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10.5 Biochemical/analytical properties 177
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alkaloids, flavonoids, lignins, steroids, glycosides, and anthraquinones (Wei et al.,
2014; Singh et al., 2017). Hydrodistillation of S. lappa root essential oil showed
higher sesquiterpenoid content (79.80%) relative to monoterpenoids (13.25%)
(Liu et al., 2012).
Principal constituents of S. lappa essential oil were dehydrocostus lactone and
costunolide (Singh et al., 2017)(Table 10.1). Costus oil/S. lappa essential oil is
the commercially valued essential oil extracted from S. lappa roots which is constituted by multiple bioactive compounds. The proportion of these constituents generally varies among plants which might be attributed to factors like phenophases,
ecotypes, chemotype, genotype, and environmental variations like temperature, relative humidity, photoperiod, and irradiance (Marotti et al., 1994).
An account of various categories of chemical constituents isolated from S. lappa
is provided in the sections below.
10.5.1 Terpenes
Monoterpenes isolated from roots include Phellandrene, Thymol, Anethole, Estragole, Citronellyl propionate, a-Pinene, b-Pinene, a-Thujene, Camphor, Camphene,
Sabinene, Myrcene, Limonene, p-Cymene, g-Terpinene, 1,8 Cineol, Menthone,
a-Terpinolene, Citronellal, Terpinen-4-ol, Linalool, Cryptone, a-Terpineol, and
Ocimene (Chang and Kim, 2008; Gwari et al., 2013).
S. lappa is an abundant source of sesquiterpenes which are roughly categorized
into three groups based on the carbocyclic skeleton: Guaiane, Eudesmane, and Germacrane (Singh et al., 2017). These have been known to biosynthesize sequentially;
however, due to instability of germacrene, half of the total sesquiterpenes are
observed in form of guaianes and 40% turn up as eudesmanes, while germacrene
constitutes the remaining fraction (Singh et al., 2017). Guaianes reportedly derived
from S. lappa include Dehydrocostus lactone (Govindan and Bhattacharaya, 1977),
Zaluzanin C, Isozaluzanin, 11b, 13-Dihydro-3-epizaluzanin C (Chhabra et al., 1998;
Kalsi et al., 1983), Lappalone (Sun et al., 2003), Cynaropicrin (Cho et al., 1998),
Saussureamine B, 12-Methoxy-dihydrodehyrocostus lactone (Dhillon et al.,
1987), Saussureamine C (Yoshikawa et al., 1993), Dihydroglucoaluzanin C, Mokko
lactone, 11,13, Saussurealdehyde, Isodehydrocostuslactone (Kalsi et al., 1983),
11,13-Epoxydehydrocostus lactone, Isodehydrocostuslactone-15-aldehyde (Kumar
et al., 1995), 11,13-Epoxyisozaluzanin C (Chhabra et al., 1997), 4b-Methoxy-
dehydrocostus lactone, 11, 13-Epoxy-3-ketodehyrocostus lactone(Chhabra et al.,
1998), Lappadilactone, 15-Hydroxydehydrocostus lactone, etc. Eudesmanes type
sesquiterpenoids include 13-Sulfodihydrosantamarine (Yin et al., 2005), Saussu real,
Saussureamine D (Yoshikawa et al., 1993), Saussureamine E (Yoshikawa et al.,
1993), 11b, 13-Dihydroreyosin, 13-Sulfodihydroreyosin, Reynosin (Cho et al.,
1998), 1b,6a-Dihydroxycostic acid ethyl ester (Sun et al., 2003), b-Costic acid
(Govindan and Bhattacharaya, 1977), a-Cyclocostunolide, Isoalantolactone,
Alantolactone, b-Cyclocostunolide (Govindan and Bhattacharaya, 1977), Hydroxyendesin-11(13)-en-12-al, Magnolialide, 4a-Hydroxy -4b
Isocostic acid, 4b-a-Costol, Santamarine ( Cho et al., 1998), Colartin and Arbusculin
-Methyldihydrocostol,
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A, etc. Some Germacrane type sesquiterpene lactones isolated from S. lappa are
Dihydrocostunolide, Saussureamine A (Yoshikawa et al., 1993), Costunolide
(Kim et al., 1999), Costunolide 15-o-bcostunolide. a-Amyrin (Yang et al., 1997a,b), 3-b-Acetoxy-9(11)-baccharene, and
