Plant Tissue Culture Technology: Sustainable Option for Mining High Value Pharmaceutical Compounds

Abstract

Production of secondary metabolites from plant tissue culture has emerged as a promising
and feasible option attracting the attention of scientists worldwide. Plant cell, tissue and
organ cultures offer an attractive alternative for homogeneous, controlled production of
secondary metabolites, throughout the year, especially when we take commercial demand
into account. They not only facilitate the de novo synthesis of novel compounds, but also
are able to produce compounds sometimes even in higher amounts than the intact plants.
Many biotechnological strategies have been experimented for enhanced production of
secondary metabolites from medicinal plants. Some of these include screening of high
yielding cell lines, media modification, precursor feeding, elicitation, large scale
cultivation in bioreactor system, hairy root culture, plant cell immobilization,
biotransformation and many others. Some of the recent developments such as metabolic
engineering of whole plants and plant cell cultures have been established as effective tools
to increase metabolites yield. The use of genetic engineering tools and elucidation of
pathways for secondary metabolism has provided the basis for the production of
commercially acceptable levels of product. In view of commercial importance of the
secondary metabolites as high value pharmaceutical compounds in recent years resulted in
a immersive interest, in secondary metabolism, and particularly in the possibility to alter
the production of bioactive plant metabolites by utilizing biotechnological tools. The plant
cell culture technology provides sustainable option for production of plant pharmaceuticals
and could be used for the large-scale production of metabolites.

Full text

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Original Research Article https://doi.org/10.20546/ijcmas.2019.802.116
Plant Tissue Culture Technology: Sustainable Option for Mining High
Value Pharmaceutical Compounds
M.K. Tripathi, Nishi Mishra, Sushma Tiwari*, Chitralekha Shyam,
Sonali Singh and Ashok Ahuja
Department of Plant Molecular Biology & Biotechnology, College of Agriculture,
RVSKVV, Gwalior 474002, MP, India
*Corresponding author
A B S T R A C T
Introduction
Plant cell culture systems are potential
renewable source of valuable medicinal
compounds, flavors, fragrances, and
colorants. Due to commercial importance of
the secondary metabolites it has resulted in an
interest in secondary metabolism. Production
of bioactive plant metabolites by means of
cell culture technology has gained interest and
number of plants has been investigated in
vitro in recent years to produce compounds of
medicinal value. This technology provide
continuous, reliable source of plant
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 8 Number 02 (2019)
Journal homepage: http://www.ijcmas.com
Production of secondary metabolites from plant tissue culture has emerged as a promising
and feasible option attracting the attention of scientists worldwide. Plant cell, tissue and
organ cultures offer an attractive alternative for homogeneous, controlled production of
secondary metabolites, throughout the year, especially when we take commercial demand
into account. They not only facilitate the de novo synthesis of novel compounds, but also
are able to produce compounds sometimes even in higher amounts than the intact plants.
Many biotechnological strategies have been experimented for enhanced production of
secondary metabolites from medicinal plants. Some of these include screening of high
yielding cell lines, media modification, precursor feeding, elicitation, large scale
cultivation in bioreactor system, hairy root culture, plant cell immobilization,
biotransformation and many others. Some of the recent developments such as metabolic
engineering of whole plants and plant cell cultures have been established as effective tools
to increase metabolites yield. The use of genetic engineering tools and elucidation of
pathways for secondary metabolism has provided the basis for the production of
commercially acceptable levels of product. In view of commercial importance of the
secondary metabolites as high value pharmaceutical compounds in recent years resulted in
a immersive interest, in secondary metabolism, and particularly in the possibility to alter
the production of bioactive plant metabolites by utilizing biotechnological tools. The plant
cell culture technology provides sustainable option for production of plant pharmaceuticals
and could be used for the large-scale production of metabolites.
