Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-014-0467-7

REVIEW

Production of secondary metabolites from cell and organ cultures:

strategies and approaches for biomass improvement
and metabolite accumulation
Hosakatte Niranjana Murthy Eun-Jung Lee
Kee-Yoeup Paek

Received: 9 November 2013 / Accepted: 5 March 2014

Springer Science+Business Media Dordrecht 2014

Abstract Plant cell and organ cultures have emerged as

potential sources of secondary metabolites, which are used
as pharmaceuticals, agrochemicals, flavors, fragrances,
coloring agents, biopesticides, and food additives. In recent
years, various strategies have been developed to assess
biomass accumulation and synthesis of secondary compounds in cultures. Biomass accumulation and metabolite
biosynthesis are two-stage events, and the parameters that
control the growth and multiplication of cultured cells/
organs and biomass accumulation are controlled in the first
stage. Parameters that assist with the biosynthesis of
metabolites are controlled in the second stage. The selection of high-producing cells or organ clones; optimization
of medium parameters such as suitable medium, salt, sugar,
nitrogen, phosphate, and plant growth regulator levels; and
physical factors such as temperature, illumination, light
quality, medium pH, agitation, aeration, and environmental
gas (e.g., oxygen, carbon dioxide, and ethylene) are

H. N. Murthy (&)
K.-Y. Paek (&)

Research Center for the Development of Advanced Horticultural
Technology, Chungbuk National University, Chongju 361-763,
Republic of Korea
e-mail: nmurthy60@yahoo.co.in
K.-Y. Paek
e-mail: paekky@chungbuk.ac.kr
H. N. Murthy
Department of Botany, Karnatak University, Dharwad 580003,
India
E.-J. Lee
Cheongsol Biotech Co. Ltd., Industry Academic Cooperation
Foundation Agribusiness Incubation Center, 205, Chungbuk
National University, Chongju 361-763, Republic of Korea

controlled in the first stage of the culture process. Elicitation, replenishment of nutrient and precursor feeding,
permeabilization, and immobilization strategies assist with
the accumulation of metabolites and can be applied in the
second stage of the culture process. By following stagespecific strategies, it is possible to produce large amounts
of biomass with an increase in the accumulation of secondary compounds.
Keywords Bioreactor cultures
Elicitation

Immobilization
Permeabilization
Plant cell cultures

Secondary metabolites
Abbreviations
ABA
Abscisic acid
BA
Benzyladenine
B5
Gamborgs medium
2,4-D
2,4-Dichlorophenoxyacetic acid
DW
Dry weight
DMSO Dimethylsulfoxide
FW
Fresh weight
GA
Gibberellic acid
HPLC
High performance liquid chromatography
HPTLC High performance thin layer chromatography
2-iP
2-Isopentenyladenine
IAA
Indole-3-acetic acid
IBA
Indole-3-butyric acid
LS
Linsmaier and Skoog medium
MS
Murashige and Skoog medium
NAA
Naphthaleneacetic acid
NMR
Nuclear magnetic resonance
SH
Schenk and Hildebrandt medium
TLC
Thin layer chromatography
PUFAs Polyunsaturated fatty acids
UV
Ultraviolet light

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Plant Cell Tiss Organ Cult

Introduction

Table 1 Strategies to enhance the production of secondary metabolites in plant cell and organ cultures

Secondary metabolites are a diverse group of organic

compounds that are produced by plants to facilitate interaction with the biotic environment and establishment of a
defense mechanism (Wink 1988; Verpoorte et al. 2002).
Most secondary metabolites such as terpenes, phenolics,
and alkaloids are classified according to their biosynthetic
origin, show different biological activities, and are used as
pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives. The production of
secondary metabolites via field cultivation of plants has
various disadvantages (e.g., low yields, and fluctuations in
concentrations due to geographical, seasonal, and environmental variations). Therefore, plant cells and cultures
have emerged as attractive alternatives for the production
of secondary metabolites (Ramachandra Rao and Ravishankar 2002). In recent years, various strategies have been
developed for use in biomass accumulation and the synthesis of secondary compounds, such as strain improvement, optimization of medium and culture environments,
elicitation, nutrient and precursor feeding, permeabilization, immobilization, and biotransformation methods
(Table 1). Biomass accumulation and the synthesis of
metabolites by cultured cells and organs is a two-step
process as follows: (1) initially cultured cells and organs
assist in the growth, multiplication, and accumulation of
biomass and (2) the synthesis of metabolites from the
biomass. In the past, biomass and secondary metabolite
production events were conducted simultaneously; however, with the adaptation of the two-stage process, it is
possible to enhance both biomass growth and metabolite
accumulation. In this article, we summarized the experimental strategies used for the production of secondary
metabolites by plant cell and organ cultures, with relevant
examples.