a-Amyrin eicosanoate are a few triterpenes which have been isolated from
S. lappa (Robinson et al., 2010).
10.5.2 Flavonoids
Multiple flavonoids isolated from S. lappa roots include Luteolin-7-O-b-Dglucoside, Apigenin-7-O-b-D-glucoside (Alaagib and Ayoub, 2015), and Rutin
which have one glucoside substituent. Flavonoids with large substituents like three
glucosides are relatively quite rare. Following acylated flavonoids have been
reported from S. lappa roots by Rao et al. (2007): Kaempferol 3-O-banosyl-(1 / 4)-a-
-O-acetyl-b-Dgluco-pyranosyl-(1 / 3)-[a-L-rhamnopyranosyl-(1 / 2)]-b-D-glu-
copyranoside, 3
tellarein 7-O-(b-O-6
-(1 / 2)]-b(1 / 2)-b-D-(6 a
pyranosyl-(1 / 3)-[bKaempferol 3-O-a-L-(2a
b-D glucopyranosyl-(1 / 3)-[a-Lrhamnopyranosyl-(1 / 2)]-b-
Rao et al. (2007) reported four new flavonoids (KSR1-4) from ethanolic extract of
powdered S. lappa roots.
L-rhamnopyranosyl-(1 / 6)-b-D-galactopyranoside 7-O-(6
0
[(3R)-3-Acetoxy-5,5-dimethylcyclopent-1-en-1-yl]-40-Omethylscu-
000
-O-acetylglucopyranosyl-(1 3)-[a-L-rhamnopyranosyl
D-glucopyranoside and Kaempferol 3-O-b-D-glucopyranosyl-
0
-O-caffeoyl) galactopyranoside 7-O-(b-D-6
L-rhamnopyranosyl-(1 / 2)]-b-D-glucopyranoside, and
0
,3a’-(E)-di-p-coumaroyl) rhamnoside 7-O-(6
D-glucopyranoside and 12-Methoxy dihydro-
D-glucopyr-
000
-O-acetyl-b-D-gluco-
000
-O-acetyl-
D-glucopyranoside.
000
10.5.3 Other constituents
A few anthraquinone compounds have also been isolated like Aloeemodin-8-O-b-Dglucopyranoside, Chrysophanol, and Rhein-8-O-b-dglucopyranoside which are
involved in inhibition of protein tyrosine phosphatase (PTP-1B) enzyme. Phytosterols like 3-Epilappasterol, Lappasterol, b-Sitosterol, Pregnenolone, Daucosterol,
and Lappalanasterol have also been reported. (E)-9-Isopropyl-6-methyl-5,9decadiene-2-one, a terpenoid C14-ketone, a-amyrin starate, b-amyrin, and lupeol
palmitates have been isolated from leaves (Pai, 1977; Bruno and Gunther, 1997).
Amino acids like Saussureamines AeE, lignan glycoside () massoniresinol-4
O-b-D-glucoside, Guaianolides like iso-zaluzanin-C, and isodehydrocostus lactone
are some other compounds reported from S. lappa.
10.6 Therapeutic attributes of Saussurea lappa
S. lappa is well known for its multiple therapeutic applications which establish its
suitability as a major ingredient in several drug formulations. Several reports have
00
-
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investigated the medicinal properties of S. lappaederived bioactive compounds as
documented below.
10.6.1 Anticancerous/antitumor properties
Costunolide, the main sesquiterpene lactone isolated from S. lappa root, was
analyzed for its potential bioactivity in inducing apoptosis in Human Leukemia cells
(HL-60). Measurement of Reactive Oxygen Species (ROS) and estimation of
mitochondrial membrane potentials validated the apoptosis inducing act ivity of
costunolide. It potentially causes mitochondrial permeability transition and release
of cytochrome C into the cytosol. Costunolide treatment causes release of
N-acetylcysteine which is responsible for causing a block in the mitochondrial alteration, production of ROS thereby leading to apoptotic death. The metabolite thus
causes stimulation of ROS-mediated transition of mitochondrial permeability and
the resultant Cytochrome C release (Lee et al., 2001). Anticarcinogenesis activity
of costunolide was also evident from reporter gene assay which is triggered by
tumor-endorsing compound phorbol ester 12-O-tetradecanoylphorbol-13-acetate.