Ke ywords
Tissue culture,
Pharmaceutical
compounds,
Secondary
metabolism
Accepted:
10 January 2019
Available Online:
10 February 2019
Article Info

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pharmaceuticals and could be used for the
large-scale cultivation of plant cells in
bioreactors and through downstreaming
processes these metabolites can be extracted
(Balandrin and Klocke, 1988). In addition to
its importance in the discovery of new
medicines, plant cell culture technology plays
an even more significant role in solving world
hunger by developing agricultural crops that
provide both higher yield and more resistance
to pathogens and adverse environmental and
climatic conditions. This paper reviews some
of the developments for the production of
some of the bioactive secondary metabolites
from medicinal plants.
Medicinal plants are the important source of
life saving drugs for the majority of the
world’s population. Biologically active
secondary metabolite compounds extracted
from plants are used as food additives,
pigments, dyes, insecticides, cosmetics and
perfumes and fine chemicals (Ahuja, 1988).
These compounds commonly referred as
secondary metabolites. Number of plant
species that are used as medicinal herbs have
been scientifically evaluated for their possible
medical applications. Due to wild crafting
owing to developmental activities natural
stands of many medicinal plants are
disappearing fast and together with
environmental and geopolitical instabilities; it
is increasingly difficult to meet the demand.
As such to face such challenges industries, as
well as scientists have to look for the
possibilities of alternative resources for the
production of plant pharmaceuticals utilizing
plant cell cultures. Advances in biotechnology
in recent years particularly methods for
culturing plant cell cultures, has provided
good strategies for the commercial processing
of plant cell cultures even rare plants and the
chemicals they provide. As such there has
been considerable interest in plant cell
cultures as a potential alternative to traditional
agriculture for the industrial production of
secondary metabolites (Rao and Ravishankar,
2000). Plant cell culture technologies were
introduced at the end of 1960s as a possible
tool for both studying and producing plant
secondary metabolites.
Different strategies using cell cultures
systems have been extensively studied with
the objective of improving the production of
bioactive secondary metabolites. Cell culture
systems could be used for the large scale
culturing of plant cells from which secondary
metabolites can be extracted. The advantage
of this method is that it can ultimately provide
a continuous, reliable source of natural
products. The major advantages of cell
cultures includes (i) synthesis of bioactive
secondary metabolites in controlled
environment, independently from climatic and
soil conditions; (ii) negative biological
influences that affect secondary metabolites
production in the nature are eliminated
(microorganisms and insects); (iii) it is
possible to select cultivars with higher
production of secondary metabolites; (iv) with
automatization of cell growth control and
metabolic processes regulation cost price can
decrease and production increase. The
objectives of many industries are to develop
plant cell culture techniques to the stage
where they yield secondary products more
cheaply than extracting either the whole plant
grown under natural conditions or
synthesizing the product. Although the
production of pharmaceuticals using plant cell
cultures have been highlighted, other uses
have also been suggested as new route for
synthesis, for products from plants difficult to
grow, or in short supply, as a source of novel
chemicals and as biotransformation systems
(Ramawat et al., 1999; Oksman-Caldentey
and Inze, 2004). Recent research results
indicate that plant cell suspension cells can be
used for recombinant protein production
under controlled conditions (Verpoorte and
Memelink, 2002). Some of the successful

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cases where callus and cell suspension
cultures have been described for production
of bioactive secondary metabolites obtained
by authors are described.
Secondary metabolites production by plant
cell cultures
For plant cell culture techniques to become
economically viable, it is important to
develop methods that would allow for
consistent generation of high yields of
products from cultured cells. Careful selection
of productive cells and cultural conditions
resulted in accumulation of several products
in higher levels in cultured cells. In order to
obtain yields in higher concentrations for
commercial exploitation, efforts have focused
on the stimulation of biosynthetic activities of
cultured cells using various methods (Rao and
Ravishankar, 2000).Culture productivity is
critical to the practical application of plant
cell culture technology to production of plant-
specific bioactive metabolites. Until now,
various strategies have been developed to
improve the production of secondary
metabolites using plant cell cultures. The
tissue culture cells typically accumulate large
amounts of secondary compounds only under
specific conditions. That means maximization
of the production and accumulation of
secondary metabolites by plant tissue cultured
cells requires (i) manipulating the parameters
of the environment and medium, (ii) selecting
high yielding cell clones, (iii) precursor
feeding and (iv) elicitation.
