal tradition of India (Kaileh
(Withania somnifera) (L) Dunal using elicitors.
et al., 2007). The drugs obtained from this plant
are used for physiological and metabolic
2. Location (S) restoration, anti-arthritic, anti-aging, anti-
cancer, cognitive function improvement in
College of Agriculture Vellayani
geriatric states and recovery from
neurodegenerative disorders (Mishra et al.,
3. (a) PC group 2000). In vitro and in vivo pharmacological
investigations have elucidated that the curative
(8) Biotechnology, Biochemistry and Plant properties have been associated with
Physiology withanolides present in the leaves and roots of
these plants (Mirjalili et al., 2009). The
(b)Thrust area withanolides constituents have been reported to
vary with different tissue types (Sangwan et al.,
2007). This causes difficulties in the
(11) Integrated Biotechnology compositional standardization of herbal
formulations and the commercial exploitation
4. Name of student of this plant (Chatterjee et al., 2010). For
product uniformity under these conditions, in
Malavika M.R. vitro shoot cultures could provide an alternative
to field grown plants for the production of
(b) Admission number therapeutically valuable compounds. Hence in
vitro cultures are applied to produce plants for
2015-09-013 secondary metabolites accumulation as well as
commercial micropropagation (Sivanandhan et
al., 2011).
5. (a) Name of major advisor
During the last few decades, several attempts
have been made to improve withanolides
Dr. M.M. Viji production through cell and organ culture, but
the product yield was small in the context of
(b) Designation industrial aspect (Sivanandhan et al., 2013). In
vitro plant secondary metabolite production can
Professor and Head be enhanced through the application of a
Department of Plant Physiology, variety of elicitors to plant cell or tissue culture
systems. Among the various elicitors, Methyl
College of Agriculture, Vellayani,
Jasmonate (MeJ) and Salicylic Acid (SA) have
Thiruvananthapuram-695522 been confirmed as effective elicitors for the
induction of secondary metabolites in plant
6. Objective cell/organ cultures. Methyl Jasmonate is
considered to be involved in a part of the signal
To standardize multiple shoot induction of transduction pathway that induces particular
Withania somnifera and to estimate enzymes to catalyze biochemical reactions to
withanolide content in multiple shoots derived form defense compounds of low molecular
under in vitro condition by incorporating weight in plants, such as polyphenols,
different elicitors. alkaloids, quinones, terpenoids, and
polypeptides (Yu et al., 2002). Salicylic Acid is
also one of the most widely studied stress-
signaling molecules and its role in influencing
plant resistance to pathogens and other stress
factors is well documented. Recently, SA
emerged as a key-signaling compound
involved in the activation of certain plant
defense responses (Kang et al., 2004).
Thidiazuron 1-phenyl-3-(1,2,3-thidiazol-5-yl
urea), (TDZ) a cytokinin that plays a role as an
anti senescence agent along with MeJ, when
added to the culture medium was reported to
overcome the problems of growth inhibition
and leaf senescence of Withania somnifera
under in-vitro conditions (Kim et al., 2004).
Among the different methods available, mainly
cell suspension culture, multiple shoot culture
and root culture are reported to be the major
in-vitro culture systems to enhance the
secondary metabolite production under in-vitro
condition. Hence the present investigation has
been formulated to standardize in vitro
propagation technique of Withania somnifera
and to estimate withanolide content in multiple
shoots derived under in vitro condition by
incorporating different elicitors.
8. Important publications on which the
study is based
Asiaticoside, a trisaccharide triterpene,
identified as the most active compound in the
plant Centella asiatica (L.), has been found to
associate with the healing of wounds and
duodenal ulcers, while the triterpene saponins
are reported to possess immunomodulatory
properties. The asiaticoside production was
found to enhance by MeJ treatment and at the
same time TDZ addition to MeJ was found to
sustain in-vitro plant growth by inhibiting the
senescence caused by MeJ treatment. On the
basis of this study it was concluded that the
accumulation of asiaticoside in whole plants is
best enhanced by a system using both MeJ and
TDZ, which might have resulted from both the
increased growth of shoots having a high
asiaticoside content and the inhibition of root
development (Kim et al., 2004).
Sivanandhan et al. (2013), stated that the
enhanced production of withanolides in shoot
culture of W.somnifera was directly influenced
by cytokinin concentration, type and
concentration of elicitors as well as the
exposure time.
A rapid and highly effective method for the
micro propagation and elite selection of
withania by auxiliary branching method using
shoot tip as explants was standardized by Baba
et al. (2013). Due to lack of optimum protocol
for in vitro multiplication of Withania
somnifera, Radika and Amutha (2013) have
developed a method of high multiplication rate
using different concentrations of growth
regulators in short period of time and enhancing
the production of rare and endangered
medicinally important ashwagandha plants.
This regeneration system could be used in mass
propagation of ashwagandha (Withania
somnifera (L.) Dunal).
Leaf explants of W. somnifera showed higher
morphogenetic potential in the presence of
Kinetin and 6-Benzylaminopurine each at 4
μM/l (Aruna et al., 2010). An efficient protocol
was developed for high regenerative capacity
and easy mass propagation from leaf explants
of Withania somnifera (Arumughan and
Gopinath 2013).
9. Outline of the technical programme
9.1 Materials
Seeds of Withania somnifera, variety Arka
aswagandha procured from IIHR Bangalore
will be used for the study.
9.2 Standardizing surface sterilization of the
seeds collected.
9.3 The seeds procured will be used to raise in
vitro mother plants.
9.4 Standardizing the media for culture
establishment.
9.5 Different explants for multiple shoot
induction taken from the in-vitro grown mother
plants are:
 Shoot tips
 Nodal segments
 Leaves
9.6 Standardization of in vitro shoot
multiplication
 Basal Media: MS
 Growth regulators:
o Cytokinin: Kinetin
o Auxin: (IAA, BAP, NAA)
o GA3
 Concentration: 0.5-3mg/l
9.7 The culture which proves to be a faster
growing line will be selected based on multiple
shoot production, subcultured for 3-4 times
and used for subsequent treatments with
different elicitors and will be maintained for a
period of around 40 days.
9.8 Standardizing/ Addition of elicitors
 Methyl Jasmonate – 0.1mM
 TDZ - 0.025mg/L
 Salicylic acid – 0.1mM
9.9 Analysis of withanolide content by HPLC
10. Main items of observations to be made
In-vitro propagation studies of Withania
somnifera
plants, preparation and sterilization of explants.
Standardization of in vitro shoot initiation and
shoot multiplication. The culture which proves
to be a faster growing line will be subcultured
and used for subsequent treatments with
different elicitors and will be maintained for
period of around 40 days.
Second six months
Standardizing the dosage of elicitors to be used
and time course studies on growth and
withanolide content of Withania somnifera
multiple shoot cultures treated with different
elicitors. Statistical analysis, writing and
submission of thesis.
14. Budget outlay (in lakhs): 0.40/-

