q NIAB 2005
ISSN 1479-2621
Biotechnological approaches for the
production of forskolin, withanolides,
colchicine and tylophorine
Sumita Jha*, Maumita Bandyopadhyay, Kuntal Narayan Chaudhuri,
Seemanti Ghosh and Biswajit Ghosh
Centre of Advanced Study in Cell and Chromosome Research, Department of Botany,
University of Calcutta, 35, Ballygunge Circular Road, Kolkata-700 019, India
Received 22 January 2005; Accepted 19 April 2005
Keywords: colchicine; forskolin; hairy roots; secondary metabolites; shooty teratomas; tissue culture;
transformed cultures; tylophorine; withanolides
Introduction
Plants are capable of synthesizing a variety of low-molecu-
lar-weight organic compounds, called secondary metab-
olites, usually with unique and complex structures. Many
of these compounds are differentially distributed among
limited taxonomic groups within the plant kingdom and,
conversely, each plant species has a distinct profile of
secondary metabolites. Plant secondary metabolites are
of tremendous importance, both for the plant itself (for
plant–environment interactions) and to humans, for their
biological activities that can have therapeutic value. Com-
pared to the main molecules found in the plants, these sec-
ondary metabolites were soon defined by their low
abundance, often less than 1% of the total carbon, or sto-
rage, usually occurring in dedicated cells or organs.
Plants are probably the best cell factories on this earth
from which more than 100 000 secondary metabolites
have been discovered, with the estimated total number in
plants exceeding 500 000 (Hadacek, 2002). Due to their
wide biological activities, plant secondary metabolites
have been used for centuries in traditional medicine.
Nowadays, they correspond to valuable compounds such
as pharmaceuticals, cosmetics, fine chemicals or more
recently, nutraceutics (Bourgaud et al., 2001).
Medicinal plants are the world’s oldest health-care pro-
ducts. Plant species are used as medicinal products in two
ways; traditional medicines, singly or in formulations,
* Corresponding author. E-mail: sumita.jha@vsnl.com or
sjbot@caluniv.ac.in
Plant Genetic Resources 3(2); 101–115
DOI: 10.1079/PGR200571
such as those prepared and dispensed by
traditional medical practitioners, which may or may not
attract a market price; and commercial products, dis-
pensed by prescription or over-the-counter sales, such
as patented or licensed medical products of allopathy or
traditional systems of medicine. The world market for
medicinal and aromatic plants (MAPs) is huge. The largest
global markets for MAPs are China, France, Germany,
Italy, Japan, Spain, the UK and the USA. Japan has the
highest per capita consumption of botanical medicines
in the world (Laird, 1999). The International Council for
Medicinal and Aromatic Plants expects world growth
during 2001 and 2002 to be approximately 8–10% a year
(Srivastava, 2000). India is a major exporter of raw MAPs
and processed plant-based drugs (Lambert et al., 1997);
75% of the total exports from India are sent to six
countries, namely France, Germany, Japan, Switzerland,
the UK and the USA. India’s booming export trade in med-
icinal plants has risen almost three-fold during the last
decade. This boom in local use and export trade is deplet-
ing many species from the wild, bringing some to the edge
of extinction. The major problem in conventional procure-
ment of MAPs is that only a few are cultivated; over 95% of
the medicinal plants used in India are collected from the
wild. Data on threatened species are rare but all over
the world, some species are becoming difficult to obtain
in sufficient amounts to meet increasing demands.
Destruction of natural habitats and technical difficulties
in cultivation are also driving the drastic reductions in
plant availability.
In the search for alternatives to production of desirable
medicinal compounds from plants, biotechnological
102
approaches, specifically plant tissue culture, has the
potential to supplement traditional agriculture in the
industrial production of bioactive plant metabolites
(Ramachandra Rao and Ravishankar, 2002). Large-scale
plant tissue culture is found to be an attractive alternative
approach to traditional methods of plantation, as it offers
a controlled supply of biochemicals independent of plant
availability (Sajc et al., 2000). In vitro propagation and
complete plant regeneration can be a viable method of
ex situ conservation of these important species. Biotech-
nological production of important phytopharmaceuticals
(secondary metabolites) can indirectly help in the conser-
vation of important plants, by reducing the demand for
raw materials from the wild.
Cell suspension culture systems could be used for
large-scale culture of plant cells, from which secondary
metabolites can be extracted. The advantage of this
method is that it can ultimately provide a continuous
and reliable source of natural products. Screening, selec-
tion and medium optimization may lead to a 20 –30-fold
increase in the case of high-producing cultures (Zhong,
2001). The culturing of differentiated cells, induction by
elicitors and metabolic engineering are new approaches
developed in the recent past for the study of biosynthesis
and production of secondary metabolites in vitro. With
the culture of differentiated cells one has in most cases
been able to get production of the desired compounds
in levels comparable to that of the plant; however the
culture of such differentiated tissues on a large scale in
bioreactors is a major constraint.
The production of high-value secondary metabolites
including pharmaceuticals and food additives through
shoot cultures, root cultures and Agrobacterium-mediated
transgenic roots obtained through biotechnological means
has the potential to supplement traditional agriculture in
the industrial production of bioactive plant metabolites.
Transformed cultures can be developed following infec-
tion with the plant pathogens Agrobacterium rhizogenes
and A. tumefaciens. Agrobacterium-mediated gene trans-
fer system has developed rapidly after the description of
the Ti plasmid and publication of the complete sequences
of T-DNAs of A. tumefaciens and A. rhizogenes, and has
been used for transformation of a large number of plants
because of the convenience of the methodology and high
probability of single-copy integration (Zupan and
Zambryski, 1995). The system is simple, inexpensive and,
in many cases, efficient transformation is achieved by co-
cultivation with Agrobacterium using different types of
explants such as leaf, root, hypocotyls, petiole and cotyle-
don (Hooykaas and Beijersbergen, 1994). Plant cell and
transgenic hairy root cultures are promising potential
alternative sources for the production of high-value sec-
ondary metabolites of industrial importance (Sevón and
Oksman-Caldentey, 2002). Transgenic hairy roots are
Sumita Jha et al.
unique in their genetic and biosynthetic stability, faster
in growth and more easily maintained. Using this
methodology a wide range of chemical compounds has
been synthesized (Shanks and Morgan, 1999; Sevón and
Oksman-Caldentey, 2002). The biotechnological appli-
cation of transformed cell cultures is promising for stable,
fast-growing high-level production of secondary metab-
olites (Jha, 1997). An advance in tissue culture, combined
with improvement in genetic engineering, specifically
transformation technology, has opened new avenues for
high-volume production of pharmaceuticals, nutraceuti-
cals and other beneficial substances (Hansen and Wright,
1999).
We have been working on production of principal sec-
ondary metabolites in in vitro cultures of a number of
economically important, indigenous medicinal plants in
our laboratory. The current status of research in this
field in selected Indian medicinal plants is summarized
in this article.
Forskolin from Coleus forskohlii Briq.
Coleus forskohlii Briq., a member of the family Lamiaceae
(Fig. 1), is a common and ancient medicinal plant of
India, and is used traditionally in Ayurvedic medicine. A
large-scale screening of medicinal plants by the Central
Drug Research Institute, Lucknow, India, in 1974,
revealed the presence of a hypotensive and spasmolytic
component of C. forskohlii that was named coleonol.
Further investigation (Saksena et al., 1985) determined
the exact chemical structure of this labdane diterpene
and its name was changed to forskolin (7b-acetoxy-
8,13-epoxy-la,6b,9a-trihydroxy-labd-14-en-11-one). This
compound is isolated principally from the roots. The
pharmacological activity of forskolin is, however, sub-
stantiating the traditional uses of C. forskohlii, which
includes lowering of blood pressure, relaxation of the
arteries and smooth muscles. It is effective against glau-
coma, abdominal colic, asthma and other allergic dis-
orders, and skin diseases, and significantly increases
lipolysis (fat burning). It is a potential cure for insomnia
and convulsions, and is anti-ageing and antioxidant
(Ammon and Müller, 1985; Marone et al., 1986;
Wysham et al., 1986; Okuda et al., 1992; Petersen, 1994;
Srivastav et al., 2002).
The basic mechanism of action of forskolin is the acti-
vation of adenylate cyclase, which increases cyclic ade-
nosine monophosphate (cAMP) in cells. Cyclic AMP is
considered to be one of the most important cell-regulat-
ing compounds. Under normal circumstances, cAMP
forms by adenylate cyclase activation due to hormonal
stimulation at the cell receptor sites. However, forskolin
seems to bypass this reaction and causes an increase in
Production of various secondary metabolites
Fig. 1. Coleus forskohlii plants and cultures as source of forskolin: (a) Structure of forskolin; (b) field plant; (c) transformed
root culture; (d) stem gall; (e) leaf gall; (f) transgenic shooty teratomas developed following infection with Agrobacterium
tumefaciens strain C58 growing on unsupplemented MS medium; (g) untransformed plants growing on unsupplemented MS
medium.
intracellular cAMP. A decreased intracellular cAMP level is
thought to be a major factor in the development of many
disease processes. Forskolin has a remarkable effect in
relaxing constricted bronchial muscles in asthmatics.