Plant cell cultures are mostly a heterogeneous
system in which individual plant cells are
different physiologically. High yielding lines
selection and screening of plant cell cultures
have been described by many researchers
(Rao and Ravishankar, 2000). Exogenous
supply of biosynthetic precursors to culture
medium is another important strategy to
increase the yield of desired products. This
approach is useful when the precursors are
inexpensive. The compound, which is an
intermediate, in or at the beginning of a
secondary metabolite biosynthetic route,
stands a good chance of increasing the yield
of the final product. Attempts to induce or
increase the production of plant secondary
metabolites by supplying precursor or
intermediate compounds, have been effective
in many cases (Anitha and Ranjitha, 2006).
Elicitors are signals triggering the formation
of secondary metabolites. Use of elicitors of
plant defense mechanisms, i.e. elicitation, has
been one of the most effective strategies for
improving the productivity of bioactive
secondary metabolites. Biotic and abiotic
elicitors based on their origin are used to
stimulate number of secondary metabolite
formation in plant cell cultures, thereby
reducing the process time to get higher yield
of secondary metabolites (Namdeo et al.,
2002; Sharma et al., 2015). Production of
some of valuable secondary metabolites using
various elicitors was reported (Namdeo et al.,
2002; Sharma et al., 2015; Harisaranraj et al.,
2009).
Steroidal Lactones Metabolism in Withania
somnifera in vitro
Withania somnifera is an important Indian
medicinal plant has received considerable
attention due to the potent biological
properties attributed to the presence has
emerged as one of the important Indian
medicinal plants due to its potent biological
properties. An efficient protocol was
established for its regeneration and mass
propagation through plant growth regulator
mediated organogenesis producing up to 1368
plantlets per explant cultured in a time frame
of 13 weeks. Withanolide contents
(Withanone, Withaferin A, Withanolide A
and Withanolide B) were analyzed in plant
parts of W. somnifera and tissue cultured lines

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grown on MS/B5 medium containing various
plant growth regulators. Withanolides were
identified by HPLC-UV (DAD) – Positive ion
electrospray ionization spectroscopy. Callus
cultures grown on B5 medium containing 2.0
mgl-1NAA yielded 17-30.8% Withanolides
producing only Withanolide A and
Withanone. The calli turned organogeneic
when placed on MS medium amended
with2.0 mgl-1 BAP in combination with1.0
mgl-1 IBA also showed the presence of
Withanolide B. MS medium supplemented
with 1.0 mgl-1 BAP supported the
multiplication of shoots and yielded
significantly higher levels of all Withanolides.
Chemical constituents of the plant comprise
of steroidal lactones (withanolides).
Modulation of Withanolides metabolism was
closely observed using different PGRs
mediated organogenesis (Sharada et al., 2007;
2008). Glycowithanolides have also been
reported from tissue cultures of Withania
somnifera (Ahuja et al., 2009).
Bacoside metabolism in Bacopamonnieri
(L.)Wettst in vitro
Bacosides have received considerable
attention as potent bioactive molecules due to
their potent biological activities. Various
studies carried out so far, most of them
pertains to in vitro regeneration of B.
monnieri plantlets, however, and none of
these reports have described potential of these
cultures or regenerated plants for bacoside
formation. As such several reports addressed
Bacoside metabolism in vitro in B. monnieri.