This compound amplifies the activity of nitric oxide synthase which has been found
to be repressed by costunolide (Fukuda et al., 2001). Another study by Choi et al.
(2009) reported the effect of the sesquiterpene lactone on telomerase activity via
analysis on MCF-7, MDA-MB-231, thereby confirming the hindering activity.
Costunolide has been observed to possess cytolytic activity and it performs via
mechanistic means to inhibit granule exocytosis and represses amplification in tyrosine phosphorylation in a dose-dependent manner (Taniguchi et al., 1995). Costunolide causes inhibition of Vascular Endothelial Growth Factor (VEGF)-induced
chemotaxis of human umbilical vein endothelial cells and causes selective inhibition
of endothelial cell proliferation activated by VEGF. Thus, by blocking signaling of
angiogenic factor pathway, it is potentially active in inhibition of angiogenesis
(Jeong et al., 2002).
Hexane extract of S. lappa comprises of dehydrocostus lactone and was found to
be effective in induction of apoptosis in human autonomous androgen prostrate cancer DU145 cell lines for inhibition of cell growth (Kim et al., 1991). Hung et al.
(2010) have also tested activity of dehydrocostus lactone against noncancer cell
lines like NCL-H
that dehydrocostus lactone actively facilitates arrest of cell cycle at G2/M stage,
leading to inhibition of cell proliferation (Choi and Kim, 2010). It also affects
cell cycle distribution, cell viability, and expression of ATP-binding cassette transporter in sarcoma cell lines. Furthermore, it led to activity of apoptosis indicators
like caspase-3, caspases 3/7, and PARP cleavage (Kretschmer et al., 2012).
Cynaropicrin derived from S. lappa was found to possess immunomo dulatory
effects coupled with nitric oxide production. It depicted repress ion against Eol-1,
Jurkat T, and U
with N-acetylalleviating cynaropicrin-mediated cytotoxicity. Cynaropicrin was observed to have
, NCL-H
460
cell lines in a dose-dependent manner. Coupling of cynaropicrin
937
L-cysteine or L-cysteine, ROS scavengers’ rottlerin is capable of
, and A
520
. Flow cytometry studies have shown
549
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higher cytotoxic potential against leukocyte-derived cancer cells as compared to fibroblasts (Cho et al., 2004). Ethanolic extract of S. lappa has also depicted apoptotic
activity against gastric cancer in a dose- and time-dependent manner (Ko et al.,
2005). Thus, main compounds involved in anticancerous activity include costuno-
lide, dehydrocostus lactone, and cynaropicrin.
10.6.2 Antibacterial properties
Various solvent extracts (ethanolic, methanolic, petroleum ether, and aqueous) have
established the antibacterial activity of S. lappa against a diversity of resistant
pathogens. Yang et al. (1998) reported the activity of ethanolic extract against five
clinical strains of Helicobacter pylori. S. lappa extract has been found to be active
against hepatitis B surface antigen (HbSAg) and other correlated antigens (Chen
et al., 1995). Bioactive constituents of S. lappa have also been found to inhibit bind-
ing and transfer of R plasmids in pathogenic microorganism, Shigella flexneri (Li
et al., 2010), and was also found to be effective against Bacillus thuringiensis,
Aspergillus, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia, Candida albicans, Proteus vulgaris, Escherichia coli, and Cornybacterium
in a concentration-dependent manner (Irshad et al., 2012, Thara and Zuhra, 2012).
Ethanolic extract showed inhibitory activity against multidrug-resistant organisms
P. aeruginosa, S. aureus, E. coli, and K. pneumonia (Hasson et al., 2013). Among
multiple organic extracts, chloroform extract presented maximum antibacterial
potential (Alaagib and Ayoub 2015).
10.6.3 Antiinflammatory activity
Sesquiterpenes are the major ingredients which possess activity for stabilization of
endosomal release and cause prevention of cell proliferation via monitoring of nitric
oxide and TNF-a levels in macrophage cells of mice (Damre et al., 2003). Methanolic extract exhibited >50% inhibition on induction of cytokine-induced neutrophil
chemotactic factors (Lee et al., 1995). Ethanolic extract showed antiinflammatory
activity via peritonitis and carrageenan induced edema in animal models (Gokhale
et al., 2002). Costunolide was found to hinder mRNA and protein expression of
interleukin-1b (Kang et al., 2004). Dehydrocostus lactone was found to possess
inhibitory activity against oxidative osteoblast damage (Choi et al., 2009). It guides
inactivation of nuclear transcription factor (NF-KB), causes inhibition of iNOS gene
expression, and reduces generation of TNF-a and nitric oxideeinduced through LPS
(Lee et al., 1995; Jin et al., 2000). Similarly, saussureamines A and B also cause
effective inhibition of NO caused by NF-kB activation and LPS (Matsuda et al.,
2003). Among sesquiterpene lactones, cynaropicrin was found to be most active
against inhibition of TNF-a (Cho et al., 1998).