Stage 1

Selection of cell lines and clones

Initiation of cell and organ cultures begins with choosing
parent plants that contain higher contents of the desired
secondary product for callus or organ induction and the
selection of high-producing cell/organ lines. Secondary
metabolite accumulation in plants is genotype specific. For
example, the concentration of triterpenoid saponin bacoside A varies among different genotypes of Bacopa monnieri, ranging from 3.53 to 18.36 mg g-1 DW (Naik et al.
2012). Similarly, the amount of quinoline alkaloid camptothecin varies among different species such as Camptotheca acuminata, Camptotheca lowreyana, Camptotheca
yunnanensis, Ervatamia heyneana, Ophiorrhiza pumila,

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Selection of cell lines or clones

Medium optimization
Influence of nutrient medium and salt strength
Influence of carbohydrate source and concentration
Influence of nitrogen source
Influence of phophate levels
Influence of growth regulator levels
Influence of inoculum density
Optimization of the culture environment
Influence of temperature
Influence of light intensity and quality
Influence of hydrogen ion concentration (medium pH)
Influence of agitation and aeration
Stage 2
Elicitation
Nutrient feeding
Precursor feeding
Permeabilization
Immobilization
Selective adsorption of metabolites or two phase systems
Biotransformation
Organ cultures as a source of secondary metabolites
Scale-up of plant cell and organ cultures

Ophiorrhiza mungos, Ophiorrhiza rugosa, Nothapodytes

foetida, and Nothapodytes nimmoniana (0.030.4 % DW;
Ramesha et al. 2008). Accumulation of camptothecin varies in different organs of the same species. For example, in
Nothapodytes nimmoniana, the leaves, stem bark, and root
bark contain 0.081, 0.23, and 0.330.77 % DW of camptothecin, respectively (Ramesha et al. 2008). Therefore,
the selection of a suitable species and specific organs for
the induction of in vitro calli, cells, or organs is essential.
The isolation and selection of cell and organ lines for
increased accumulation of biomass and metabolites is
extremely important. In the past, cell-line selection was
carried out by visual screening, if the secondary product
was a pigment. For example, enhanced anthocyanin production by clonal selection and visual screening has been
reported in Euphorbia millii and Daucus carota (Yamamoto and Mizuguchi 1982; Dougall 1980). However, selection via analysis of the growth of cell lines or root clones
(e.g., adventitious or hairy roots) in suspension cultures
and, subsequently, quantification of the desired product is
superior to visual selection techniques. A growth kinetic
analysis is then conducted in some cases. For example, in
Orthosiphon stamineus, two cell lines capable of producing
a higher amount of rosmarinic acid via cell suspension

Plant Cell Tiss Organ Cult

culture were previously identified (Liang et al. 2006).

Quantification of metabolites by chromatographic and
spectroscopic evaluations is followed by screening for
high-yielding cell lines (Matsumoto et al. 1980; Zenk
1978). Sophisticated modern quantification techniques
such as UVVis spectrophotometry, TLC, HPTCL, HPLC,
and NMR provide quantitative information on cells and
organs under culture conditions. For example, TLC, UV,
and HPLC analyses are used for quantification of metabolites in cell suspensions and in vitro regenerated organs of
Hypericum perforatum (Pasqua et al. 2003).