The clonal propagation of B. monnieri
through shoot tips and auxillary buds
described here provided a strategy to clonally
propagate plants and have more homogenous
bacoside content and maintain genetic
integrity of elite clone. Multiple shoot
forming capability retained on long term
basis. Bacoside analysis of clonally
propagated plants was carried out by means of
HPLC and LC-MS showed Bacoside A3 and
A2 as major bacosides. Their structure and
preferred confirmation were determined on
the basis of spectroscopic data. The total
bacosides content was comparable and
essentially the same as detected for mother
plant. The total bacosides ranged between
2.30 to 2.70 % on dry weight basis. The
foliage collected from field grown clonally
propagated plants and naturally grown plants
at 2 stages of development; vegetative and
reproductive stages, were harvested and dried
at 50±2C overnight. The dried samples (10 g
each) of powdered plant material were soxhlet
extracted with methanol (150 ml) for 4 h at
room temperature. The extract was
concentrated to 60 ml under vacuo in water
(90 ml) and successively extracted with n-
hexane (100 ml x 3) and n-butanol (50 ml x
4). Butanol extracts were dried under vacuo
to obtain total bacosides. Thin layer
chromatography (TLC) and HPLC were used
for identification and quantification of
bacosides. The presence of bacoside (A3 +
A2) was additionally confirmed by LC-MS.
HPLC – quantitative analysis of bacosides
was performed by HPLC. Calibration curves
for bacoside A2 and A3 were prepared on the
basis of standard mixture. The concentrations
of bacoside A2 and A3 were in the ratio of
41:9 as determined by HPLC at 210 nm.
Efficient calibration coefficients were
obtained for these two standards. The values
for the calibration coefficients were 0.99985
and 0.99782, respectively. Bacoside A3 and
A2 eluted at retention times of 8.452 and
9.470 minutes which exhibited molecular ion
peaks respectively at m/z 951 [M+Na]+ and
921[M+Na]+in the positive mode (Ahuja et
al., 2005; Sharma et al., 2015).
In vitro plumbazin production from
cultured tissue of Plumbago zeylanica
Plumbagin is an important bioactive molecule
known for its broad range of pharmacological

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activities, such as anticancer, antimicrobial,
antifertility and insecticidal. Natural
occurrence of plumbagin occurs in several
plant species of the family Plumbaginceae
and Droseraceae. Plumbaginceae is found in
Africa, many parts of Asia and Europe while
Droseraceae (sundew) family is found in
many temperate and tropical regions of the
world. Roots of Plumbago species are the
main source of plumbagin production.
Plumbago zeylanica L., belongs to family
Plumbaginaceae, is a rambling subscandent
perennial herb or under shrub. The roots of
Plumbago zeylanica L are used extensively in
China and other Asian countries for the
treatment of cancer, rheumatoid arthritis,
dysmenorrheal and contusion of extremities.
The root stimulates the secretion of sweat,
urine and bile and has a stimulant action on
the nervous system. Extract of the root is
given internally or applied to the sodium uteri
causes abortion.
Production of plumbagin by plant cell
cultures is receiving more attention because
native plants such as Plumbago sp. and
Drosophyllum sp. produce only small
amounts of this compound after 2-6 years of
growth (Kitanov and Pashankov, 1994).
Production of plumbagin from P. rosea cell
cultures have been reported (Komaraiah et al.,
2001). But these cultures produced plumbagin
in very small amount and not found suitable
for commercial exploitation. Plant cell
cultures could be a potential source of a wide
variety of valuable pharmaceuticals, however,
only a few commercial processes based on
plant cell cultures exist at the moment. The
main drawback of cultured plant cells is lower
yields, stability of the cell lines, inconsistency
in the production and the storage of the
metabolites within the cells or vacuoles.
Recovery of products from cultures needs
harvesting and extraction of the cell
suspension. Cell suspension culture may be
used for whole or partial synthesis of
secondary plant products. Although a few
studies have been conducted in some
laboratories of worldwide to produce
secondary metabolites in Plumbago zeylanica
but reports are not encouraging. Experiments
were conducted to quantify secondary
metabolite production in calli obtained from
nodal segment and leave disc cultures and cell
clumps/embryoid acquired from cell
suspension cultures of Plumbago zeylanica.