The antispasmodic action of forskolin supports the long
time use of C. forskohlii in the treatment of asthma, intes-
tinal colic, uterine cramps, painful urination, angina and
hypertension. Forskolin’s ability to relax smooth muscle
in bronchial asthma is most probably due to an increase
in cAMP, although forskolin has other anti-allergic activi-
ties such as inhibiting the release of histamine. Psoriasis is
a very common skin disease that seems to be caused by a
relative decrease in cAMP. Preliminary studies have indi-
cated that forskolin may be of great benefit to individuals
with psoriasis. A new application for C. forskohlii is redu-
cing body fat while maintaining lean body mass. Lipoly-
sis, the breakdown of stored fat, is actually regulated by
cAMP. Forskolin not only enhances lipolysis, but it may
also inhibit fat storage from occurring (Ammon and
Müller, 1985; Marone et al., 1986; Wysham et al., 1986;
Okuda et al., 1992; Petersen, 1994; Srivastav et al., 2002).
The demand for forskolin is mainly satisfied by large-
scale indiscriminate collections of C. forskohlii from wild
habitats due to lack of organized cultivation; the natural
populations of this species in the wild have declined
rapidly. Since C. forskohlii hitherto is the only known
plant source for forskolin, this has led to a severe depletion
of the plant (Vishwakarma et al., 1988), and it is listed as
one of the endangered plant species in India (Sharma
et al., 1991). Forskolin occurs at an average content of
103
0.1% of the dry weight of tubers, but the content varies
substantially in different populations, from 0.01 to 0.44%
(Vishwakarma et al., 1988).
Micropropagation and in vitro culture for
production of forskolin
The first step towards prevention of extinction of this
rare species is micropropagation, and propagation of
C. forskohlii through shoot multiplication has been
reported by Sen and Sharma (1991a) and Bhattacharyya
and Bhattacharya (2001). There are some reports on
in vitro cultures of C. forskohlii tissues for production
of forskolin. Mersinger et al. (1988) demonstrated that
suspension cultures under natural growth conditions
did synthesize forskolin and other labdane diterpenoids.
Suspension cultures, however, lost their capacity to pro-
duce secondary metabolites after 3–4 years and new cul-
tures had to be established.
The interest in root cultures was based on the occur-
rence of forskolin in root tuber. Krombholz et al. (1992)
established root cultures of C. forskohlii and reported a
close correlation between root differentiation and forsko-
lin production. Sen et al. (1992), on the other hand,
demonstrated the production of forskolin in shoots and
shoot-forming callus but not, or only traces, in rhizogenic
callus and root cultures, thus showing that root differen-
tiation and forskolin production are not necessarily
coupled. In untransformed root cultures, Sen et al.
(1993) reported enhancement in forskolin accumulation
104
by supplementation of basal media with chloro-deriva-
tives of indoleacetic acid (IAA). The highest amount of
forskolin (0.09%) was accumulated at 50 mg/l 4-Cl-IAA
after 60 days of culture.
It is evident that cell and organ culture systems may be
suitable to produce forskolin and that a close correlation
exists between differentiation and forskolin production
in vitro.
Forskolin synthesis in transformed cultures
Transformed cell and organ cultures have proved valuable
in the study of different aspects of secondary metabolism as
the transformed cultures are more stable genetically and
biochemically over long periods in culture. Agrobacterium
tumefaciens-mediated transformation in Coleus forskohlii
for forskolin production was first reported by Mukherjee
et al. (1996). Various tumorous cultures were developed
and maintained in long-term culture following infection
of C. forskohlii with wild-type A. tumefaciens strains C58
and N2/73. The different tumorous culture lines estab-
lished, namely gall calluses, cell suspension cultures, rhizo-
genic calluses, and rooty and shooty teratomas,
represented different levels of cellular organization and
differentiation. Axenic tumour callus, cell suspensions,
and shooty and rooty teratomas were well maintained on
basal medium without growth regulators.
The unorganized tumour callus (line GCO), growing
on B5 agar medium with 3% sucrose, when transferred
to B5 media containing 100 mg/l casein hydrolysate
(CH), turned rhizogenic in nature after 2 weeks. The
lower concentrations of CH (i.e. 10–50 mg/l) did not
induce any root initials. Rapid subculturing (at 10 –12-
day intervals) of this rhizogenic line (line GCO-RCH)
was essential, otherwise the calluses turned brown and
growth rate decreased.
Another callus line (LC-DK) was induced from leaves of
shooty teratoma. When leaves of shooty teratomas were
cultured on B5 medium supplemented with 2,4-dichloro-
phenoxyacetic acid (2,4-D) and kinetin, callus was induced
within 15 days. This callus (line LC-DK) was maintained on
the same medium by subculturing at 21-day intervals.
When LC-DK calluses were subcultured on medium con-
taining 6-benzyladenine (BA) and kinetin, calluses
became highly rhizogenic, showing development of
numerous whitish roots. This line (LC-BK-RCH) also was
fast growing and was subcultured every 2 weeks, otherwise
long gaps between subcultures stopped growth comple-
tely. The growth patterns of various transformed tissue cul-
ture lines and forskolin production at different levels of
organization were studied (Mukherjee et al., 1996).
Tumour callus line GCO and line LC-DK (callus induced
from leaves of shooty teratomas) differed in their
Sumita Jha et al.
morphology and growth rate. Line GCO being faster grow-
ing with a 5.3-fold biomass increase in 9-day period com-
pared to only a 2.8-fold increase in biomass in line LC-DK
in an 18-day period. Both callus lines were capable of
accumulating forskolin (lines GCO and LC-DK, 0.002 and
0.024%, respectively). Rhizogenic callus (line GCO-RCH)
was obtained from tumorous callus and grown in the
absence of phytohormones but in the presence of casein
hydrolysate (100 mg/l). Line GCO-RCH is a very fast-grow-
ing cell line that resulted in a 14.5-fold increase in biomass
in a 28-day period. Forskolin accumulation was 0.011%
during this period. Another rhizogenic line, LC-BK-RCH,
showed higher levels of forskolin accumulation (0.037%)
as compared to the line GCO-RCH, although growth rate
(3.5-fold biomass increase in a 15-day period) was lower
than that of the line GCO-RCH. Shooty teratomas did not
accumulate forskolin under any cultural conditions
(Mukherjee et al., 1996).
Transformed cell suspension culture
The crown gall and gall-derived cell suspension cultures
infected with wild-type Agrobacterium tumefaciens Ti
plasmid have been used for production of some specific
secondary metabolites such as quinoline alkaloids from
Cinchona ledgeriana (Payne et al., 1987), isoflavonoid
glucosides from Lupinus polyphyllus (Berlin et al., 1989)
and cardenolides from Digitalis lanata (Moldenhauer
et al., 1990). We developed a stable reproducible high-
yielding, hormone autotrophic gall-derived transformed
cell suspension culture from Coleus forskohlii for forsko-
lin production (Mukherjee et al., 2000a,b, 2003).
Gall suspension cultures capable of growing and pro-
ducing forskolin in hormone-free basal medium were
established. These cultures were creamish in colour, fast
growing and produced 0.002% forskolin that is similar
to its originating tumorous callus line GCO (Mukherjee
et al., 1996). Five fast-growing cell lines were selected
(GSO-5, GSO-10, GSO-12, GSO-18, GSO-24), by employ-
ing the single-cell plating technique, which were of dis-
tinct morphology and varied in forskolin synthesizing
capacity (Table 1). Among the five lines, line GSO-5
was less friable, showed moderate growth rate but maxi-
mum forskolin content (0.021%). Line GSO-12 showed
very fast growth with higher cell friability but was not
selected as the forskolin content in this line was low
(0.003%). Cell growth in suspension was found to be
influenced significantly by carbon source, initial cell den-
sity, and light or dark conditions. Optimal cell growth
(20-fold increase in biomass in a 42-day period) was
obtained when the cells were grown in dark conditions
in B5 media containing 3% sucrose as sole carbon
source with an initial cell density of 1.5 £ 105 cells/ml.
Production of various secondary metabolites 105

Table 1. Selective criteria used for callus line selection to establish cell
cultures (after 6 weeks; Mukherjee et al., 2000a)

Forskolin
Cell line Colour Texture Growth rate content (%)
GSO (control) Cream Friable High 0.002
GSO-5 Cream Non-friable High 0.021
GSO-18 Light brown Non-friable Very high 0.017
GSO-12 White Highly friable Very high 0.003
GSO-10 Yellow Friable High 0.01
GSO-24 Light brown Friable High 0.007

Forskolin accumulation was maximum (0.021%) in the
stationary phase of cell growth. These suspension cul-
tures showed continuous and stable production of for-
skolin. Forskolin was not found to be excreted into the
medium at any time of the culture period studied.
Although all the suspension culture lines established in
this study were capable of producing forskolin in hor-
mone-free media, line GSO-5 was found to accumulate
forskolin with content almost comparable to roots of in
vivo-grown plants and the potential for synthesizing for-
skolin has been retained by this cell line for more than
7 years.
