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and group II containing 5-OH and 6,7-epoxy groups (withanolide A-like)), and their
ratio was found to be a marker for discriminating leaf samples from the W. somnifera
plants growing in different areas (Namdeo et al., 2011). In an attempt to study the
pattern of the accumulation of three important bioactives viz., withaferin A, 12deoxywithastramonolide, and withanolide A, using LC-ESI-MS-MS (liquid
chromatographyeelectrospray ionizationetandem mass spectrometry) in four
different organs (root, stem, fruits, and leaves) of Ashwagandha, it was found that
withaferin A was highest in the leaves, whereas 12-deoxywithastramonolide and
withanolide A were highest in the roots (Gajbhiye et al., 2015).
Besides the difference in the metabolic profile of different organs, the difference
among the different chemotypes of W. somnifera was analyzed using HR-MAS
NMR spectroscopy (Bharti et al., 2011). Further, when the withanolide profiles of
different W. somnifera accessions were examined by scoring as presence and
absence of major withanolides and were processed for UPGMA-based dendrogram
construction, a high level of phytochemical diversity was recorded in various accessions (as discrete chemotypes) . The different accessions were clustered together
with respect to their characteristic profile of major withanolides and represented
as withaferin A, withanone, withanolide D, or withanolide A rich groups, or the accessions lacking a specific withanolide like withanone minus chemotypes and withaferin A minus chemotypes. Some accessions were found to be rich in 17-hydroxy
withaferin A too (Chaurasiya et al., 2009).
Moreover, the differential alterations in profile of primary and secondary metabolites in Ashwagandha fruits at seven different stages of development have also been
recorded using both one- and two-dimensional NMR spectroscopy. The fruits from
1 week after fertilization until maturity were classified in seven stages; and a qualitative as well as quantitative analysis of metabolites were made in order to find out
the critical stage for harvesting the fruits for obtaining significant amount of the
desired bioactive ingredients with the required pharmacological activity. During
the early stages of fruit development, a relatively higher concentrations of alanine,
aspartate, caffeic acid, choline, phosphocholine, sucrose, and withanolides were
observed, whereas during the maturation phase, accumulation of citrate and withanamides was recorded (Sidhu et al., 2011). The study, therefore, suggested a higher
metabolic activity during the initial stage of fruit development and a metabolic
rerouting in the later stages.
15.8 Crop improvement interventions
Various efforts have been made in this crop to collect, evaluate, and characterize the
germplasm and utilize the interspecific variability in view of the crop improvement.
The hybridization among different chemotypes has also been tried. Recently, many
biotechnological interventions like in vitro manipulations, elicitation (both in vitro
and in vivo), and the genetic transformation have also been tried for the enhanced
production of bioactives. All these attempts have been discussed below.
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15.8.1 Breeding efforts
Furthermore, there have been a few attempts to improve the crop using conventional
breeding approaches like development of hybrids, etc. One major step in the classical breeding is characterization/screening of germplasm for the desired characters.
In this regard, several attempts have been made to study the chemotypic variability
in the germplasm (discussed in Section 15.6.1) from different parts of the world. In
India, the systematic germplasm collection efforts for Ashwagandha are in place
since inception of the AICRPMAP (All India Coordinated Project on Medicinal
and Aromatic Plants) of ICAR (Indian Council of Agricultural Research) in 1969
and a total of 665 accessions of this crop are maintained at different AICRPMAP
centers and its headquarters at DMAPR (Directorate of Medi cinal and Aromatic
Plants Research) (Venugopal et al., 2018). The hybrid between Israel chemotypes
and an Indian chemotype of W. somnifera, new substituted withanolides, was found
in the hybrid plants (Bessalle et al., 1987; Kirson et al., 1977; Nittala and Lavie,
1981). Similarly, three new withanolides were recorded from the hybrids between
a South African and an Israeli chemotype (Eastwood et al., 1980). However, Kumar
et al. (2011a) found the probl ems like very low seed set and a poor percentage germi-
nation was recorded in the hybrids. Venugopal et al. (2018) have discussed the major
breeding objectives of W. somnifera viz., development of dwarf, high root yielding
plants, uniform crop canopy, nonspreading plant architecture, high withanolide content per unit biomass, resistance toward major pests and pathogens, resistance toward abiotic stresses, increasing starchefiber ratio, and early maturity.