Higher plumbazin content was detected in
one-month-old friable callus
(0.428mg/100gm), cell clumps/embryoids
(0.357 mg.l-1) as well as in two-months-old
rhizogenic calli (1.257 mg per gm) on MS
culture medium amended with 3.0 mgl-1 2,4-D
in combination with 0.5 mgl-1 IBA. Linearly
increased plumbagin concentration in both
callus and cell suspension culture filtrate was
recorded with increased concentration of 2, 4-
D (Patidar et al., 2015).
Glychyrhizin and related terpenoids
Simultaneous qualitative and quantitative
assessment of eight flavonoids and two
terpenoids was performed in fourteen in vitro
raised morphogenic cultures of
Glycyrrhizaglabra. Our study revealed that
the spectrum and production of ten
compounds, under investigation, was higher
in organized tissue than the undifferentiated
mass, however, aerial portions of the in vitro
raised plants (leaf and stem) were found to be
devoid of glycyrrhizin. Additionally, an
interesting correlation was revealed between
glycyrrhizin accumulation and various
differentiation stages of the plant. We also
evaluated cytotoxic effect of the extracts
against panel of human cancer cell lines in
vitro, among which, pancreatic cell line
(MIA-PaCa-2) was found to be sensitive to all
the fourteen extracts investigated. Notably,
extracts with higher glycyrrhizin content
displayed cell inhibition activity of the order
of 44% against breast cancer cell line.

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Overall, our findings demonstrated that the
metabolite spectrum of varied in vitro raised
morphogenetic lines, at different stages of
maturation, might offer a powerful tool to
understand the regulatory aspects of the
concerned metabolite pathway and their
consequent role in differentiation. Results
presented here have revealed that the
phytochemical profiling was found associated
with the organogenesis (Gupta et al., 2013).
Recently simultaneous qualitative and
quantitative assessment of eight flavonoids
and two terpenoids were performed in
fourteen in vitro raised morphogenic cultures
of Glycyrrhiza glabra. Our study revealed
that the spectrum and production of ten
compounds, under investigation, were higher
in organized tissue than the undifferentiated
mass, however, aerial portions of the in vitro
raised plants (leaf and stem) were found to be
devoid of therapeutically relevant
triterpenoid, glycyrrhizin. A correlation was
observed between cell maturation,
morphological differentiation and
glycyrrhizin accumulation. Mature stolons (4
months) were characterized by the maximum
accumulation of glycyrrhizin (8.60 g/mg) in
in vitro plantlets. The cytotoxic effect of the
extracts evaluated against a panel of human
cancer cell lines (in vitro) indicated that the
pancreatic cell line (MIAPaCa-2) were
sensitive to all the fourteen extracts
investigated (Saima et al., 2015).
Amarogentin and amaroswerin
Chemical investigations of various in vitro
developed morphotypes revealed that
proliferating shoot cultures produce bioactive
molecules amarogentin and amaroswerin
equal to the parental plants. As the herb is
directly being used by the industry without
any downstream process of extraction of
active principal, the shoot cultures seem to
have potential for direct use in the industry.
Studies are being carried out to explore
possibility for an alternative supply route
through biotechnological production of
biomass/product using shoot cultures in a
bioreactor. Present study is aimed at to
develop procedure for a. development of
shoot cultures of Swertia chirayita; b.
culturing shoot material in tissue culture
under conditions that organogenically
produce a proliferating of shoot biomass; and
c. standardization of the conditions for
harvesting said shoots and/or leafy material
while at green, actively-growing, non-
senescent stage and produce desired amount
of amarogentin and amaroswerin (Sushmaet
al., 2009).