Scaling up of forskolin production has been studied by
growing cell suspension culture in 1 litre flasks contain-
ing 160 ml of hormone-free B5O medium (3% sucrose).
Cells were also grown in 100, 250 and 500 ml flasks con-
taining 20, 40 and 80 ml of media, respectively, for com-
parative studies. It was found that, unlike other cultures,
those grown in 1 litre flasks grew linearly with time and
did not cease growth even after a 56-day period, and
average increase in biomass in this last phase (between
42 and 56 days) was compared to only 2.63% in the
other three cultures. When accumulation in 1 litre flasks
(0.067 mg/l/day) was compared to that of 100, 250 and
500 ml flasks, it was found that productivity of 6-week-
old cultures was highest in the 500 ml flask containing
80 ml of basal medium (0.094 mg/l/day) as compared to
0.067 mg/l/day of the 1 litre flask (Table 2) (Mukherjee
et al., 2000a).
Table 2. Growth and forskolin production of suspension
cultures at different flask volume (after 6 weeks; Mukherjee
et al., 2000a)
Flask size Biomass yield Productivity
(ml) (g dry wt/l) (mg/l/day)
150 10 0.05
250 12.5 0.063
500 19 0.094
1000 13.6 0.067
Role of different growth regulators on forskolin
synthesis in transformed cultures
Forskolin synthesis at different degrees of organization in
transformed cultures of C. forskohlii was studied and
compared with those of untransformed cultures
(Mukherjee et al., 2000b). The effect of casein hydrolysate
on transformed cultures was examined to illustrate the
relationship between levels of organization and forskolin
synthesis in vitro. It was found that casein hydrolysate at
2.0 g/l significantly enhanced forskolin content (2.3 mg/g
cell dry wt) in rhizogenic tumorous line GCO-RCH-2. In
rooty teratoma line RC-ST-2/4, forskolin content increased
to 1.7 mg/g cell dry wt in the presence of 2.5 g/l casein
hydrolysate.
Plants transformed with Agrobacterium always show
changes in their endogenous hormone concentration
due to the integration of the T-DNA region, which con-
tains auxin and cytokinin biosynthetic genes. Thus, we
have studied the extent to which phytohormones can
influence secondary metabolism in transformed cell and
organ cultures of C. forskohlii (Mukherjee et al., 2003).
The role of auxins, cytokinins and gibberellin has been
studied for their influence on growth, morphology and
forskolin synthesis in genetically transformed cell cul-
tures. In rhizogenic line GCO-RCH-10, very thin, white
pointed root tips were found in the rhizogenic mass in
the presence of IAA or naphthaleneacetic acid (NAA)
but root tips became swollen and blunt in the presence
of indolebutyric acid (IBA) at various concentrations
(0.5, 1.0, 2.0 mg/l). On the other hand, rhizogenic cul-
tures were deformed, became yellowish in colour and
tended towards callusing in media containing 2,4-D. All
the auxins enhanced forskolin production in this rhizo-
genic line (GCO-RCH-10) although the forskolin content
was highest (0.77 mg/g dry wt) in the presence of 2.0 mg/
l IBA. In root line RC-ST-2/16, IAA and IBA enhanced for-
skolin production at all concentrations while NAA and
2,4-D suppressed forskolin production. Maximum
forskolin (1.83 mg/g dry wt) was produced in the pre-
sence of 0.5 mg/l IAA. In general, auxin conjugates
106
favoured moderate growth in all the three cell lines
without affecting their morphology remarkably. In IAA –
phenylalanine (0.20 mg/l) supplemented medium, maxi-
mum forskolin synthesis occurred, namely 0.81 and
1.07 mg/g dry wt in rhizogenic line GCO-RCH-10 and
shoot line RC-ST-2/16, respectively (Mukherjee et al.,
2003).
Transformed hairy root cultures
Agrobacterium rhizogenes-mediated transformation for
initiation of hairy root culture and forskolin production
was reported by Krombholz et al. (1992). They studied
forskolin production in root cultures established follow-
ing infection with A. rhizogenes and compared growth
and forskolin production of hairy root cultures with
untransformed root cultures. The hairy roots grew rela-
tively slowly on hormone-free media and showed accel-
erated growth only in auxin-supplemented media. The
forskolin yield was about the same as in untransformed
root cultures but due to better growth in auxin-
supplemented media, productivity of the transformed
root cultures was much higher.
Sasaki et al. (1998) reported high forskolin production
in hairy roots induced by infection with the A. rhizogenes
MAFF 03-01724 strain. Hairy root clone B9 grew well in
woody plant liquid medium and showed a high forskolin
yield (ca 1.3 mg per 100 ml flask) after 5 weeks of culture.
The time course of growth and forskolin production of
the clone B9 cultured in woody plant liquid medium
was also examined. Rapid growth started at week 2 and
continued until week 5. The highest forskolin yield (ca
1.6 mg per 100 ml flask) was obtained at week 5. Pro-
ductivity was thus much higher than that previously
reported.
Molecular cloning
Engprasert et al. (2004) reported molecular cloning and
functional expression of geranylgeranyl pyrophosphate
from Coleus forskohlii Briq. Isopentenyl diphosphate, a
common biosynthetic precursor to the labdane diterpene
forskolin, has been biosynthesized via a non-mevalonate
pathway. Geranylgeranyl diphosphate (GGPP) synthase
is an important branch-point enzyme in terpenoid bio-
synthesis. Therefore, GGPP synthase is thought to be a
key enzyme in the biosynthesis of forskolin. The open
reading frame for full-length GGPP synthase encodes a
protein of 359 amino acids, 1077 nucleotides long with
calculated molecular mass of 39.3 kDa. Alignments of
C. forskohlii GGPP synthase amino acid sequences
revealed high homologies with other plant GGPP
Sumita Jha et al.
synthases. Several highly conserved regions, including
two aspartate-rich motifs, were identified. Transient
expression of the N-terminal region of C. forskohlii
GGPP synthase–green fluorescent protein (GFP) fusion
protein in tobacco cells demonstrated subcellular localiz-
ation in the chloroplast. Carotenoid production was
observed in Escherichia coli harbouring pACCAR25DcrtE
from Erwinia uredovora and plasmid-carrying
C. forskohlii GGPP synthase. These results suggested
that cDNA encoded functional GGPP synthase.
Furthermore, C. forskohlii GGPP synthase expression
was strong in leaves, decreased in stems and very little
expression was observed in roots. This investigation pro-
posed that the forskolin was synthesized via a non-meva-
lonate (1-DX) pathway. GGPP synthase is thought to be
involved in the biosynthesis of forskolin, which is primar-
ily synthesized in the leaves and subsequently accumu-
lates in the stems and roots.
Withanolides from Withania somnifera (L.) Dunal.
Withania is a small genus of shrubs belonging to the
family Solanaceae, distributed east of the Mediterranean
region, extending to South Asia, and also to Africa.
W. somnifera, also known as ashwagandha (Fig. 2b),
Indian ginseng and winter cherry, is a very valuable
and popular Ayurvedic and Unani drug, which is even
compared with ginseng in view of the various therapeutic
activities attributed to it (Sharma and Dandiya, 1992).
The plants are propagated from seeds, which are sown
in early spring. Harvesting starts in January and continues
till March. The entire plant is uprooted for roots. The
commercial drug consists of dried roots of W. somnifera
(Anonymous, 1976).
The major biochemical constituents of ashwaganda
root are steroidal alkaloids and steroidal lactones in a
class of constituents called withanolides, or withasteroids.
Withasteroids (Fig. 2a) are a group of naturally occurring
C-28 steroidal lactones, built on an intact or rearranged
ergostane framework, found generally in many solanac-
eous genera, and specifically in the genus, Withania
(Ray and Gupta, 1994).
Withaferin A, the first known member of this group,
was isolated from the well-known Indian medicinal
plant Withania somnifera, and its structure was fully elu-
cidated by Lavie et al., in 1965. The structural novelty and
interesting biological activities elicited by this compound
led to a thorough chemical investigation of the plant, and
numerous compounds with similar structural features
were subsequently isolated and characterized. Such C-
28 steroidal lactones, characterized by a 9-C side-chain
and a six-membered ring lactone, were designated as
Production of various secondary metabolites 107

Fig. 2. Withania somnifera plants and cultures as sources of withaferin A and withanolide D: (a) chemical structure of witha-
nolides; (b) field-growing plant; (c) multiple shoot cultures growing on MS basal medium with 2 mg/l BA; (d) induction of
transformed roots on leaf explants following infection with Agrobacterium rhizogenes strain A4; (e) an electrophoretogram
showing the presence of agropine and mannopine in different A. rhizogenes-transformed root cultures (lane 1, standard
agropine and mannopine; lanes 2 and 3, untransformed roots; lanes 4 –6, transformed hairy roots).

withanolides, after the generic name of the plant of origin
(Ray and Gupta, 1994).
At present, 12 alkaloids, 35 withanolides and several
sitoindosides from this plant have been isolated and stu-
died. Much of ashwaganda’s pharmacological activity has
been attributed to two main withanolides: withaferin A,
and withanolide D which is isomeric to withaferin A
(Anonymous, 2004).