Medium optimization
Several chemical and physical factors have been recognized, which could influence biomass accumulation and
synthesis of secondary metabolites in plant cell and organ
cultures. Some of the key constituents include the type of
culture medium, suitable salt strength of the medium, types
and levels of carbohydrates, nitrate levels, phosphate levels, and growth regulator levels (Dornenburg and Knorr
1995; Ramachandra Rao and Ravishankar 2002; Stafford
et al. 1986).
Influence of nutrient medium and salt strength
The media formulations of Gamborg (B5 1968), Linsmaier
and Skoog (LS 1965), Murashige and Skoog (MS 1962),
and Schenk and Hildebrandt (SH 1972) have been widely
used for establishing plant cell and organ cultures. The B5
medium that was originally used for callus and suspension
cultures of soybean differs from the MS medium. The
nitrogen supplement concentration in the B5 medium is
considerably lower than that in the MS medium; thus, it is
not suitable for the growth of Withania somnifera cell
suspension cultures (Praveen and Murthy 2010). Therefore,
the selection of a suitable medium is essential for establishing cell and organ cultures. In addition, appropriate salt
concentration is crucial for the growth of the isolated cells
and organs. For instance, with ginseng adventitious root
cultures, maximum biomass was obtained in a 0.75strength MS medium. In terms of ginsenoside production, a
0.5-strength MS medium resulted in higher ginsenoside
content and yield (Sivakumar et al. 2005a). A full-strength
MS medium was suitable for cell suspension cultures of
Gymnema sylvestre for biomass accumulation and gymnemic acid production (Nagella et al. 2011). Among the
0.25-, 0.5-, 0.75-, 1.0-, 1.5-, and 2.0-strength MS media
tested, the full-strength (1.0) medium for W. somnifera cell
suspension cultures was found to yield greater biomass
accumulation and withanolide A production (Praveen and
Murthy 2010).

Influence of the carbohydrate source and concentration

Plant cell cultures are usually grown by using a single
simple sugar or a combination of simple sugars such as
glucose, fructose, maltose, and sucrose. Sugars in the
medium act as energy sources and supply inorganic nutrients. In cell suspension cultures of Gymnema sylvestre, the
use of several sugars was tested, and sucrose was found to
be the ideal carbohydrate source for biomass accumulation
(11.56 g L-1 DW) and gymnemic acid production
(9.95 mg g-1 DW) (Nagella et al. 2011). Wang and
Weathers (2007) verified the effect of equimolar concentrations (30 g L-1) of individual sugars such as sucrose,
glucose, or fructose on artemisinin production from the
hairy root cultures of Artemisia annua and reported a
dramatic improvement in the accumulation of artemisinin
in the medium supplemented with glucose. They also tested
a mixture of sugars, including sucrose (27 g L-1) plus
glucose or fructose (3 g L-1), which was found to reduce
the production of artemisinin. Similarly, supplemental
concentration of a carbohydrate in the medium greatly
affected biomass and metabolite production. For example,
of the various levels of sucrose (18 % w/v) tested in
Gymnema sylvestre cell cultures, a 3 % sucrose concentration favored the accumulation of biomass, whereas the
highest amount of gymnemic acid (10.1 mg g-1 DW) was
shown to accumulate with a 4 % sucrose concentration
(Nagella et al. 2011). In Ginkgo biloba cell cultures, a 3 %
sucrose concentration was suitable for biomass accumulation, whereas higher concentrations (i.e., 5 and 7 %
sucrose) favored the production of ginkgolides and bilobalides (Park et al. 2004). In Bacopa monnieri shoot
cultures, a 2 % sucrose concentration was found to be the
optimal range tested (06 %, w/v) for biomass accumulation, and the sucrose-free medium was shown to accumulate the maximum amount of bacoside-A (Naik et al. 2010).
The osmotic stress created by sucrose alone and the other
osmotic agents was found to regulate anthocyanin production in Vitis vinifera cell suspension cultures (Do and
Cormier 1990). The dual role of sucrose as a carbon source
and osmotic agent has been observed in Solanum melongena (Mukherjee et al. 1991). Recently, sugars have been
recognized as signaling molecules that affect the growth,
development, and metabolism of cultured cells (Wang and
Weathers 2007; Praveen and Murthy 2012); therefore, a
suitable carbohydrate source and concentration should be
identified for the production of secondary metabolites in
cell and organ cultures.
Influence of nitrogen source
Nitrogen concentration was found to affect biomass growth
and metabolite accumulation in cell and organ suspension