15.8.2 Biotechnological interventions
One of the major objectives for the biotechnological interventions employed for
improvement of W. somnifera is the increased production of bioactives (like withanolides, etc.), that can be achieved by a number of in vitro and in vivo manipulations
listed in Fig. 15.3 and discussed in detail under the subsequent sections.
15.8.2.1 In vitro propagation of Ashwagandha
Because of the problems associated with the crop (discussed in Section 15.5), like low
percentage of seed germination and seed viability, attack by various pests/pathogens,
and availability of different chemotypic variants in the crop, the plant tissue culture
(PTC) in an important alternative, that can benefit the herbal industry by means of
a constant supply of the consistent chemotype, expected to meet with the increasing
demand of Ashwagandha. Further, the utilization of in vitroeraised plantlets enhances
the productivity per unit area. PTC is, therefore, an important tool for conserving and
exploiting the commercially important medicinal plants like Ashwagandha. This plant
grows well in IndoeHimalayan region and the conventional methods of harvesting
may lead to the habitat destruction; thus, the in vitro methods have been tried extensively for its propagation and conservation. Further, various PTC-based approaches
like tissue and organ cultures, cell suspension/callus cultures, somatic embryos, and
somaclonal variations/hairy root cultures are considered good alternatives to the conventional methods for getting high value bioactives.
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FIGURE 15.3
A flow chart describing the major strategies for enhancing the production of bioactives in
Withania somnifera, both in vitro and in vivo.
A variety of explants such as seeds, cotyledonary nodes, nodal segments, and
axillary buds taken from field-grown mature plants as well as from the in vitroe
propagated plantlets have been tested for mass multiplication of W. somnifera.
Out of tested explants, enhanced caulogenesis have been observed in cultures initiated from nodal segments or shoot tip. Almost all type of basal tissue culture media
starting from MS to Gamborg B5, Nitsch medium, WPM (woody plant medium),
SH, and their different modifications have been tested by various researchers for
the in vitro culture establishment. However, the optim um bud break, shoot proliferation, and maturation have been observed in MS medium (Shasmita and Naik,
2017). In most of the studies conducted in W. somnifera, sucrose at a concentration
of 3% has given better results for shoot multiplication than the other carbon sources.
The in vitro propagation of W. somnifera has been reported by many researchers
using different explants viz., shoot tips and axillary buds (Autade et al., 2016; Baba
et al., 2013; Rani et al., 2014), hypocotyl, cotyledon (Kumar et al., 2013), seed (Supe
et al., 2006), cotyledonary leaf segments (Rani et al., 2003a), callus from leaves
(Arumugam and Gopinath 2013), and the nodal areas (Kumar et al., 2011b).