Micropropagation and tissue culture for production
of withanolides
Micropropagation and tissue culture studies through
shoot multiplication from seedling explants was reported
by Sen and Sharma (1991b). Kulkarni et al. (1996) devel-
oped an efficient protocol for in vitro propagation of
shoot buds from leaf explants in Murashige and Skoog’s
(1962) basal medium supplemented with IAA and BA.
Shoots were elongated and rooted in the presence of
low levels of BA and 100% in vitro-grown plantlets
could be established in potted soil. However, the witha-
nolide content of the tissue culture-derived plants was
not reported. Micropropagated plants of W. somnifera
from Italy showed low contents of withanolides and the
presence of withanolide J (Vitali et al., 1996). Rani and
Grover (1999) reported shoot regeneration from callus
cultures induced from axillary leaves, axillary shoots,
hypocotyls and root segments. Organogenesis and
regeneration of whole plants of the Indian chemotypes
were reported (Ray and Jha, 2002) (Fig. 2c) and withano-
lide analysis in plants transferred to soil revealed a
chemotype-specific withanolide profile in regenerated
plants. Manickam et al. (2000) reported regeneration of
shoots from stem callus in MS medium supplemented
with IAA and BA. Rani et al. (2003) reported callus
induction from hypocotyls, root and cotyledonary leaf
explants, and regeneration of shoots from induced callus.
Untransformed cell and organ culture for
withanolide production
Tissue culture for biomass production and withasteroid
accumulation is an important biotechnological approach
and very few studies have been carried out on this
aspect in W. somnifera. The earliest report on phyto-
chemical investigation of W. somnifera tissue cultures
by Yu et al. (1974) revealed that undifferentiated suspen-
sion cultures did not synthesize withanolides. Roja et al.
(1991) established multiple shoot cultures from axillary
meristems in MS medium containing BA, rooted those
shoots in MS medium supplemented with coconut milk
(10%) and reported callus formation in MS medium sup-
plemented with 2,4-D. Callus cultures failed to synthesize
withanolides. Multiple shoot culture synthesized signifi-
cant levels of withanolides and their concentrations
108
differed with different exogenous growth hormones
used. Withanolides identified in shoot cultures were with-
anolide I, withanolide G, traces of withanolide D and
withanolide E. Ray and Jha (2001) reported induction
of multiple shoots from single shoot-tip explants in MS
medium supplemented with BA, and detected withano-
lide D and withaferin A in the regenerated shootlets.
They also reported that supplementation of the solid
regeneration medium with 4% sucrose increased accumu-
lation of both withaferin A and withanolide D, while
culture in liquid regeneration medium containing 10%
coconut milk favoured not only an increase in the
number of microshoots induced per explant but also
withaferin A accumulation. Similar studies involving
multiple shoot induction from nodal explants were per-
formed by Furmanowa et al. (2001) who reported immu-
nomodulatory action of the methanolic extract of
greenhouse-transplanted regenerated plants.
Transformed cell and organ cultures
There are two types of root cultures, untransformed root
cultures and hairy root cultures. Unlike other solanaceous
species, there are no reports on untransformed root cul-
tures of W. somnifera. Transformed root cultures were
established following infection of shoots with A. rhizo-
genes strain LBA 9402 (Fig. 2d) (Ray et al., 1996). The
hairy root cultures grew axenically in the absence of
growth hormones. The transgenic nature of the roots was
confirmed by opine assay (Fig. 2e). The root cultures pro-
duced several withanolides, of which withanolide D was
isolated and identified. Transformed root clone HR1
showed a 20-fold increase in growth with a withanolide
D yield of 0.30 mg/g dry wt tissue. While transformed
root cultures could be maintained in long-term culture,
all attempts to maintain untransformed root cultures
failed (Ray, 2000). Vitali et al. (1996) reported that withano-
lides were absent in hairy root cultures obtained following
infection of Italian W. somnifera with A. rhizogenes strain
LBA 9402. Further studies on A. rhizogenes-mediated trans-
formation on different genotypes of W. somnifera in our
laboratory have led to establishment of hairy root lines dif-
fering in root morphology and high withanolide content.
Agrobacterium tumefaciens-mediated transformation
of Withania somnifera was reported by Ray and Jha
(1999). Transformed organ cultures were also established
following infection with wild-type nopaline and octopine
strains of A. tumefaciens. The oncogenic strains had
different virulence in the two genotypes studied, the
main difference being in the nature of galls formed and
their morphological competence. Galls obtained follow-
ing infection with nopaline strain N2/73 spontaneously
developed shooty teratomas of altered phenotype. The
Sumita Jha et al.
shooty teratomas grew in unsupplemented basal
medium and were able to synthesize both the major with-
anolides of the parent plants. Withanolide synthesis in
shooty teratomas was much higher (0.1% withaferin A
and 0.08% withanolide D) than in non-transformed
shoot cultures. The rooty teratomas developed following
infection with octopine and nopaline strains of
A. tumefaciens accumulated withanolide D but not with-
aferin A. Gall callus and untransformed callus did not
show the presence of principal withanolides of Indian
chemotypes withanolide D and withaferin A (Ray and
Jha, 1999). Thus shoot differentiation seems to be a pre-
requisite for the synthesis of withaferin A. Although the
molecular mechanism of shooty teratoma formation is
not yet completely clear, shooty teratoma form after inte-
gration of part of the A. tumefaciens Ti plasmid into the
plant genome in an analogous way to hairy root induc-
tion by A. rhizogenes and the expression of bacterial
genes affecting the response of the plant cells to auxin
and cytokinin may need to be manipulated before
shooty teratomas can be induced in a wide number of
plant species for secondary metabolite production
(Subroto et al., 1995).
Tylophorine from Tylophora indica (Burm. f) Merrill
Tylophora indica (Burm. f) Merrill (previously known as
T. asthmatica Wight et Arn.), of the Asclepiadaceae
family, is an important indigenous medicinal plant in
India. It is the main source of phenanthroindolizidine
alkaloid tylophorine (C24H27NO4) (Gellert, 1982).
T. indica (Fig. 3) has been used in ethno-medical prac-
tices over the centuries in certain parts of India and in
South-East Asia; its medicinal use was first documented
in the Bengal pharmacopoeia in 1884 when, because of
its emetic properties, European practitioners in India
used it as substitute for ipecac (Cephaelis ipecacuanha).
Both roots and leaves are described as dried crude drug
in several pharmacopoeias (Anonymous, 1976).
The leaves are emetic, diaphoretic and expectorant
(Chopra et al., 1958), and used to treat bronchial
asthma (Nadkarni, 1954), bronchitis and rheumatism
(Chopra et al., 1958). They are also used to destroy
vermin (Nadkarni, 1954). The roots have a sweetish
taste, aromatic odour and brittle fracture. They possess
stimulant, emetic, cathartic, expectorant, stomachic and
diaphoretic properties and are used for the treatment of
asthma, bronchitis, whooping cough, dysentery, diar-
rhoea, rheumatism and gout (Anonymous, 1976). The
roots have bacteriostatic properties (Bhutani et al., 1985).
Dymock et al. (1891) were the first to report the presence
of alkaloids in T. indica. Karnick (1975) reported that the
leaves had a higher total alkaloid content (0.12–0.50%)
Production of various secondary metabolites
Fig. 3. Tylophora indica plants and cultures as sources of tylophorine: (a) 12-year-old field plant (bar ¼ 50 mm); (b) root-
derived shoot organogenic nodular meristemoid cultures on basal MS medium (BM) supplemented with 2 mg/l BA
(bar ¼ 6 mm); (c) root-derived friable embryogenic callus cultures on BM supplemented with 2 mg/l BA (bar ¼ 6 mm); (d)
12-week-old plantlets regenerated from root explants cultured on BM (bar ¼ 12 mm); (e) transformed (pRiA4) root cultures
on BM (bar ¼ 10 mm); (f) structure of tylophorine.
compared to roots (0.09–0.35%) and stems (0.07–0.37%),
and the alkaloid content in all three organs was highest
during the flowering period and in the rainy season. Tylo-
phorine was also detected in other plant species (Table 3).
The phenanthroindolizidine alkaloids including tylo-
phorine show profound cytotoxicity due to their inhibition
of protein and DNA synthesis (Rao et al., 1997, 1998; Rao
and Venkatachalam, 2000; Stærk et al., 2002). Thus, many
of the alkaloids such as tylophorine (Donaldson et al.,
1968; Nacci et al., 1973), tylophorinidine (Rao et al.,
1971), tylocebrine (Gellert and Rudzats, 1964) from
T. crebrifolia and pergularine from Pergularia pallida
(Rao et al., 1999) are known to be antineoplastic.
Tylophorine and many of the other phenanthroindolizi-
dine alkaloids show equal toxicity for drug-sensitive and
multi-drug-resistant target cells, which contrasts with
many front-line antineoplastic drugs such as Catharanthus
alkaloids and taxol (Stærk et al., 2002).