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Plant Cell Tiss Organ Cult

cultures. The plant-tissue culture media such as MS, LS,

SH, and B5 contain both nitrate and ammonium as nitrogen
sources. However, the ratio of ammonium to nitrate and
overall levels of total nitrogen have been shown to markedly affect biomass accumulation and the production of
secondary plant products. For example, the effects of
macroelements were examined by varying the levels of
NH4NO3, KNO3, CaCl2, MgSO4, and KH2PO4 in the MS
medium at 0.059, 1.09, 1.59, and 2.09 each for the shoot
cultures of Bacopa monnieri. The results showed that the
optimum number of shoots (99.33 shoots per explant) and
biomass (0.150 g DW), and the highest production of
bacoside A (17.9 mg g-1 DW) were obtained with 29
strength NH4NO3 (Naik et al. 2011). In another experiment, the effect of nitrogen supplements was tested (i.e.,
NH4?/NO3- at 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/
18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80,
14.38/28.20, and 14.38/37.60 mM), and the results showed
that shoot biomass and bacoside A content were optimum
when the NO3- concentration was higher than that of
NH4? (i.e., a ratio of 14.38/37.60 mM). Reduced levels of
NH4? and increased levels of NO3- promoted the production of gymnemic acid and withanolide A (Praveen and
Murthy 2013; Praveen et al. 2011; Praveen and Murthy
2011). Reduced nitrogen improved the production of capsaicin in Capsicum annuum (Ravishankar et al. 1988) and
anthraquinones in Morinda citrifolia (Zenk et al. 1975).
However, the complete elimination of nitrate in cultures of
Chrysanthemum cinerariaefolium induced a twofold
increase in pyrethrin accumulation in the second phase of
the culture process (Rajashekaran et al. 1991).
Influence of phosphate levels
The phosphate concentration in the medium can have a
notable effect on the production of secondary metabolites
in plant cell and organ cultures. An increase in the phosphate level was shown to stimulate the synthesis of digitoxin in Digitalis purpurea (Hagimori et al. 1982). Liu and
Zhong (1998) reported that the highest rate of saponin
production was achieved at an initial phosphate concentration of 1.04 and 1.25 mM in Panax ginseng and Panax
quinquefolius, respectively. In the production of rosmarinic
acid by Lavandula vera suspension cultures (Ilieva and
Pavlov 1996), gymnemic acid from Gymnema sylvestre
cell cultures (Praveen and Murthy 2011), and solamargine
from Solanum paludosum multiple shoot cultures (Badaoui
et al. 1996), twice the amount of phosphate levels in a
standard MS medium (1.25 mM) yielded notable increases
in metabolite accumulation. On the other hand, there have
been a number of reports showing that phosphate limitation
could improve the production of secondary metabolites.
For example, increases in the synthesis of caffeine and

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anthocyanin were reported to occur alongside phosphate

deprivation in cell suspension cultures of Coffea arabica
(Bramble et al. 1991) and Vitis vinifera (Dedaldechamp
et al. 1995), respectively.
Growth regulators
Cell, adventitious root, or shoot cultures generally require
an exogenous supply of growth regulators for the growth
and proliferation of biomass and metabolite accumulation.
Hairy root cultures are genetically transformed roots via
Agrobacterium rhizogenes mediation and have the ability
to grow in the absence of plant growth regulators (Giri and
Narasu 2000). However, recent experimental results have
shown that the exogenous application of growth regulators
also influences growth and metabolite accumulation in
hairy root cultures (Vanhala et al. 1998; Weathers et al.
2005). In general, the plant growth regulator type and
concentration are crucial factors in cell and organ growth,
proliferation, and metabolite accumulation (DiCosmo and
Towers 1984). The type and concentration of auxin or
cytokinin, or the auxin to cytokinin ratio, dramatically
altered both biomass growth and product formation in
cultured cells (Mantell and Smith 1984). Auxins, indole
acetic acid (IAA), and naphthalene acetic acid (NAA) have
been shown to enhance the production of anthocyanins in
populous and carrot suspension cultures, nicotine in
tobacco suspension cultures, and anthraquinones in noni
suspension cultures (Seitz and Hinderer 1988; Sahai and
Shuler 1984, Zenk et al. 1975). 2,4-Dichlorophenoxyacetic
acid (2,4-D) has also been shown to exhibit a stimulatory
effect on the accumulation of carotenoids and anthocyanins
in carrot (Mok et al. 1976) and oxalis (Meyer and van
Staden 1995), respectively.
Among the cytokinins, the addition of benzyladenine
(BA) has been shown to improve the production of saponins in ginseng, and kinetin stimulated the production of
anthocyanin in slender golden weed but inhibited the production of anthocyanins in populous (Mok et al. 1976;
Seitz and Hinderer 1988). Moreover, 2-isopentenyladenine
(2-iP) inhibited root growth but stimulated artemisinin
production in A. annua (Weathers et al. 2005).
The effect of gibberellins (GA) has been shown to be
species specific; for example, Vanhala et al. (1998)
observed that GA3 decreased the accumulation of hyoscyamine in henbane. In contrast, GA stimulated the production of artemisinin in A. annua and coumarin content in
Cichorium intybus (Liu et al. 1997; Bais et al. 2001).
Ethylene stimulated artemisinin production in plantlet
cultures of A. annua (Fulzele et al. 1995) and enhanced the
growth of the hairy roots of Hyoscyamus muticus (Biondi
et al. 1997). Data is lacking on the effects of exogenous
abscisic acid (ABA) on cell and organ cultures; typically,