Utilization of the axillary shoot buds for multiplication in the micropropagation
serves as a safer strategy to reduce the chances of somaclonal variations and confirming the clonal fidelity of regenerated plants. The vital role played by PGRs alone
or in combination like cytokinins for breaking the dormancy of axillary buds, and
initiating caulogenesis, as well as synergistic combinations of auxins and cytokinins
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for multiplication, elongation, and proliferation of shoots has been stud ied and are
well established in W. somnifera. The high frequency of shoot bud induction was
observed using MS medium fortified with BAP (6-benzene aminopurine). Further,
the synergistic effect of BAP and NAA (1-nephtalene acetic acid) in the refinement
of shoot regeneration efficiency has been obtained in W. somnifera (Fatima et al.,
2015). The cytokinins viz., BAP, kinetin, 2-iP (iso-pentyl adenine), and TDZ (thidia-
zuron) alone or in combination with the auxins IAA (indole-3-acetic acid), IBA
(indole-3-butyric acid), NAA, and 2, 4-D (2, 4-dichlorophenoxy acetic acid) have
been tried for the varied responses in its micropropagation. BAP alone or in combination with other cytokinin or auxins in lower proportions have been found optimum
in shoot multiplication from the meristems (Shasmita and Naik, 2017). In a study on
the micropropagation of W. somnifera involving the use of thidiazuron has resulted
in proliferation of shoots but it also established the adverse effect of its longer exposure resulting in distorted shoots, reduced elongat ion, and even death of primary
shoots (Fatima and Anis, 2011). Apart from the use of the above PGRs, which
have also been used in various other medicinal plan ts, the effect of the PGRs that
are used relatively lesser frequencies for the micropropagation has been tested as
an additive in shoot proliferation and has been resulted in shoot elongation (Sang-
wan et al., 2008; Sivanesan and Murugesan, 2008). Addition of the other growth fac-
tors viz., polyamines, reduced nitrogen sources like amino acids, and coconut milk
was reported to have a profound effect on the in vitro caulogenesis (Ray and Jha,
2001; Sivanandhan et al., 2011, 2015). The one-step elongation and rooting has
also been established in W. somnifera (Kulkarni et al., 2000), by reducing the con-
centration of sucrose or the PGRs.
Moreover, the effect of type of culture vessel and the sealing material of culture
vessel on organogenesis and regeneration potential of different explants has also
been evaluated and found to be of utmost importance, for an in vitro regeneration
protocol. The glass culture tubes were found to be more efficient in direct organogenesis in comparison to the plastic petri plates, which resulted only in the callus
formation. Th is differential response may be because of the accumulation of
ethylene (a PGR) in the glass tubes that could have induced the shoots. Cotton plugs
in comparison to parafilm sealing were recorded better sealing agent for the regeneration, as they can allow the free gas exchange (Kulkarni et al., 1996). Indirect
organogenesis via callus formation is also a good means of mass multiplication, somatic embryogenesis, and has been well established in W. somnifera using the nodal
segment, leaf explant, in vitroegrown seedlings, internode, and hypocot yl and
epicotyl explants. 2,4-D has been used for the callus initiation either alone or in combination with BAP or kinetin in MS basal medium and after callus maturation shoot
induction has been done using BAP alone or in combination with auxins (Manickam
et al., 2000; Rani et al., 2003b; Udayakumar et al., 2013b). The suitability of leaf
explant for inducing embryogenic callus over internodal segments has been established by Rani et al. (2004) and Sharma et al. (2010). The in vitro rhizogenesis of
microshoots has also been a deciding factor in establishing a successful micropropa gation protocol and extensively studied in W. somnifera. Auxins alone or in
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combination with cytokinins are having the morphogenic effect in rooting the microshoots, although the cytokinins like BAP have also been found to initiate rooting in
W. somnifera (Kulkarni et al., 1996, 2000). Various additives like polyamines,
adenine sulfate, and ammonium nitrate have the varied effect on in vitro rooting.
Polyamines, like putrescine and the additives adenine sulfate and ammonium nitrate,
promoted rooting when included in medium. In W. somnifera the cost and time of
tissue culture protocol have been reduced considerably by introducing ex vitro rooting of microshoots, thereby omitting the in vitro rhizogenesis and acclimatization
(Fatima and Anis, 2011).
The tissue cultureeraised shoot propagules and the nodal segment of
W. somnifera have been successfully encapsulated in alginate beads (sodium alginate and calcium chloride based) for the production of synthetic seeds (Fatima
et al., 2013; Singh et al., 2006). Encapsulated somatic embryos or vegetative prop-
agules including shoot apices, axillary buds, or nodes, etc., based synthetic seeds
may also be used in germplasm conservation of the different chemotypes as well
as for exchange of axenic plant material between the laboratories.