10.6.4 Hepatoprotective properties
Acetone extract and costunolide were found to possess choleretic effect which could
inhibit ulcers in mice (Yamahara et al., 1985 ). Costunolide and dehydrocostus
10.6 Therapeutic attributes of Saussurea lappa 181
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lactone were found to be active against Human Hematome Hep3B cells and HBsAg,
thus proving their potential in development of HBV drugs (Chen et al., 1995). Use of
aqueous methanolic extract in a dose-dependent manner caused absence of parenchymal congestion, improved architectural detail, and decreased apoptotic cells
and cellular swelling in hepatic damage repair (Yaeesh et al., 2010). Shao et al.
(2005) reported choleretic effect and enhanced bile flow in rats. Examination of
S. lappa extract depicted induction of gall bladder contraction in dogs as well
(Liu et al., 2008).
10.6.5 Antiulcer and cholagogic properties
S. lappa is a major constituent of the popular antiulcer formulation, UL-409, which
might be attributed to inflection of defensive factors via improved gastric cytoprotection (Mitra et al., 1996; Venkataranganna et al., 1998). In chronic superficial
gastritis patients, decoction perfusion of S. lappa into patient’s stomach resulted
in increased endogenous motilin release and fastened gastric emptying (Chen
et al., 1994). Herbal formulat ion of S. lappa was tested for antiulcer activity in Wis-
tar rats which caused reduction in gastric ulceration induced by aspirin and alcohol
(Mitra et al., 1996). Sutar et al. (2011) reported potential of the ethyl acetate extract
against duodenal and gastric ulceration in rats. An apparent protective activity
against acute damage to gastric mucosa of rats has also been observed (Wang,
2004). Saussureamines A, B, and C have been found to be active in repair of gastric
damage inflicted by ethanol and hydrochloric acid, while saussureamine A is active
in inhibition of stress-induced gastric ulcers in mice (Yoshikawa et al., 1993). In
addition to saussureamines, dehydrocostus lactone and costunolide have also been
known in treatment of gastric ulcers in mice (Matsuda et al., 2000).
10.6.6 Immunomodulatory properties
High doses of S. lappa extract have been shown to be immunomodulatory in cellular
and humoral arms of immune system (Pandey, 2012). Dehydrocostus lactone and
costunolide have also been observed to be active as inhibitors of cytotoxic T
lymphocyte (CTL) activity. Costunolide prevents increase in tyrosine phosphorylation thereby inhibiting the killing potential of CTLs. Guaianolide moiety was also
observed to exhibit substantial inhibitory activity toward CTLs and initiation of
intercellular adhesion molecule-1 (Taniguchi et al., 1995; Yuuya et al., 1999).
10.6.7 Cardiovascular properties
S. lappa extract has been observed to cause lowering of blood pressure and prevention of blood coagulation while causing reduction in triglycerides and cholesterol in
blood (Upadhyay et al., 1996). Aqueous decoction of S. lappa fortifies the fibrin content of blood (Yu, 1986) and costus oil has been reported to depict hypoglycemic
effect (Gupta and Ghatak, 1967; Wang, 1997). Costunolide and dehydrocostus
lactone present in the volatile oil are involved in inhibition of ADP-induced platelet
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coagulation (Hou et al., 2008). S. lappa extract is a source of betulinic acid, betulinic
acid methyl ester, dehydrocostus lactone, mokko lactone, and anthraquinones which
are reported to be active against obesity and hypertension associated with Type-II
diabetes (Li et al., 2006; Choi et al., 2009, 2012).
10.6.8 Bronchitis
Alkaloid fraction isolated from S. lappa is nontoxic and exhibits noticeable spasmolytic effect on the tracheal and smooth (intestinal) muscle of pig lungs (Dutta et al.,
1960). Tincture Saussurea petroleum ether extract and tincture Saussurea were eval-
uated for activity in bronchitis. Since tincture in petroleum ether extract has been
reported to induce bronchoconstriction in guinea pigs, tincture Saussurea holds potential for development of drugs for asthma and chronic bronchitis (Sastry and Dutta,
1961).