Detailed studies on the pharmacological effects of tylo-
phorine (Gopalakrishnan et al., 1979) established its
effect on the respiratory, cardiovascular and central
nervous system. Tylophorine is toxic to Paramoecium
Table 3. Natural sources of tylophorine
Plant species Reference
Tylophora indica Govindachari et al., 1954
Tylophora crebrifolia Gellert et al., 1962
Tylophora tanakae Abe et al., 1995
Vincetoxicum officinale Pailer and Streicher, 1965
Cyanchum vincetoxicum Wiegrebe et al., 1969
Pergularia pallida Mulchandani and
Venkatachalam, 1976
109
caudatum in dilutions $5 £ 1024, and is lethal to frogs
at 0.4 mg/kg body weight dosage. Its toxicity for mice
and guinea pigs is very low (Govindachari et al., 1961).
Cell and tissue culture for production of tylophorine
Tissue culture in T. indica started with the study of mor-
phogenetic potential of stem callus cultures (Rao et al.,
1970) and the study of somatic embryo development in
them (Rao and Narayanaswamy, 1972). Later, alkaloid
productivity was studied in stem and root callus cultures
(Benzamine and Mulchandani, 1973) and irradiated stem
callus cultures (Benzamine and Mulchandani, 1976) but
phenanthroindolizidine alkaloids could not be detected.
Benzamine et al. (1979) studied alkaloid productivity in
tissue cultures and regenerated plants and were the first
to detect alkaloids in callus cultures. Several other reports
on in vitro propagation using different explants, culture
systems and regeneration pathways are also known
(Table 4), but none of them have studied alkaloid pro-
ductivity. Jayanthi and Mandal (2001), using random
amplified polymorphic DNA (RAPD) analysis, have con-
firmed the genetic integrity of callus-regenerated plants.
We have recently reported complete plant regeneration
from root explants of T. indica via shoot organogenesis
and somatic embryogenesis, from nodular meristemoid
(NM) and friable embryogenic callus (FEC), respectively
(Chaudhuri et al., 2004). We now have developed a pro-
tocol for the extraction of tylophorine and its analysis by
high performance liquid chromatography (HPLC), based
on the modification of the method of Abe et al. (1995,
2001). We detected and quantified tylophorine (Fig. 3)
110
Table 4. In vitro propagation in Tylophora indica
Plant regeneration
Protoplast-mediated regeneration
Axillary shoot multiplication
Somatic embryogenesis from stem callus
Shoot organogenesis from leaf callus
Somatic embryogenesis from leaf callus
Shoot organogenesis from root nodular meristemoid
Somatic embryogenesis from root callus
in samples extracted from about 100 mg dry wt tissue
(Chaudhuri et al., 2005).
The HPLC analysis of extracts of root-derived NM and
FEC cultures and the regenerated plants (Chaudhuri et al.,
2004) revealed that they synthesized tylophorine. The
alkaloid content decreased with the loss of structural
organization of the root tissue. In the NM (after 4 weeks
of growth in MS medium supplemented with 2 mg/l BA),
the tylophorine content was lower (a 35% decrease) than
the root explants. The completely disorganized FEC
(under growth conditions similar to NM), showed
almost half the tylophorine content of the NM from
which they were induced. However, on increasing the
growth period from 4 to 12 weeks, the tylophorine con-
tent in the FEC increased 2.7 times. Although the tylo-
phorine productivity in FEC cultures appeared to
decrease in the absence of BA, in BA-supplemented
cultures the concentration of BA had very little effect
(Table 5). In the in vitro-regenerated plantlets, tylophor-
ine content was higher compared to both the above cul-
tures, but lower than field plants. The tylophorine
productivity of in vitro plantlets showed a 21-fold
increase on extending the growth period from 12 to 24
weeks (Table 6). This is mostly due to the increase in
shoot and root biomass. Analysis of regenerated plants
(both in vitro and in the field) indicated that the tylo-
phorine content in the different organs has remained
stable (Table 7).
Table 5. Effect of BA on tylophorine accumulation in root-
derived friable embryogenic callus in Tylophora indica
BA concentration (mg/l) Tylophorine productivity (mg)a
0.0 0.06
1.0 0.41
2.0 0.43
5.0 0.41
Friable embryogenic callus (FEC) was grown on solid MS
medium in 150-ml flasks for 12 weeks with 16/8 h light/
dark photoperiod; 1 g FEC was used as inoculum per flask.
a a
Dry wt biomass per flask (g) £ tylophorine content (mg/g
dry wt).
Sumita Jha et al.
Reference
Mhatre et al., 1984
Sharma and Chandel, 1992
Rao et al., 1970; Rao and Narayanaswamy, 1972
Faisal and Anis, 2003
Manjula et al., 2000; Jayanthi and Mandal, 2001
Chaudhuri et al., 2004
Chaudhuri et al., 2004
Transformed culture
We have recently reported Agrobacterium rhizogenes-
mediated transformation and the establishment of
pRiA4-transformed different root clones in T. indica
(Chaudhuri et al., 2005). Tylophorine was detected in
all the transformed root clones and the tylophorine con-
tent and productivity significantly varied between the
different transformed root clones. The tylophorine con-
tent of transformed root clones was 1.2–1.5 times more
than that of the roots of non-wild-type plantlets. These
root clones also showed high productivity in contrast
with wild-type root cultures, with the biomass pro-
ductivity ranging from 1.54 to 3.20 g per flask and tylo-
phorine productivity ranging from 0.16 to 0.30 mg per
flask (inoculated with 200 mg of root tips and cultured
on MS solid for 4 weeks in the dark). Analysis of different
factors contributing to productivity showed that this
increase was mostly due to the enhancement of the bio-
mass production due to the genetic transformation-
induced autonomous growth of transformed roots. The
productivity of transformed roots (analysed in the root
tissue) increased 2.4 times further in liquid cultures due
to increase in both biomass yield (1.7 times) and alkaloid
level (1.4 times). Interestingly, tylophorine was detected
Table 6. Effect of growth period on tylophorine accumu-
lation in in vitro plantlets of Tylophora indica
Tylophorine
Growth period Organ productivity (mg)a
12 weeks Root 0.07
Shoot 0.03
Total 0.10
24 weeks Root 0.46
Shoot 1.65
Total 2.12
Plantlets were grown on solid MS medium in 250-ml flasks
with 16/8 h light/dark photoperiod; one shoot tip (2 cm) was
used as inoculum per flask.
Dry wt biomass per flask (g) £ tylophorine content (mg/g
dry wt).
Production of various secondary metabolites
Table 7. Tylophorine content in tissue culture-derived
plants and source plants of Tylophora indica
Tylophorine content
Plant Organ (% dry wt)
Mother plant Root 0.10
(field; 12 years old) Stem 0.06
Leaf 0.21
Regenerated plant (field; Root 0.11
1 year old) Stem 0.05
Leaf 0.20
Tissue samples of field plants were collected in the months
of July and August.
in the chloroform extracts of the liquid culture medium.
Time-course studies showed that the tylophorine level
excreted into the medium remained more or less stable,
about 13% of the total amount produced.
Colchicine from Gloriosa superba L.
Gloriosa (Liliaceae) is a small genus of ornamental climb-
ing herbs native to tropical Asia and Africa, commonly
known as Glory Lilies or Climbing Lilies (Anonymous,
1956). Clewer et al. (1915) isolated a mixture of alkaloids
consisting mainly of colchicine from dried tubers of Glor-
iosa superba (Fig. 4f). Bellet and Gaignault (1985) com-
pared the relative colchicine content of the genera
Colchicum and Gloriosa. On dry mass basis, Colchicum
yielded 0.62% colchicine and 0.39% colchicoside, while
Gloriosa yielded 0.9% and 0.82%, respectively. Colchicine
is a tropolone type of neutral alkaloid. The chemical
name for colchicine or acetyltrimethylcolchicinic acid is
(S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxo-
benzo[a ]heptalen-7-yl)acetamide (C22H25NO6; molecular
weight 399.43; m.p. 151–1528C) (Fig. 4a).
In the 19th century it was realized that gout and urate
metabolism were linked and that gout is caused by depo-
sition of microcrystals of urate in the joints. Colchicine
therapy diminishes the metabolic activity of leucocytes,
resulting in reduced phagocytosis of urate microcrystals,
therefore interrupting the cycle of new crystal deposition.
Colchicine has an inhibitory effect on the growth of cer-
tain tumours in plants and animals. It is reported to mark-
edly increase the susceptibility of cancer cells to X-rays,
presumably due to its action on mitosis. It is an antimito-
tic agent that suppresses cell division by inhibiting karyo-
kinesis. It inhibits spindle formation by arresting the
polymerization of tubulin proteins and thereby checks
karyokinesis (Andreu et al., 1998). Colchicine has been
widely used in plant breeding to induce polyploidy.
Gloriosa can be a commercially viable source of colchi-
cine, provided that it can be propagated at a fast rate.
111
In horticultural practice, vegetative propagation of
Gloriosa is commonly used but the growth is very slow
(Krause, 1986). Multiplication rate is low as only two
plants are produced per corm per year. It takes four or
five vegetative cycles to complete a reproductive phase
(Samarajeewa et al., 1993). There is great scope for
plant biotechnology for mass propagation as well as col-
chicine production through plant cell and tissue culture.