Besides the above concise compilation of the efforts made in PTC of Ashwagandha, such as micropropagation, organogenesis, and somatic embryogenesis
etc., the detailed reviews on advancements in PTC-based techniques developed in
Ashwagandha are also available elsewhere (Singh et al., 2017b; Pandey et al.,
2017). However, the attempts made to enhance the productions of bioactives (mainly
withanolides) using various PTC-based methods and strategies to enhance their
in vitro production are discussed in more details in the subsequent section.
15.8.2.2 In vitro production of bioactives
With the increasing demand for bioactive constituents like withanolides, attempts
have been made recently to enhance their in vitro production, which could be a
cost-effective and time saving method to produce the adequate amounts. An important approach for the enhancement of bioactives is the in vitro elicitation by some
chemical compounds, microbial cultures, and/or microbe free culture filtrates in tissue culture. However, in order to successfully commercialize the in vitro production
of plant secondary metabolites are identification of appropriate elicitor, optimization
of the concentration of the elicitors, and scaling up of the process for the commercialization of the production of bioactive metabolites at industrial level. Further,
some native fungal endophytes (isolated from W. somnifera plant, itself) have also
been recorded to enhance the in vitro production of bioactives. The importance of
endophytes is more than the other microbial elicitors since they can be used as effective bioinoculants in the field conditions to increase the production of bioactives,
alone or in combination with other biocontrol agents or biofertilizers. In this section,
an attempt has been made to discuss the recent advances in this field.
In an experiment to evaluate the elicitation potentiality of a rhizosphere fungi,
Piriformospora indica with known secondary metaboliteeenhancing activities in
some othe r medicinal plants was tried (Ahlawat et al., 2016). In this experiment,
when a range of concentrations of the fungal cell homogenate, culture filtrate, and
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the individual fungal culture discs were added into the cell suspension and callus
cultures at different time intervals, it was recorded that the maximum enhancement
in withaferin A production was achieved with 3% fungal homogenate, followed by
3% culture filtrate and culture disc, respectively. Further, a concurrent increase in the
expression of a number of genes involved in withanolide pathway was recorded,
among which the fold increase was highest in hmgr gene, considered to be coding
for a rate-limiting enzyme HMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase) in the withanolide pathway. Further, when the elicitation potential of the cell
homogenates of P. indica was compared with that of three other fungi (viz.,
A. alternata, Fusarium solani, and Verticillium dahlia) in the cell suspension culture
of W. somnifera, it was found that the 3% P. indica cell homogenate was most effective followed by 5% V. dahliae,3%A. alternata, and 3% F. solani cell homogenate,
respectively (Ahlawat et al., 2017). Further, the scaling up of the process was optimized in the study from a shake flask to a lab-scale bioreactor level, thereby leading
to the increased production of the three key withanolides (viz., withanolide A, withaferin A, and withanone).
A native endophytic fungus (isolated from W. somnifera leaves), Aspergillus ter-
reus strain 2aWF, was also found effective in eliciting the production of withanolide
A in root cell suspension cultures of W. somnifera, when applied as 1% mycelial
extract or as 5% culture filtrate with a concurrent upregulation of key genes of the
withanolide pathway (Kushwaha et al., 2019a). The culture filtrate was found to
be more effective in early and effective elicitation than the mycelial extract. Using
the culture filtrate significantly elicited the withanolide A within 6 h, whereas the
mycelial extract elicited the maximum withanolide A after 24 h.
When a biotic (chitosan) and an abiotic (aluminum chloride) elicitation was evaluated for their potential to enhance withanolide production in adventitious root cultures, it was found that their responces are variable in magnitude. Under the similar
culture conditions, the biotic elicitor (100 mg/L) stimulated higher production of all
withanolides in comparison to the abiotic elicitor (Sivanandhan et al., 2012).