10.6.9 Anticonvulsant properties
S. lappa petroleum ether extract has also been known to be effective against picrotoxin and pentylenetrazole-induced convulsions in mice by causing elevation of
seizure threshold via GABAergic receptors (Ambavade et al., 2009). Alcoholic
extract of S. lappa also reportedly exhibits significant activity against epilepsy
(Gupta Pushpraj et al., 2009).
10.6.10 Antiparasitic properties
S. lappa extract has been known to be effective against nematodal infections via oral
administration in rabbits infected with Clonorchis sinensis (Rhee et al., 1985). It also
significantly reduces the percentage of fecal eggs of nematodes in children naturally
infected with worms (Akhtar and Riffat, 1991).
10.6.11 Antihyperlipidemic properties
S. lappa aqueous extract shows significant hypolipidemic activity as tested through
administration in rabbits (Upadhyay et al., 1996). Ethanolic extract is also involved
in causi ng reduction in triglyceride levels coupled with an increase in HDL-C level
in serum as well as tissues (Anbu et al., 2011).
10.6.12 Antidiarrheal properties
Methanolic extract of S. lappa was observed to exhibit substantial antidiarrheal activity in a dose-dependent manner. In fact, it was reported to have effects similar to
the popular drug, loperamide in causing reduction of diarrhea stool (Hemamalini
et al., 2011). Methanolic extract is also effective in diarrhea induced by castor oil
in rats (Negi et al., 2013).
10.6 Therapeutic attributes of Saussurea lappa 183
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10.6.13 Angiogenesis activity
Costunolide has been known to suppress endothelial cell proliferation. Chemotaxis
induced by VEGF of endothelia was observed to be suppressed effectively by
S. lappa. Similarly, in vivo methods have also reported inhibition of VEGFstimulated neovascularization in mouse cornea (Jeong et al., 2002; Thara and Zuhra,
2012; Saleem et al., 2013).
10.6.14 Spasmolytic activity
S. lappa is also capable of relaxing contractions induced by carbachol owing to activity of sesquiterpene lactones. These compounds have been recognized for their
role in stimulation of Sgc which induces extrusion of K
of intrinsic calcium ions via activation of cyclic GMP and PKG pathways, thus
relaxing the smooth muscles (Hsu et al., 2009).
þ
ions leading to reduction
10.6.15 Antimycobacterial activity
Investigation of in vitro antimycobacterial activity was investigated where costunolide as well as dehydrocostus lactone in whole oil and fractions depicted activity
against Mycobacterium tuberculosis H37Rv strain. Both compounds reportedly
exhibit synergistic activity as the mixture proved to be more effective as compared
to pure compounds (Luna-Herrera et al., 2007).
10.6.16 Synthesis of nanoparticles
Recently, S. lappa has been successfully used in synthesis of silver nanoparticles.
Nanoparticles offer a favorable option for usage in air/water treatment, optics,
catalysis, mirrors, photography, medicine, drug delivery, electronics, clothing,
food packaging, and electronics (Prabhu and Poulose, 2012). Nanoparticles have
significant usage in nanobiotechnology as they enhance biomedical, optical, environmental, catalytic, and electrical properties, their applications and performance
(Stark et al., 2015; Tonga et al., 2014). Silver nanoparticles due to their optical properties have been extremely successful in scientific applications like nanophotonics,
sensors, photo-thermal therapy, medicine, and biological activity (El-Nour et al.,
2010). Silver nanoparticles are the most widely used nanoparticles in biotechnology
and can be effectively synthesized using either conventional but hazardous chemical
and physical methods or green chemistry/biological methods which are natural,
single-step, and eco-friendly. Plant extracts are preferable over other biological
methods for uniform and controlled synthesis under natural conditions (El-Nour
et al., 2010). Secondary metabolites procured from medicinal plants can be utilized
for synthesis and capping of nanoparticles (Abdu l Majeed Almashhedy and Al-
Kawaz 2016). Mahapatra et al. (2018) proposed a rapid fabrication method for
synthesis of silver nanoparticles (AgNPs) using root extract of S. lappa (RESL).