Cell and tissue culture of Gloriosa superba for
production of colchicine
There are few reports on micropropagation of Gloriosa
sp. Since the active principle is mainly concentrated in
the tubers, multiplication of tubers in vitro is essential.
In vitro tubers have several advantages; they are hardier,
easier to handle, can be transported dry; there is no dor-
mancy period—thereby year-round cultivation is poss-
ible. These in vitro-generated plantlets could serve as a
source of cultures for studying the relationship between
secondary metabolite accumulation and tissue differen-
tiation in this plant species.
MS basal medium was most effective in inducing in
vitro tubers (six tubers in 24 weeks) from tuber explants
as compared to medium supplemented with growth reg-
ulators. These in vitro-generated tubers developed
healthy green shoots within 6 days of initiation and
rooted in basal media with variation in root initiation
and size depending on the strength of inorganics used
in the medium (Fig. 4b). Best results were obtained in
half-strength MS medium with reduced nitrates, which
gave rise to an average of eight roots per tuber after 15
days in culture. The in vitro-generated tubers were
found to accumulate 0.03% colchicine when analysed
after 6 months in culture (Ghosh and Jha, 2005).
There have been several attempts to initiate callus and
organ cultures of Gloriosa for plant regeneration. Levels
of colchicine extracted from Gloriosa callus, malformed
roots and entire plantlets show an increase that can be
directly related to the amount of differentiation in culture
(Finnie and Van Staden, 1994). Very recently, Sivakumar
and Krishnamurthy (2004) reported regeneration of
healthy plants derived from six categories of explants
from both in vivo- and in vitro-raised plants, namely
roots, corm buds (dormant and non-dormant), young
leaves, stems, pedicels and shoot tips from aerial shoots.
Colchicine production in cell cultures has been
reported earlier, however, the amount of colchicine
recorded in the tissues cultures of Colchicum and
Gloriosa were 10–25 times lower than those found in
plants growing in vivo (Hayashi et al., 1988; Finnie and
Van Staden, 1991). Colchicine is formed from phenyl-
alanine, tyrosine and methionine (Herbert and Knagg,
112 Sumita Jha et al.

Fig. 4. Gloriosa superba plants and cultures as sources of colchicine: (a) chemical structure of colchicine; (b) in vitro tuber
growing on basal MS medium; (c) tuber-derived callus culture on MS medium supplemented with 2,4-D and kinetin;
(d) excised root organ culture in liquid MS medium supplemented with NAA and BA; (e) rhizogenic culture derived from
callus on MS medium supplemented with NAA and BA; (f) flowering branch of Gloriosa superba L.

1986). In cell cultures of Colchicum, feeding of some pre-
cursors, e.g. phenylalanine and tyrosine, had no effect on
colchicine formation, however feeding with p-coumaric
acid, tyramine and demecolcine did increase alkaloid for-
mation in Colchicum cell cultures (Yoshida et al., 1988).
We studied the effect of precursor feeding on colchi-
cine production in root cultures of Gloriosa superba.
Excised root cultures of G. superba reached 7.5 g
dry wt/l and accumulated 240 ^ 40 mg colchicine/g cell
dry wt after 4 weeks of growth. While all precursors
(except trans-cinnamic acid) enhanced colchicine con-
tent of root cultures without adversely affecting root
growth, treatment with p-coumaric acid and tyramine
(each at 20 mg/l) increased colchicine content to
1.9 mg/g cell dry wt (Ghosh et al., 2002).
Callus cultures were also established from tuber
explants following supplementation of MS medium with
high 2,4-D and low kinetin under complete darkness
(Fig. 4c). Growth was higher in the callus line maintained
under dark conditions but colchicine could not be
detected in these calluses. Rhizogenic calluses induced
from this callus showed accumulation of colchicine
(0.005%) after 4 weeks of growth (Fig. 4e). Root organ
cultures initiated from root-tip explants on MS medium
supplemented with NAA and BA or kinetin under
complete darkness accumulated 0.037% colchicine after
5 weeks in culture (Fig. 4d). Thus root cultures accumu-
lated maximum amounts of colchicine comparable to the
colchicine content of in vitro tubers. Secondary product
formation is an expression of a particular state of cell
differentiation, which in turn can be influenced by par-
ticular signals. In some cases, initiation of morphological
differentiation represents such a triggering signal. Under
normal physiological conditions formation of the
enzymes of secondary pathways is integrated in the pro-
grammed expression of genes during cell specialization
as well as tissue and organ development. Colchicine con-
tent of different in vitro cultures revealed a close corre-
lation between root differentiation and colchicine
accumulation (Ghosh and Jha, 2005).
Conclusions
The productivity of the culture systems (transformed/
untransformed) needs to be improved significantly and
to be shown to be competitive with field plants for pro-
duction of target secondary metabolites on an industrial
scale. The lack of understanding of the molecular mech-
anism of regulation of secondary metabolism is the main
bottleneck in attempts for further study on improving the
yield of target compounds.
Production of various secondary metabolites
References
Abe F, Iwase Y, Yamauchi T, Honda K and Hayashi N (1995)
Phenanthroindolizidine alkaloids from Tylophora tanakae.
Phytochemistry 39: 695 –699.
Abe F, Yamauchi T, Honda K, Omura H and Hayashi N (2001)
Sequestration of phenanthro-indolizidine alkaloids by an
Asclepiadaceae-feeding danaid butterfly, Ideopsis similis.
Phytochemistry 56: 697 –701.
Ammon HPT and Müller AB (1985) Forskolin: from Ayruvedic
remedy to a modern agent. Planta Medica 51: 473 – 477.
Andreu JM, Perez-Ramirez B, Gorbunoff MJ, Ayala D and Tima-
sheff SN (1998) The role of the colchicine ring A and its
methoxy groups in the binding to tubulin and microtubule
inhibition. Biochemistry 37: 8356 –8368.
Anonymous (1956) The Wealth of India (Raw Materials) IV.
New Delhi: CSIR, p. 139.
Anonymous (1976) The Wealth of India (Raw Materials) X. New
Delhi: CSIR, pp. 398 –400, 582 –585.
Anonymous (2004) Withania somnifera—monograph. Alterna-
tive Medicine Review 9: 211 –214.
Bellet P and Gaignault JC (1985) Le Gloriosa superba L. et la
production de substances colchiciniques. Annales Phar-
maceutiques Françaises 43: 345 – 347.
Benzamine BD and Mulchandani NB (1973) Studies in biosyn-
thesis of secondary constituents in tissue cultures of
Tylophora indica. Planta Medica 23: 394 – 397.
Benzamine BD and Mulchandani NB (1976) Effect of gamma
irradiation on biosynthetic potential of callus cultures of
Tylophora indica. Planta Medica 29: 37– 40.
Benzamine BD, Heble MR and Chadha MS (1979) Alkaloid syn-
thesis in tissue culture and regenerated plants of Tylophora
indica Merr. (Asclepiadaceae). Zeitschrift für Pflanzenphy-
siologie 92: 77– 84.
Berlin J, Martin B, Nowak J, Witte L, Wray L and Strack D (1989)
Effects of permeabilization on the biotransformation of
phenylalanine by immobilized tobacco cell cultures. Zeits-
chrift für Naturforsch 44: 249 – 254.
Bhattacharyya R and Bhattacharya S (2001) In vitro multipli-
cation of Coleus forskohlii Briq.: an approach towards
shortening the protocol. In Vitro Cellular & Developmental
Biology—Plant 37: 572 – 575.
Bhutani KK, Sharma, GL, Sarin AN, Kaur R, Kumar V and Atal CK
(1985) In vitro amoebicidal and bactericidal activities in
medicinal plants. Indian of Pharmaceutical Sciences 47:
65 –67.
Bourgaud F, Gravot A, Milesi S and Gontier E (2001) Production
of plant secondary metabolites: a historical perspective.
Plant Science 161: 839 – 851.
Chaudhuri KN, Ghosh B and Jha S (2004) The root: a potential
new source of competent cells for high-frequency regener-
ation in Tylophora indica. Plant Cell Reports 22: 731 – 740.
Chaudhuri KN, Ghosh B, Tepfer D and Jha S (2005) Genetic
transformation of Tylophora indica with Agrobacterium
rhizogenes A4: growth and tylophorine productivity in
different transformed root clones. Plant Cell Reports 24:
25 –35.
Chopra RN, Chopra IC, Handa KL and Kapur LD (1958) Indigen-
ous Drugs of India. Calcutta: UN Dhur & Sons.
Clewer HWB, Green SJ and Tutin F (1915) The constituents of
Gloriosa superba. Journal of the Chemical Society 107:
835– 846.
Donaldson GR, Atkinson MR and Murray AW (1968) Inhibition
of protein synthesis in Ehrlich ascites-tumor cells by the
113
phenanthrene alkaloids tylophorine, tylocrebrine and cryp-
topleurine. Biochemical and Biophysical Research
Communications 31: 104–109.
Dymock W, Warden CJH and Hooper D (1891) Pharmacogra-
phia Indica: A History of Principal Drugs of Vegetable
Origin Met with British India, Vol 2. Dehra Dun: Bishen
Singh Mahendra Pal Singh.