Further, the nitrogen source plays a critical role in the accumulation of withanolides
in vitro. It was found that withanolide A production was maximum when the NH
NO
ratio was 14.38/37.60 mM (Nagella and Murthy, 2011).
3
The higher concentrations of total withanolides were obtained in shake-flask culture as well as in the bioreactor using precursor feeding and elicitation, than the control treatments. Maximum withanolides content was recorded in the combined
treatment of chitosan (an elicitor) and squalene (withanolide precursor) in the bioreactor (Sivanandhan et al., 2014).
þ
/
4
15.8.2.3 Genetic transformation
Genetically transformed plants, plant cells, or the organ cultures are attractive systems to study the production of bioactives and also to get their enhanced production.
Several attempts have been made for the genetic transformation for the crop
improvement/improved production of bioactives in Ashwagandha. In the recent decades, the hairy root system based on Agrobacterium rhizogenes inoculation has
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been successfully used as an effective method of producing secondary metabolites.
The transformed root cultures of Ashwagandha using wild-type A. rhizogenes strain
LBA 9402 were able to grow axenically in vitro, without exogenous PGRs, which
were able to synthesize the bioactives. The withanolide D production in these transformed root cultures was higher than the untransformed cultures (Ray et al., 1996).
However, withaferin A was not detected in these transformed cultures, which is usually present in the field-grown plants.
Kumar et al. (2005) also reported that the transformed hairy roots induced by
infecting the leaf explants of W. somnifera using a wild-type A. rhizogenes strain
showed the presence of withanolides and the enhancement of the secondary metabolites. This is the first detection of withaferin A in hairy root cultures and was first
reported by Bandyopadhyay et al. (2007). The accumulation of withaferin A was
recorded in rooty callus line obtained by the tra nsformation using A. rhizogenes
strains LBA 9402 and A
in the transformed root line WSKHRL-1. Murthy et al. (2008) recorded faster
growth, increased biomass, and a 2.7-fold increase of withanolide A content in
A. rhizogenes strain R1601 transformed roots than the nontransformed cultured
roots. Further, the differential performance of different A. rhizogenes strains and nature of explant has also been reported for hairy root induction in W. somnifera (Sar-
avanakumar et al., 2012). It was also recorded that the addition of acetosyringone is
effective in enhancing the transformation frequency. In another experiment,
different elicitors were tried to induce the production of bioactives in
W. somnifera, and it was recorded that both chitosan and nitric oxide increased withaferin A content, acetyl salicylic acid stimulated the accumulation of both withaferin
A and withanolide A, and the elicitation by triadimefon highly increased withaferin
A content (Doma et al., 2012). The role of heat treatment and sonication in
enhancing the rate of A. rhizogenesemediated transformation of leaf segment
explant was also reported (Thilip et al., 2015, 2020). In an experiment to try various
elicitors for enhancing withaferin A production in hairy root cultures, Thilip et al.
(2019) recorded that the elicitation by chitosan (100 mg/L) was most useful and
the production was increased by 4.03-folds than the control.
There have been several attempts to optimize the A. tumefaciensemediated
methods of genetic transformation, knowledge gain from which can now be utilized
in successful metabolic engineering efforts as well as in the functional genomics
studies in this medicinal plant. In one of the initial attempts to develop the transformed organ cultures of Ashwagandha using different wild-type strains of
A. tumefaciens, it was found that some of the galls obtained following infection
of A. tumefaciens strain N2/73 were able to spontaneously develop shooty teratomas
that were grown in unsupplemented basal medium. Further they are able to synthesize more amount of two major native bioactives, i.e., withaferin A and withanolide
D, than the untransformed shoot cultures (Ray and Jha, 1999). Transformation using
A. tumefaciens strain LBA4404 having binary vector pIG121Hm was successfully
used by Pandey et al. (2010), the gusA reporter gene with intron under the transcriptional control of the CaMV (Cauliflower Mosaic Virus) 35S promoter. It was
with the maximum accumulation of withaferin A content
4
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recorded that the leaf segments from two-and-a-half-month-old greenhouseegrown
seedlings were more efficient in transformation than the in vitroegrown shoots and
the second expanded leaf from the shoot tip had the highest efficiency toward transient transformation.