10.1 Response of different explants of

Withania somnifera to shoot induction. Place : Vellayani
 Percentage of shoot induction. Date : 14-03-2019
 Average number of shoots per culture Signature of the student
 Average shoot length (cm)
Name and signature of members of the
10.2 Effect of different growth regulators on advisory committee
multiple shoot production
 Percentage of shoot induction. 1. Dr. M.M. Viji
 Average number of shoots per culture Professor and Head,
 Average shoot length (cm) Department of Plant Physiology
College of Agriculture Vellayani
10.3 Time course studies on growth of Thiruvananthapuram 695522
multiple shoot cultures (raised from uniform
sized in-vitro derived shoot cultures) will be 2. Dr. Beena R.
treated with different elicitors. Assistant Professor
 Average shoot length (cm) Department of Plant Physiology
College of Agriculture Vellayani
10.4 Withanolide concentration at different Thiruvananthapuram 695522
time intervals on the multiple shoot cultures
subjected to different elicitors. 3. Dr. Swapna Alex
Professor & Head
 Withanolide content (µg/g) Department of Plant Biotechnology
College of Agriculture Vellayani
Thiruvananthapuram-695522
11. Facilities
a) Existing facilities: Adequate. 4. Dr. K.B.Soni
b) Additional facilities required: Nil Professor
Department of Plant Biotechnology
12. Duration of the study College of Agriculture, Vellayani,
Thiruvananthapuram - 695 522
One year

13. Time frame of study Place : Vellayani

Date : 14-03-2019
First six months
Signature of Head of the department
Collection of literature, review writing and
studying about Withania sp., standardizing
media required, raising of in-vitro mother
Appendix (Reference)
Arumugam, A. and Gopinath, K. 2013. In vitro
regeneration of an endangered medicinal
plant Withania somnifera using four
different explants. Plant Tissue Cult.
Biotechnol. 23(1): 79-85.
Baba, I. A., Alia, A., Saxena, R. C., Itoo, A.,
Kumar, S., and Ahmad, M. 2013. In vitro
propagation of Withania Somnifera (L.)
Dunal (ashwagandha) an endangered
medicinal plant. Int. J. Pharm. Sci.
Invent. 2(3): 6-11.
Chatterjee, S., Srivastava, S., Khalid, A.,
Singh, N., Sangwan, R. S., Sidhu, O. P.,
Roy, R., Khetrapal, C. L., and Tuli, R.
2010. Comprehensive metabolic
fingerprinting of Withania somnifera leaf
and root extracts. Phytochemistry 71(10):
1085-1094.
Joshi, A. G. and Padhya, M. A. 2010. Shoot
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Sivanandhan, G., Mariashibu, T. S., Arun, M.,
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polyamines on the efficiency of
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M., Dev, G. K., Arjunan, A.,
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Selvaraj, N., and Ganapathi, A. 2013.
Effect of culture conditions, cytokinins,
methyl jasmonate and salicylic acid on the
biomass accumulation and production of
withanolides in multiple shoot culture of
Withania somnifera (L.) Dunal using
liquid culture. Acta. Physiol. Plant. 35(3):
715-728.
Yu, K. W., Gao, W., Hahn, E. J., and Paek, K.
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