Engprasert S, Taura F, Makoto M and Shoyama Y (2004) Molecu-
lar cloning and functional expression of geranylgeranyl
pyrophosphate synthase from Coleus forskohlii Briq. BMC
Plant Biology 4: 18.
Faisal M and Anis M (2003) Rapid mass propagation of
Tylophora indica Merrill via leaf callus culture. Plant Cell,
Tissue and Organ Culture 75: 125– 129.
Finnie JF and Van Staden J (1991) Isolation of colchicine from
Sandersonia aurantiaca and Gloriosa superba. Variation
in alkaloid levels of plants grown in vivo. Journal of
Plant Physiology 138: 691– 695.
Finnie JF and Van Staden J (1994) Gloriosa superba L. (Flame
lily): micropropagation and in vitro production of colchi-
cine. In: Bajaj YPS (ed.) Biotechnology in Agriculture and
Forestry, Vol. 26. Medicinal and Aromatic Plants VI.
Berlin: Springer-Verlag, pp. 146– 166.
Furmanowa M, Gajdzis-Kuls D, Ruszkowska J, Czarnocki Z,
Obidoska G, Sadowska A, Rani R and Upadhyay SN
(2001) In vitro propagation of Withania somnifera and iso-
lation of withanolides with immunosuppressive activity.
Planta Medica 67: 146– 149.
Gellert E (1982) The indolizidine alkaloids. Journal of Natural
Products 45: 50 –73.
Gellert E and Rudzats R (1964) The antileukemia activity of tylo-
crebrine. Journal of Medicinal Chemistry 7: 361– 362.
Gellert E, Govindachhari TR, Lakshmikantham MV, Ragade IS,
Rudzats R and Viswanathan N (1962) The alkaloids of
Tylophora crebriflora: structure and synthesis of tylocrebr-
ine, a new phenanthroindolizidine alkaloid. Journal of the
Chemical Society 1008 – 1014.
Ghosh B, Mukherjee S, Jha TB and Jha S (2002) Enhanced col-
chicine production in root cultures of Gloriosa superba by
direct and indirect precursors of the biosynthetic pathway.
Biotechnology Letters 24: 231– 234.
Ghosh S and Jha S (2005) Colchicine accumulation in in
vitro cultures of Gloriosa superba. In: D’ Souza L, Anuradha
M, Nivas S, Hegde S and Rajendra K (Eds). Biotechnology
for a Better Future. Mangalore: SAC Publications,
pp. 92– 98.
Gopalakrishnan C, Shankaranarayan D, Kameswaran L and
Natarajan S (1979) Pharmacological investigations of tylo-
phorine, the major alkaloid of Tylophora indica. Indian
Journal of Medical Research 69: 513– 520.
Govindachari TR, Pai BR and Nagarajan K (1954) Chemical
examination of Tylophora asthmatica Part I. Journal of
the Chemical Society 5192: 2801.
Govindachari TR, Lakshmikantham MV and Rajadurai S (1961)
Chemical examination of Tylophora asthmatica—V. Struc-
ture of tylophorinine. Tetrahedron 14: 284–295.
Hadacek F (2002) Secondary metabolites as plant traits: current
assessment and future perspectives. CRC Critical Reviews
in Plant Science 21: 273–322.
Hansen G and Wright MS (1999) Recent advances in the trans-
formation of plants. Trends in Plant Science 4: 226– 231.
Hayashi T, Yoshida K and Sano K (1988) Formation of alkaloids
in suspension cultured Colchicum autumnale. Phytochem-
istry 27: 1371 –1374.
114
Herbert RB and Knagg E (1986) The biosynthesis of phen-
ethylisoquinoline alkaloid, colchicine from cinnamalde-
hyde and dihydrocinnamaldehyde. Tetrahedron Letters
27: 1099 –1102.
Hooykaas PJJ and Beijersbergen AGM (1994) The virulence
system of Agrobacterium tumefaciens. Annual Review of
Phytopathology 32: 157 –179.
Jayanthi M and Mandal PK (2001) Plant regeneration through
somatic embryogenesis and RAPD analysis of regenerated
plantlets in Tylophora indica (Burm. f.) Merrill. In Vitro
Cellular & Developmental Biology—Plant 37: 576 –580.
Jha S (1997) Genetic transformation for production of secondary
metabolites. In: Ramawat KG and Merillon JM (eds) Bio-
technology: Secondary Metabolites. New Delhi: Oxford &
IBH Publishing Co., pp. 305 – 329.
Karnick CR (1975) Phytochemical investigations of some
Tylophora species found in India. Planta Medica 27:
333– 336.
Krause J (1986) Production of Gloriosa tubers from seeds. Acta
Hortica 177: 353–360.
Krombholz R, Mersinger R, Kreis W and Reinhard E (1992)
Production of forskolin by axenic Coleus forskohlii roots
cultivated in shake flasks and 20-l glass jar bioreactors.
Planta Medica 58: 328 – 333.
Kulkarni AA, Thengane SR and Krishnamurthy KV (1996) Direct
in vitro regeneration of leaf explants of Withania somni-
fera (L) Dunal. Plant Science 119: 163 –168.
Laird SA (1999) The botanical medicine industry. In: Kate K and
Laird SA (eds) The Commercial Use of Biodiversity: Access
to Genetic Resources and Benefit-sharing. London: Earth-
scan, pp. 78 –116.
Lambert J, Srivastava J and Vietmeyer N (1997) Medicinal Plants:
Rescuing a Global Heritage. World Bank Technical Paper
355. Washington, DC: World Bank.
Lavie D, Glotter E and Shvo Y (1965) Constituents of Withania
somnifera Dun: the structure of withaferin A. Journal of the
Chemical Society 7517.
Manickam VS, Elango Mathavan R and Antonisamy R (2000)
Regeneration of Indian ginseng plantlets from stem
callus. Plant Cell, Tissue and Organ Culture 62: 181 – 185.
Manjula S, Job A and Nair GM (2000) Somatic embryogenesis
from leaf derived callus of Tylophora indica (Burm. f.)
Merrill. Indian Journal of Experimental Biology 38:
1069 – 1072.
Marone G, Columbo M, Triggiani M, Vigorita S and Formisano S
(1986) Forskolin inhibits the release of histamine from
human basophils and mast cells. Agents Actions 18:
96 –99.
Mersinger R, Dornauer H and Reinhard E (1988) Formation of
forskolin by suspension cultures of Coleus forskohlii.
Planta Medica 54: 200 – 204.
Mhatre M, Bapat VA and Rao PS (1984) Plant regeneration in
protoplast cultures of Tylophora indica. Journal of Plant
Physiology 115: 231 – 235.
Moldenhauer D, Fuerst B, Diettrich B and Luckner M (1990) Car-
denolides in Digitalis lanata cells transformed with Ti-pla-
mids. Planta Medica 56: 435 –438.
Mukherjee S, Ghosh B and Jha S (1996) Forskolin synthesis in
in vitro culture of Coleus forskohlii Briq. transformed with
Agrobacterium tumefaciens. Plant Cell Reports 15: 691 – 694.
Mukherjee S, Ghosh B and Jha S (2000a) Establishment of for-
skolin yielding transformed cell suspension cultures of
Coleus forskohlii as controlled by different factors. Journal
of Biotechnology 76: 73– 81.
Sumita Jha et al.
Mukherjee S, Ghosh B and Jha S (2000b) Enhanced forskolin
production in genetically transformed cultures of Coleus
forskohlii by casein hydrolysate and studies on growth
and organisation. Biotechnology Letters 22: 133– 136.
Mukherjee S, Ghosh B and Jha S (2003) Higher production of
forskolin in genetically transformed cultures of Coleus for-
skohlii Briq. induced by growth regulators. Journal of Plant
Biochemistry and Biotechnology 12: 81 –85.
Mulchandani NB and Venkatachalam SR (1976) Alkaloids of
Pergularia pallida. Phytochemistry 15: 1561– 1563.
Murashige T and Skoog F (1962) A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physio-
logia Plantarum 15: 473–497.
Nacci V, Stefancich G, Giuliano, R, Filachoni G, Artico M, Dolfini
E and Morasca L (1973) [Research on substances with anti-
neoplastic activity. LII. Tylophorine analogs. I. Synthesis
and cytostatic and cytotoxic activity of 4-(3,4-dimethoxy-
phenyl) piperidine derivatives.] Farmaco-Edizione
Scientifica 28: 399– 410 [in Italian].
Nadkarni AK (1954) Indian Materia Medica, Vol. 1. Bombay:
Popular Prakashan.
Okuda H, Morimoto C and Tsujita T (1992) Relationship
between cyclic AMP production and lipolysis induced by
forskolin in rat fat cells. Journal of Lipid Research 33:
225– 231.
Pailer M and Streicher W (1965) Alkaloids from Vincetoxicum
officinale. Monatschefth für Chemie 96: 1094 –1102.
Payne J, Hamill JD, Robins RJ and Rhodes MJC (1987) Pro-
duction of hyoscyamine by ‘hairy root’ cultures of Datura
stramonium. Planta Medica 53: 474– 478.