Plant Cell Tiss Organ Cult

ABA inhibits the growth and accumulation of secondary

metabolites. For example, ABA inhibited hyoscyamine
accumulation in the hairy root cultures of H. muticus
(Vanhala et al. 1998), with no adverse effect on biomass. In
Lotus corniculatus, ABA application stimulated growth but
inhibited the accumulation of tannin (Robbins et al. 1996).

Influence of inoculum density

Inoculum density is an important factor for plant cell and
organ suspension cultures, which can influence growth,
biomass accumulation, and metabolite formation (Kanokwaree and Doran 1997; Mavituna and Buyukalaca 1996).
There is a critical minimum inoculum size below which cell
or organ growth will typically fail. There have been
numerous studies reporting on the effect of inoculum density
of cultured cells on biomass and metabolite accumulation
(Berlin et al. 1986; Lee and Shuler 2000; Matsubara et al.
1989; Moreno et al. 1993a). In suspension cultures of Perilla
frutescens, a maximum DW cell density of 38.3 g L-1 was
obtained at an elevated inoculum size of 50 g of wet cells
per liter; in addition, anthocyanin production was enhanced
23-fold (Zhong and Yoshida 1995). In adventitious root
suspension cultures of various inoculum quantities of ginseng (i.e., 2.5, 5.0, 7.5, and 10.0 g L-1), the optimal density
of dry biomass (10.5 g L-1) and ginsenosides (5.4 mg g-1
of DW) was achieved using 5.0 g L-1 of inoculum.
Increased inoculum quantities (i.e., 7.5 and 10.0 g L-1)
caused reductions in biomass and ginsenoside accumulation.
Inoculum density is also known to have an effect on the
induction of enzymes involved in general phenylpropanoid
metabolism when cells are transferred to a fresh medium.
This is called the transfer effect or dilution effect.
Hahlbrock and Wellman (1973) reported that the phenylalanine ammonia-lyase induced via transfer to a fresh medium
decreased with increased inoculum size. Therefore, inoculum density may affect secondary metabolism. Morphology
of the roots is another factor that influences biomass growth
and the synthesis of secondary compounds (Folk and Doran
1996; Jeong et al. 2009) in root suspension cultures. For
example, certain adventitious root inocula (i.e., chopped at
13 and 46 mm or unchopped) were responsible for the
lower DW and ginsenoside yield. The root inocula chopped
to 710 mm resulted in a higher yield of 10 g L-1 of DW
and the highest ginsenoside content (i.e., 5.5 mg g-1 of
DW) (Jeong et al. 2009).

Optimization of the culture environment

Culture environmental conditions such as light, temperature, medium pH, and essential gases of the medium have

been examined for their effect upon biomass growth and

secondary metabolite accumulation in cell and organ
cultures.
Influence of temperature
A temperature range of 1725 C is typically used for the
maintenance of cultured cells and organs; however, each
plant species may exhibit optimum growth and metabolism
under different temperature regimes. Since the early
development of plant biotechnology, the effects of temperature have been investigated. Morris (1986) studied the
Catharanthus roseus cell line C87 and found that its
maximum growth rate occurred at 35 C, and its maximum
dry weight yield (0.47 g g-1) was realized at 25 C.
Scragg et al. (1988) investigated the Catharanthus roseus
cell line ID1 at 20, 25, and 30 C; the maximum biomass
yield of 0.65 g g-1 was reported at 25 C. Courtois and
Guern (1980) found that the optimum temperature of 16 C
was required for the production of ajmalicine, and Morris
(1986) reported an optimum temperature of 25 and 20 C
for serpentine and ajmalicine production, respectively. In
addition, Toivonen et al. (1992) estimated an optimum
temperature of 25 C for the production of alkaloids from
the cell suspension cultures Catharanthus roseus. Shohael
et al. (2006) studied the effect of low (i.e., 12 and 16 C)
and high (30 C) temperatures and reported that they
caused a significant decrease in biomass and a reduction in
phenolics and flavonoids. In addition, low temperature
boosted the accumulation of eleutheroside E in the somatic
embryos of Eleutherococcus senticosus, which was correlated with an increase in oxidative stress. Yu et al. (2005)
studied the growth of the hairy roots of ginseng under
different temperature regimes (i.e., 13/20, 20/13, 25/25,
and 30/25 C for 16/8 h day and night cycles); the highest
hairy root biomass was obtained with the cultures incubated at 20/13 C. However, total ginsenosides was optimum (10.5 mg g-1 of DW) in the cultures incubated at
25/25 C.
Influence of light intensity and quality
Light is an energy source that affects biomass growth and
secondary metabolite accumulation in cultured cells and
organs. Chan et al. (2010) investigated the effects of different environmental factors such as light intensity and
irradiance (continuous irradiance and continuous darkness)
on cell biomass yield and anthocyanin production in cultures of Melastoma malabathricum. Moderate light intensity (300600 lx) induced an increase in the accumulation
of anthocyanins; the cultures exposed to 10 days of continuous darkness exhibited the lowest pigment content,
while the cultures exposed to 10 days of continuous