Reserpine and Ajmalicinemetabolism in
Rauvolfia serpentina
Rauvolfia serpentine is an erect evergreen,
woody perennial shrub and commonly known
as sarpagandha. Major constitutes of
sarpagandha roots are reserpine,
rescinnamine, deserpidine and yohimbine
(Klyshnichenko et al., 1995). According to
Ayurveda, the roots and whole plants are used
for the treatment of cardio vascular disorder,
snake bite, rheumatism, hypertension,
insanity, epilepsy and hypochondria infusion,
decoction and extract of the roots are
employed to increase uterine contraction for
expulsion of foetus, to treat painful affection
of bowels, diarrhoea, dysentery, cholera and
colic value of sarpagandha root depends on
total alkaloid content and proportion of
reserpine and ajmalcine alkaloids present in it.
Reserpine has remarkable physiological
activities, which have led to its extensive use
in the treatment of hypertension, nervous and
mental disorders. It is also used in headache
and asthma. Ajmalicine has remarkable
physiological activities, which have led to its
extensive use as anti-hypertensive, anti-
bacterial and sedative in drugs (Rojaet al.,
1990).

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Experiments were conducted to quantify
secondary metabolite production in callus and
cell suspension culture of Rauvolfia
serpentina. Reserpine and ajmalicine were
detected in one-month-old callus as well as in
cell suspension cultures. MS medium
supplemented with 1.0 mgl-1 2,4-D in
combination with 0.5 mgl-1 IBA indicates the
highest recovery of reserpine content in both
callus and liquid suspension medium of one-
month age. Increasing concentration of 2,4-D
in liquid medium drastically decreased
reserpine content. Linearly decreased
ajmalicine concentration in both callus and
cell suspension culture was recorded with
increased concentration of 2,4-D.
Embryogenic cell suspension culture of R.
serpentine may be proved quite useful and
convincing tool to improve the yield of
secondary metabolites reserpine and
ajmalicine in in vitro. Both alkaloids may be
further produced in commercial scale by
bioreactor cultivation (Uikey et al., 2014).
Volatile terpenoids
The biosynthetic capacity of in vitro
proliferating shoots and regenerated callus
clones has been evaluated for essential oil
production. On evaluation it was found that
the essential oil isolated from foliage of
proliferating shoots and regenerated plantlets
was a complex mixture with 49 components,
25 of which were identified, corresponding to
80% of the total oil content. The analysis of
the identified constituents included
monoterpene hydrocarbon (43%), oxygenated
monoterpene (31%), sesquiterpene
hydrocarbons (7.4%) and oxygenated
sesquiterpenes (4.0%). The major constituents
were myrcene, limonene, (E)-linalool, (Z)-
ocimene and caryophyllene oxide (Ahuja et
al., 2005).Recently reported study revealed
comparative similarity of volatile constituents
of naturally grown and micropropagated
plants (Ahuja et al., 2016).
The production of chemicals and
pharmaceuticals using plant cell cultures has
made great strides building on advances in
plant science. The use of genetic and rDNA
technology tools and regulation of pathways
for secondary metabolism have provided the
basis for the production of commercially
acceptable levels of products. However,
despite progress strategies are still needed to
develop an information based on a cellular
and molecular level for the most of the
molecules. Because of the complex and
incompletely understood nature of plant cells
in in vitro cultures, case-by-case studies have
been used to explain the problems occurring
in the production of secondary metabolites
from cultured plant cells. As such focused
approach depending upon nature of
compound and resource plant and culture type
needs to be taken into consideration for
successful application of tissue culture to
harvest appreciable level of compound for
production at commercial level. Knowledge
concerning pathway dissection at molecular
level is required to be developed for each
compound to harvest the benefit of system
biology and metabolic approaches for
production at commercial level.
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How to cite this article:
Tripathi, M.K., Nishi Mishra, Sushma Tiwari, Chitralekha Shyam, Sonali Singh and Ashok
Ahuja. 2019. Plant Tissue Culture Technology: Sustainable Option for Mining High Value
Pharmaceutical Compounds. Int.J.Curr.Microbiol.App.Sci. 8(02): 1002-1010.
doi: https://doi.org/10.20546/ijcmas.2019.802.116