For a reproducible and efficient transformation protocol development for a
particular plant, several other factors also require optimization. One of the initial efforts in W. somnifera was made by Ishnava et al. (2012), to optimize the infection
treatment by A. tumifaciens, time duration, PGR treatment, and sucrose concentration with an aim to increase production of bioactives. Further, it was recorded that
out of three approaches tried for the development of an efficient transformation system in W. somnifera, the microprojectile bombardmenteassisted agro-infection was
found better than A. tumefaciensemediated transformation and microprojectile
bombardment alone (Patel et al., 2014). Transgenic W. somnifera plants generated
by A. tumefaciensemediated genetic transformation that were overexpressing
WsSQS (one of the withanolide pathway genes) led to a 1.5- to 2-fold increas e in
the total withanolide (Patel et al., 2015). Gen etic transformation is also an important
tool for the function genomics studies. The transgenic lines containing the constructs
(in RNAi vector pGSA backbone) which impairs the normal functioning of a WsCAS
(W. somnifera cycloartenol synthase gene) suppressed the withanolide biosynthesis
in W. somnifera but an enhanced level of withanolide production was recorded in the
CAS overexpressing transgenic lines. This suggested a key position of CAS in the
withanolide biosynthetic pathway (Mishra et al., 2016). Similar gene silencing
studies using RNAi were tried to find the possible roles of SMT1(sterol-C24methyltransferase type 1) and SGTL1 (a sterol glycosyltransferases from class 1)
(Pal et al., 2019; Saema et al., 2015).
15.8.2.4 Enhanced production of bioactives in vivo
There are a number of methods reported/suggest ed for the enhancement of the concentration of bioactives in the Ashwagandha plan ts in field conditions. They include
the foliar application of PGRs, endophytes treatment, elicitor’s treatment, imposition of stress conditions, photoperiod, nutritional status, age of the plant at the harvest, season of the harvest, etc.
It has been found that the drought stress induces the concentration of a bioactive,
i.e., withaferin A in Ashwagandha (Kannan and Kulandaivelu, 2011). Sanchita et al.
(2015) also recorded the increase in the production of different withanolides viz., the
withaferin A, 12-withastromonolides, and withanolide A by 42.7%, 78%, and 71%,
respectively, under drought stress. However, a decrease in the concentration of these
bioactives has been recorded under biotic stress, i.e., leaf spot disease by A. alternata
(Pati et al., 2008).
Singh et al. (2020) reported the increased production upon the foliar applications
of PGRs (jasmonic acid and salicylic acid SA) and chitosan as the elicitors.
Endophytes are also effective in production of secondary metabolites under field
conditions. Three native fungal viz., A. terreus strain 2aWF (2aWF), Penicillium
oxalicum strain 5aWF (5aW F), and Sarocladium kiliense strain 10aWF (10aWF)
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were reported to enhance withanolides content in both leaves and roots and a corresponding upregulation of key withanolide biosynthetic pathway genes was also reported (Kushwaha et al., 2019b). When the compatibility of these inherent fungal
endophytes was tested with the combined treatment with the biocontrol agent, Tri-
choderma viride, it was recorded that the coinoculation treatments increased the production of secondary metabolite, mainly withanolide A in both leaf and root tissues
of W. somnifera (Kushwaha et al., 2019c). Treatment with microbial consortium
consisting various PGPRs suggested that this treatment increases the withaferin A
content of W. somnifera (Rajasekar and Elango, 2011).