Petersen M (1994) Coleus spp.: in vitro culture and the pro-
duction of forskolin and rosmarinic acid. In: Bajaj YPS
(ed.) Biotechnology in Agriculture and Forestry, Vol. 26.
Medicinal and Aromatic Plants VI. Berlin: Springer-
Verlag, pp. 69– 92.
Ramachandra Rao S and Ravishankar GA (2002) Plant cell cul-
tures: chemical factories of secondary metabolites. Biotech-
nology Advances 20: 101– 153.
Rani G and Grover IS (1999) In vitro callus induction and regen-
eration studies in Withania somnifera. Plant Cell, Tissue
and Organ Culture 57: 23– 27.
Rani G, Virk GS and Nagpal A (2003) Callus induction and plant-
let regeneration in Withania somnifera (L.) Dunal. In Vitro
Cellular & Developmental Biology—Plant 39: 468–474.
Rao KN and Venkatachalam SR (2000) Inhibition of dihydrofo-
late reductase and cell growth activity by the phenanthroin-
dolizidine alkaloids pergularinine and tylophorinidine: the
in vitro cytotoxicity of these plant alkaloids and their
potential as antimicrobial and anticancer agents. Toxicology
in Vitro 14: 53 –59.
Rao KN, Bhattacharyya RK and Venkatachalam SR (1997)
Inhibition of thymidylate synthase and cell growth by the
phenanthroindolizidine alkaloids pergularinine and tylo-
phorinidine. Chemico-Biological Interactions 106:
201– 211.
Rao KN, Bhattacharyya RK and Venkatachalam SR (1998) Thymidy-
late synthase activity in leukocytes from patients with chronic
myelocytic leukemia and acute lymphocytic leukemia and its
inhibition by phenanthroindolizidine alkaloids pergularinine
and tylophorinidine. Cancer Letters 128: 183–188.
Rao KN, Bhattacharyya RK and Venkatachalam SR (1999) Inhi-
bition of thymidylate synthase by pergularinine, tylophori-
nidine and deoxytubulosine. Indian Journal of
Biochemistry and Biophysics 36: 442–448.
Production of various secondary metabolites
Rao KV, Wilson RA and Cummings B (1971) Alkaloids of
Tylophora indica and T. dalzelli. Journal of Pharmaceuti-
cal Sciences 60: 1725 –1726.
Rao PS and Narayanaswamy S (1972) Morphogenetic investi-
gations in callus cultures of Tylophora indica. Physiologia
Plantarum 27: 271 – 276.
Rao PS, Narayanaswamy S and Benjamin BD (1970) Differen-
tiation ex ovulo of embryos and plantlets in stem tissue
cultures of Tylophora indica. Physiologia Plantarum 23:
140– 144.
Ray AB and Gupta M (1994) Withasteroids, a growing group of
naturally occurring steroidal lactones. In: Herz W, Kirby
GW, Moore RE, Steglich W and Tamm Ch (eds) Progress
in the Chemistry of Organic Natural Products, Vol. 63.
Berlin: Springer-Verlag, pp. 1 –106.
Ray S (2000) Tissue culture and Agrobacterium mediated trans-
formation in Withania somnifera for production of second-
ary metabolites. PhD Thesis, University of Calcutta.
Ray S and Jha S (1999) Withanolide synthesis in cultures of
Withania somnifera transformed with Agrobacterium
tumefaciens. Plant Science 146: 1 –7.
Ray S and Jha S (2001) Production of withaferin A in shoot cul-
tures of Withania somnifera. Planta Medica 67: 432 – 436.
Ray S and Jha S (2002) Regeneration of Withania somnifera
plants. Journal of Tropical Medicinal Plants 3: 89– 95.
Ray S, Ghosh B, Sen S and Jha S (1996) Withanolide production
by root cultures of Withania somnifera transformed with
Agrobacterium rhizogenes. Planta Medica 62: 571 – 573.
Roja G, Heble MR and Sipahamalani AT (1991) Tissue cultures of
Withania somnifera: morphogenesis and withanolide syn-
thesis. Phytotherapy Research 5: 185 – 187.
Sajc L, Grubisci D and Vunjak-Novakovic G (2000) Bioreactors
for plant engineering: an outlook for further research.
Biochemical Engineering Journal 4: 89– 99.
Saksena A, Green MJ, Shue HJ and Wong JK (1985) Identity of coleo-
nol with forskolin: structure revision of a base-catalysed
rearrangement product. Tetrahedron Letters 26: 551–554.
Samarajeewa PK, Dassanayake MD and Jayaawardena SDG
(1993) Clonal propagation of Gloriosa superba L. Indian
Journal of Experimental Biology 31: 719 –720.
Sasaki K, Udagawa A, Ishimaru H, Hayashi T, Alfermann AW,
Nakanishi F and Shimomura K (1998) High forskolin pro-
duction in hairy roots of Coleus forskohlii. Plant Cell
Reports 17: 457–459.
Sen J and Sharma AK (1991a) In vitro propagation of Coleus forskoh-
lii Briq. for forskolin synthesis. Plant Cell Reports 9: 696–698.
Sen J and Sharma AK (1991b) Micropropagation of Withania
somnifera from germinating seeds and shoot tips. Plant
Cell, Tissue and Organ Culture 26: 71– 73.
Sen J, Sharma AK, Sahu NP and Mahato SB (1992) Production of
forskolin in in vitro cultures of Coleus forskohlii Briq.
Planta Medica 58: 324 – 327.
Sen J, Sharma AK, Sahu NP and Mahato SB (1993) Forskolin pro-
duction in untransformed root culture of Coleus forskohlii.
Phytochemistry 34: 1309– 1312.
Sevón N and Oksman-Caldentey K-M (2002) Agrobacterium
rhizogenes-mediated transformation: root cultures as a
source of alkaloids. Planta Medica 68: 859 – 868.
115
Shanks JV and Morgan J (1999) Plant hairy root culture. Current
Opinions in Biotechnology 10: 151–155.
Sharma K and Dandiya PC (1992) Withania somnifera Dunal:
present status. Indian Drugs 29: 247.
Sharma N and Chandel KPS (1992) Effect of ascorbic acid on
axillary shoot induction in Tylophora indica (Burm. f.)
Merrill. Plant Cell, Tissue and Organ Culture 29:
109– 113.
Sharma N, Chandel KPS and Srivastav VK (1991) In vitro propa-
gation of Coleus forskohlii Briq., a threatened medicinal
plant. Plant Cell Reports 10: 324 –327.
Sivakumar G and Krishnamurthy KV (2004) In vitro organoge-
netic responses of Gloriosa superba. Russian Journal of
Plant Physiology 51: 713–721.
Srivastav SK, Chaubey M, Khatoon S, Rawat AKS and Mehrotra S
(2002) Pharmacognostic evaluation of Coleus forskohlii.
Pharmaceutical Biology 40: 129– 134.
Srivastava R (2000) Studying the information needs of medic-
inal plant stakeholders in Europe. TRAFFIC Dispatches
15: 5.
Stærk D, Lykkeberg AK, Christensen J, Budnik BA, Abe F and
Jaroszewski JW (2002) In vitro cytotoxic activity of phenan-
throindolizidine from Cynanchum vincetoxicum and
Tylophora tanakae against drug-sensitive and multidrug-
resistant cancer cells. Journal of Natural Products 65:
1299 – 1302.
Subroto MA, Hamill JD and Doran PM (1995) Growth kinetics
and stoichiometry of Mentha citrata shooty teratomas
transformed by Agrobacterium tumefaciens. Biotechnology
Letters 17: 427– 432.
Vishwakarma RA, Tyagi BR, Ahmed B and Husain A (1988) Vari-
ation in forskolin content in the roots of Coleus forskohlii.
Planta Medica 54: 471– 472.
Vitali G, Conte L and Nicoletti M (1996) Withanolide compo-
sition and in vitro culture of Italian Withania somnifera.
Planta Medica 62: 287– 288.
Wiegrebe W, Faber L, Brockmann H, Budzikiewicz H and
Krüger U (1969) Alkaloids from Cynanchum vincetoxicum
(L) Pers. Justus Liebigs Annals of Chemistry 721: 154–162.
Wysham DG, Brotherton AF and Heistad DD (1986) Effects of
forskolin on cerebral blood flow: implications for the role
of adenylate cyclase. Stroke 17: 1299– 1303.
Yoshida K, Hayashi T and Sano K (1988) Colchicine precursors
and the formation of alkaloids in suspension cultured
Colchicum autumnale. Phytochemistry 27: 1375 – 1378.
Yu PLC, El-Olemy MM and Stohs SJ (1974) A phytochemical
invstigation of Withania somnifera tissue cultures. Lloydia
37: 593– 597.
Zhong JJ (2001) Biochemical engineering of the production of
plant-specific secondary metabolites by cell suspension
cultures. In: Scheper Th (ed.) Advances in Biochemical
Engineering/Biotechnology, Vol. 72. Berlin: Springer-
Verlag, pp. 1– 26.
Zupan J and Zambryski P (1995) Transfer of T-DNA from Agro-
bacterium to the plant cell. Plant Physiology 107:
1041 – 1047.