An interesting flower-like morphology was observed for these nanoparticles and
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had a mean diameter of 4000e5000 nm. Surface Plasmon Resonance revealed the
optical absorption band peak at 440 nm. In the biofabrication process, diffractogram
planes depicted silver as the chief constituent. Presence of root phytoconstituents
gave the appearance of crystalline peaks. Capping phenomenon for these bionanoparticles was validated using UV, X-ray diffraction, and scanning electron microscopy (SEM). Riaz et al., (2018) also reported the biogenic synthesis of AgNPs
using aqueous and methanol extracts of S. lappa roots. Characterization of these
nanoparticles was performed using UVevisible spectroscopy, SEM, as well as
FT-IR. Antimicrobial activity of the synthesized nanoparticles was also observed
against E. coli (11.0 mm) and P. aeruginosa (9.0 mm), where aqueous extract
exhibited better potency. Recently, two more research groups reported synthesis
of AgNPs using aqueous root extracts of S. lappa. Groach et al. (2019) synthesized
spherical nanoparticles ranging in size from 7.13 to 24.0 nm, while (Ashwini and
Kumar, 2019) synthesized cube-shaped nanoparticles having a size range of
500 nme2 mm. Antibacterial activity was successfully observed against E. coli
and Bacillus cereus, thereby proving their antimicrobial potential (Ashwini and
Kumar, 2019; Groach et al., 2019). These studies have successfully proven the
potential of S. lappa extract in synthesis of nanoparticles which can be promising
candidates for usage in biomedicine.
10.7 Conservation status
Medicinal plants are under a constant threat owing to unregulated harvesting of their
natural habitat for domestic as well as industrial purposes. Well-planned and effectively utilized strategies aimed at conservation of these plants remain the sole hope
for preventing their extinction. These become especially relevant for popular herbs
like S. lappa which have a well-recognized and documented potential in curative and
preventive medicine. Butola and Samant attempted to study the distribution, diversity, habitat preference, endemism, nativity, status, as well as indigenous uses of
Saussurea species in the Indian Himalayan Region (Butola and Samant, 2010).
They recognized S. lappa as the most commercially viable species of Saussurea
genus. It was first listed in Appendix II of CITES (Convention on International Trade
in Endangered species of Wild Fauna and Flora) on July 01, 1975, and 10 years later,
it was uplisted to Appendix I (Bano et al., 2018). According to IUCN, S. lappa has
been categorized as a critically endangered species. Export of S. lappa has been
prohibited due to inclusion in category of red list species according to Appendix I
of CITES, 2003. Jammu and Kashmir which hosts its prime natural habitat has
enforced a special Act called “The Kuth Act, 1978” for regulating the trade of
S. lappa (Jain, 2001). It has also been included in the negative list of exports as
imposed by Ministry of Commerce, Government of India along with listing in the
“Schedule VI” of the Wildlife Protection Act, India. Trade of this valuable herb
has been strictly prohibited under Foreign Trade Development Act 1992. Although
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the species is cultivated in regions like Lahaul valley, low and fluctuating market
price limits the practice to a few scattered villages (Kuniyal et al., 2005).
Following in vitro propagation and ex situ strategies have been employed for
conservation of the species.
10.7.1 In vitro propagation of Saussurea lappa
Genetic conservation is particularly important for endangered plant species li ke
S. lappa owing to fear of their extinction and loss of valuable metabolites. Although
germplasm storage in form of seeds appears to be the most lucrative option for conservation, it is not much feasible in case of plants which do not produce seeds or ones
with low seed viability as in case of S. lappa (30%) (Wealth of India, 1972). The
traditional methods intended for their maintenance are risky, laborious, and expensive. Tissue culture is the most suitable alternative for large-scale conservation in
vegetative state which offers an option of production on demand as well. Arora
and Bhojwani (1989) initiated the first attempt on in vitro propagation of S. lappa
using different explants derived from aseptic plants. Among different seedling explants, maximum shoot regeneration was observed in case of leaves followed by cotyledons. However, roots and hypocotyl appeared to lack morphogenetic potential.