The production of withanaloids is also influenced by photoperiod. It was
recorded that under the long photoperiod-treated condition the concentration of
withanolides was higher in Ashwagandha (Sharma and Puri, 2020). However,
another study conducted to examine low light stress (25%, 50% and 75% shade
along with the control, for 30 days) on withanolide production in W. somnifera,
the highest withanolide accumulation was recorded under 75% (Jacob et al.,
2014). Further, it was found that the expression of withanolide pathway gene
WsFPPS is enhanced in the response to mechanical injury (Gupta et al., 2011). It
has also been recently recorded that the seasonal variations may also influence the
withanolide biosynthesis (Mishra et al., 2020); therefore, choosing the appropriate
season for the sowing and harvesting of Ashwagandha is useful for the consistent
quality of the herbal products thereof.
Moreover, the proper nutrient status of the plant can lead to the enhanced production of the bioactives. It was found that the application of a nitrogenous fertilizer,
ammonium sulfate, improved the production of withaferin A (Pal et al., 2017).
The use of organic amendments like caster cake and vermicompost along with microbial consortium was able to enhance the total withanolide content in Ashwagandha (Basak et al., 2020). Further, the leaves of Ashwagandha plants cultivated
using vermicompost and vermicompost leachate showed the higher concentration
of the withanolides viz., withanone, withanolide A, and withaferin A (Kaur et al.,
2018). Therefore, the organic farming/adding organic amendments or the integrated
nutrient management of this medicinal plant may be good choice to get the enhanced
amount of bioactives in field-grown plants.
15.9 Conclusions and future perspectives
Recently, there has been a global shift in consumer behavior to go back to nature in
search of the needs of mankind including primary healthcare. Therefore, an
increasing number of efforts are being made to look at the potential of the medicinal
herbs like Ashwagandha in primary healthcare system in a scientific and systematic
way. A number of bioactives with a wide range of pharmaceutical properties have
been identified from it. Due to its increasing demand, it has been gradually understood that exploiting the wild-grown plants of Ashwagandha in a way may lead to
loss of its genetic variability and even it may lead to habitat destruction. Therefore,
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efforts were made by different researchers to collect the germplasm, evaluate its chemotypic and genotypic variability in the natural populations, and identify the elite
genotopes/chemotypes and promote them for cultivation. Further, researchers
have also came across various problems in the cultivation of Ashwagandha and identified some important pests and diseases limiting its cultivation to meet the market
demand. However, the harvesting of roots of Ashwagandha from field is a laborintensive process. Therefore, the in vitro approaches were also tried to meet the demand of bioactives via alternative methods like cell/organ culture (like hairy roots)
and other in vitro methods. Further, these methods can be scaled up in bioreactors to
meet the requirements of bioactives. Efforts have also been made to increase the production of bioactives through in vitro elicitation and/or precursor feeding. With the
increased knowledge of pathway genes accumulate very recently and availability of
ESTs and transcriptome data in public domain, more advancement in enhancing the
production capacity of bioactives through methods like genetic transformation are
also being adopted. Further, the increasing availability of sequence-based markers
will help in marker-assisted selection of elite genotypes and will lead to commercialization of improved varieties. In this chapter, we tried to present the efforts made in
the improvement of W. somnifera using cell cultureebased methods with/without
the integration of recent knowledge gained through various OMICS approaches
(like genomics, transcriptomics, proteomics, and metabolomics) till date and to
identify the knowledge gaps hampering the further improvement of the crop. A
comprehensive knowledge in a broad holistic view of these diverse strategies will
help in paving the way to identify these research gaps in the crop improvement
and will motivate the workers to work on them.
Acknowledgments
Authors acknowledge the support from DAV University administration during the preparation
of the manuscript. Further, the financial assistance from CSIR (Council for Scientific and Industrial Research), Government of India, is duly acknowledged.
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