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irradiance exhibited the highest pigment content. The

stimulatory effect of light on the formation of secondary
compounds has been reported by various authors, including
flavonoids in Petroselinum hortense (Kreuzaler and Hahlbrock 1973), anthocyanins in Centaurea cyanus (Kakegawa et al. 1991), betalains in red beet (Shin et al. 2004), and
artemisinin in A. annua (Liu et al. 2002). However, light
has an inhibitory effect on the accumulation of secondary
metabolites such as nicotine and shikonin in Lithospermum
erythrorhizon (Tabata et al. 1974), and monoterpenes in
Citrus limon (Mulder-Krieger et al. 1988). In some species
such as Fragaria ananassa (Nakamura et al. 1999) and
sweet potato (Konczak-Islam et al. 2000), cell cultures
have been reported to produce anthocyanin under the dark
condition. Yu et al. (2005) studied the effect of fluorescent
light, metal halide light, blue light, red light, and blue plus
red light on biomass growth and the synthesis of ginsenosides in ginseng hairy root cultures and reported that
hairy root growth was stimulated by red-light-incubated
cultures when compared to dark-incubated cultures. Fluorescent irradiation enhanced the accumulation of ginsenosides (5.3 mg g-1 of DW). They also noticed the
differential accumulation of the Rb and Rg groups of
ginsenosides in dark- and light-grown cultures; Rb-group
ginsenosides were highest in the dark-grown cultures
(4.5 mg g-1 of DW), and the Rg group ginsenosides were
optimal in the light-grown cultures (5.3 mg g-1 of DW).
These results suggest that it is possible to manipulate
secondary metabolite accumulation by varying the light
and dark regimes.
Influence of hydrogen ion concentration (medium pH)
The medium pH is usually set between 5 and 6 (before
autoclaving), and extreme pH values are avoided. The
concentration of hydrogen ions in the medium changes
under culture conditions due to nutrient uptake or the
accumulation of metabolites. For example, a decrease and
increase in media pH caused by ammonia assimilation and
nitrate uptake, respectively, have been reported by
McDonald and Jackman (1989). In W. somnifera hairy root
cultures, the initial pH of the medium (i.e., 5.8) favored the
accumulation of biomass (12.1 g L-1 of DW), and a
medium pH of 6.0 favored the accumulation of withanolide
A in the roots (13.84 mg g-1 of DW; Praveen and Murthy
2012). In hairy root cultures of Tagetes patula, a medium
pH of 5.7 was suitable for the growth and accumulation of
thiophene (Mukundan and Hjortso 1991). In Panax ginseng
hairy root cultures, the medium pH values of 6.0 and 6.5
favored both biomass accumulation and ginsenoside production (Sivakumar et al. 2005b). Changing the medium
pH, which results in changes in membrane permeability
and the release of a secondary product into the culture