Shoot multiplication of 3.5-folds was observed every 3 weeks on MS (Murashige
and Skoog) media containing benzylaminopurine and gibberellin. Roots were
observed with 90% efficiency on MS having 0.5 mM napht haleneacetic acid. Interestingly, shoot cultures preserved in the dark at 5
intervening subculture showed 100% viability which presented a promising method
for long-term storage. It was observed that shoots stored at cold temperature
exhibited higher rates of multiplication in culture room conditions relative to untreated shoots. Later, role of TDZ (thiadiazuron) in achieving direct organogenesis
was achieved by culturing shoot tips of 2-week-old seedlings on MS þ TDZ
(0.45 mM) media (Johnson et al., 1997). Among N6-benzyladenine-(BA) and TDZ
containing media, latter was found to be more effective for induction of callusfree multiple shoots. Liquid medium was observed as a better culture media as
compared to agar-solidified media. Shoots developed roots on MS þ Nap ththaleneacetic acid media (NAA, 1.07 mM). The micropropagated plantlets were transferred
to soil and 90% plants showed survival after the process of hardening. Verma et al.
(2012) reported callus induction using root explants using 2.4-D and BAP during an
initiative intended for in vitro propagation of 23 overexploited medicinal plants in
India. Later, Warghat et al. (2016) optimized the plant regeneration under in vitro
as well as in vivo conditions. On evaluation of multiple combinations of auxins as
well cytokinins, 2, 4-dichlorophenoxyacetic acid (2, 4-D) (3 mg/L) and kinetin
(Kin) (5 mg/L) were found to be most effective for callus induction in all tested explants. Explants used in the study included leaf, cotyledonary leaf, hypocotyl,
epicotyl, stem, and root. Root and stem explants exhibited earlier response and better
callus frequencies. Maximum number and length of shoots and roots were derived
C for 12 months, even without any
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on medium having kinetin (2 mg/L) and indole-3-butyric-acid (2 mg/L) in the callus
derived from root explant. Regenerated plants were transferred to a potting mixture
(sand:soil:perlite (1:1:1)) for hardening followed by multiplication. 100% survival
rate was observed for plants in greenhouse conditions, while well-developed healthy
plants showed a survival rate of 80% in open conditions of herbal garden. Another
study tested the callus induction potential of seedling, root, lamina, and petiole
(Zaib-Un-Nisa et al., 2019). Callus appeared in shoot and seedling explants after
3 days, while lamina explant took the maximum time of 15e20 days. Recently,
Sharma et al. (2019) developed an efficient protocol for in vitro multiplication of
in vitroegrown seedling-derived shoot tip explants for generation of genetically uniform plants. MS medium supplemented with TDZ (1.14 mM) and NAA (2.68 mM)
proved to be an optimal media combination for highest average shoot regeneration
potential (73.33%) where maximum average number of shoots (11.4) and average
shoot length (4.17 cm) was achieved. Maximum rooting of microshoots (77.78%)
was observed on MS þ indole butyric acid (2.46 mM) with average root number
of 6.0 and average length of 3.07 cm. In vitro propagation via direct organogenesis
as well as via callus induction have been established in S. lappa. However, future
studies need to be planned for obtaining high content of secondary metabolites
via tissue culture for establishing a supply system for medicinal purposes.
10.7.2 Ex situ strategies
1. S. lappa rhizomes were collected from its natural habitat and cut into small
pieces having two to three active buds and were placed a day later in experimental plots (Sher et al., 2010) to evaluate their growth routine. However, the
strategy was not very successful as the planted rhizomes had a very poor
sprouting efficiency (Sher et al., 2010).
2. A comprehensive analysis was undertaken for assessing seed germination,
seedling analysis, and survival percentage of S. lappa in higher and lower Himalayan altitudes in Uttarkashi (Parmar et al., 2012). A significant increase in
root length was observed in polyhouse conditions at higher altitude. Overall,
highest percentage of seed germination potential and survival percentage was
noticed for high altitudinal places. Plant morphology at higher altitudes is
indicative of adaptability measures employed for thermoregulation. Woolly
hairs of these plants are densest at low temperature which prevent frost damage
and UV damage. The study thus emphasized the relevance of natural habitat
conditions for better growth and survival (Parmar et al., 2012).
3. Few reports have attempted to increase longevity of seed viability in S. lappa.
Sharma et al. (2014) analyzed the physiological status of seeds procured from
Lahaul for ambient long-term storage (66 months). The study demonstrated that
seed viability and germination could be enhanced via chilling and GA
pretreatments. The seed viability was observed to maintain completely for at
least 18 months and further, even beyond 30 months, 82% viability retention
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