123

medium, is a technique that has been utilized in many

culture systems (Mukundan et al. 1998; Saenz-Carbonell
et al. 1993). For example, betalains normally accumulate in
the roots of Beta vulgaris but are released into the medium
at a pH of 5.5 (Mukundan et al. 1998). Up to 50 % of the
total pigment was released at the time of exposure, and,
subsequently, the roots continued to grow and accumulate
betalains. When the roots were exposed to pH 2 for 10 min,
they failed to grow, suggesting that the low pH value
induced mature pigment cell lysis. The short duration of
pH exposure was beneficial for the continuous production
of pigment from the cells and cultured roots.
Influence of agitation and aeration
Agitation is an important parameter that should be controlled in flask-scale to large-scale bioreactor cultures. The
mixing of cultures promotes better growth by enhancing
the transfer of nutrients from the liquid and gaseous phases
to the cells/organs and the dispersion of air bubbles for
effective oxygenation. Although plant cells have higher
tensile strength in comparison to microbial cells, their
shear sensitivity to hydrodynamic stresses restricts the use
of extreme agitation for efficient mixing. The high shear
rate and shear time that accompany adequate mixing
reduce the mean aggregate size; however, they also have an
adverse effect on cell viability. Plant cells, therefore, are
often grown in stirred tank bioreactors at very low agitation
speeds. Shifting from cell cultures to organ cultures such as
adventitious or hairy root, shoot, and embryo cultures for
the production of secondary metabolites may be advantageous for overcoming rheological problems (Baque et al.
2012; Murthy et al. 2008a).
Aeration is another important factor that should be
controlled in bioreactor cultures for optimization of biomass growth and secondary metabolite production
(Chattopadhyay et al. 2002; Georgiev et al. 2009). Aeration
of plant cell cultures fulfills three main functions, including
the maintenance of aerobic conditions, desorption of volatile products, and removal of metabolic heat (Georgiev
et al. 2009). The oxygen requirements of plant cells are
comparatively lower than those of microbial cells because
of their low growth rates. However, the oxygen supply has
been shown to significantly affect secondary metabolite
formation in cell cultures (Gao and Lee 1992; Schlatmann
et al. 1997). The effects of oxygen supply within 20.8, 30,
40, and 50 % have been studied by Thanh et al. (2006a) by
using ginseng cell cultures, and 40 % oxygen supply was
found to be beneficial for cell biomass production and
saponin yield. In some cases, high oxygen concentration
was shown to be toxic to the cells metabolic activities and
may decrease nutrient availability (carbon dioxide) from
the culture broth (Georgiev et al. 2009). Carbon dioxide is

Plant Cell Tiss Organ Cult

often considered an essential nutrient in plant cell cultures

and has a positive effect on growth. The effects of carbon
dioxide supply within 0.03, 1.0, 2.5, and 5.0 % in ginseng
cell cultures were analyzed by Thanh et al. (2006b). A 1 %
carbon dioxide supply was shown to improve biomass
accumulation; however, supplementation of carbon dioxide
was not beneficial for saponin accumulation. The beneficial
effect of carbon dioxide on secondary metabolite production has also been observed in cell cultures of Thalictrum
minus (Kobayashi et al. 1991), T. rugosum (Kim et al.
1991), and Stizolobium hassjoo (Huang and Chou 2000).

Elicitation
Secondary metabolites accumulate in plant cells in
response to various biotic (e.g., pathogens or insects) and
abiotic (e.g., temperature, salinity, water, radiation, heavy
metal, and mineral) stresses (Ramakrishna and Ravishankar 2011). These varied stress conditions have been designated as elicitors (Dornenburg and Knorr 1995), and
elicitation has been widely used for the overproduction of
secondary metabolites in plant cell and organ cultures
(Dornenburg and Knorr 1995; Ramakrishna and Ravishankar 2011). The elicitors of fungal, bacterial, and yeast
origins (e.g., polysaccharides, glycoproteins, inactivated
enzymes, purified crudlan, xanthan, and chitosan) and salts
of heavy metals have been reported to induce the overproduction of various secondary metabolites. Signaling
molecules such as methyl jasmonate and salicylic acid have
also been widely used for the increased accumulation of
secondary metabolites in cell and organ cultures (Kim et al.
2004; Dong et al. 2010; Shohael et al. 2008; Thanh et al.
2005; Yu et al. 2002). Elicitor concentrations, duration of
exposure, and age or stage of the culture at the time of
elicitor treatment are also important factors influencing the
successful production of biomass and secondary metabolite
accumulation. For example, Yu et al. (2002) studied the
effect of jasmonic acid (i.e., at concentrations of 0, 1.0, 2.0,
5.0, and 10.0 mg L-1) on ginseng adventitious root cultures; an increase in concentration resulted in a decrease in
both fresh and dry biomass. However, ginsenoside content
increased with increasing concentrations, and a 5.2-fold
increment was reported. Since a significant decrease in the
biomass was observed, the two-step methodology was
employed (e.g., growth of the adventitious roots in cultures
for 25 days without an elicitor, followed by the addition of
jasmonic acid [2 mg L-1]), and increases in the total ginsenosides and Rb-group ginsenosides by 5- and 5.6-fold,
respectively were noted. By following the two-step methodology, Yu et al. (2002) showed that it was possible to
achieve both biomass growth and ginsenoside accumulation. Similarly, jasmonates have been used to elicit an

increase in the accumulation of taxol in the cell cultures of

Taxus chinensis (Ketchum et al. 1999), saikosaponins in
the root cultures of Bupleurum falcatum (Aoyagi et al.
2001), and eleutherosides in the embryo cultures of
Eleutherococcus senticosus (Shohael et al. 2007).
Recently, polyunsaturated fatty acids (PUFAs) have been
shown to possess biological activities in tissue cultures. For
instance, exogenous PUFAs increased the accumulation of
secondary metabolites in the suspension cultures of Solanum
esculentum, Tinospora cordifolia, Erythrina crista-galli, and
Eschscholzia californica (Gundlach and Zenk 1998). In
addition, elicitation with a-linolenic acid enhanced the
activity of lipoxygenase, a key enzyme involved in oxylipin
biosynthesis (Wasternack 2007). Linoleic and a-linolenic
acids were used as elicitors in the range 020 lM with
adventitious root cultures of Panax ginseng. The results
showed that the effects of linoleic and a-linolenic acids were
concentration dependent; for instance, linoleic acid and alinolenic acid significantly decreased and increased root
biomass growth, respectively (Wu et al. 2009). The content
of protopanaxadiol and protopanaxatriol ginsenosides was
elevated with the addition of a-linolenic acid. Similarly, in
the cell cultures of Agrostis tenuis, Rauvolfia serpentina, and
Nicotiana tabacum, the addition of a-linolenic acid induced
the accumulation of jasmonic acid, and thus, facilitating the
biosynthesis of pentacyclic oxylipins (Gundlach and Zenk
1998).

Nutrient feeding
The medium/nutrient feeding strategy is one of several
approaches utilized to improve the yields of secondary
metabolites after optimizing the chemical and physical
parameters for the large-scale cultivation of cells/organs
(Zhong 2001; Jeong et al. 2008). For instance, various
nutrients in the culture medium were exhausted by 40 days
of culture for ginseng adventitious root cultures. With the
objective of meeting the nutrient requirements of ginseng
adventitious root cultures, and enhancing biomass and
ginsenoside production, Jeong et al. (2008) replenished the
cultures with 0.75- and 1.0-strength media at 10 and
20 days after culture initiation. The cultures that were
replenished with fresh medium (1.0-strength MS medium
after 20 days of culture) showed a 27.45 and 8.25 %
increase in dry biomass (28.66 g L-1 with replenishment
treatment) and ginsenoside content (4.93 mg g-1 DW),
respectively. A similar, positive effect of the medium
exchange technique has been reported for adventitious root
cultures of Echinacea purpurea (Wu et al. 2007a), cell
cultures of Lithospermum erythrorhizon (Srinivasan and
Ryu 1993), and cell suspension cultures of Taxus chinensis
(Wang et al. 2001).

123

Plant Cell Tiss Organ Cult

A major disadvantage of batch processes is that a significant amount of time is required for system and media
sterilization, and filling, emptying, and cleaning of the
system. Thus, to improve the cost effectiveness of the plant
cell culture process, various operational modes have been
developed, including fed-batch, repeated fed-batch, semicontinuous, and continuous cultivation by biochemical
engineers; information on these modes has been well
documented by Georgiev et al. (2009). Fed-batch operation
involves the continuous or intermittent addition of one or
more nutrients to the initial medium after the start of cultivation or during the batch process. Continuous cultivation
includes variants without feedback control (e.g., chemostats, where the substrate is fed at a constant rate) and with
feedback control (e.g., trubidostats, where the turbidity of
the culture is kept constant by adjusting the rate at which
the substrate is fed, and auxostats, where the pH or dissolved oxygen of the medium is maintained at a set value).
Perfusion cultivation is carried out by continuously feeding
fresh medium to the bioreactor and constantly removing
the cell-free medium while retaining the biomass in the
reactor.

Precursor feeding
Many plant cell cultures have also been used to convert
precursors into products by utilizing preexisting enzyme
systems. For example, the addition of loganin, tryptophan,
and tryptamine enhanced the production of secologanin
(Contin et al. 1999) and indole alkaloids (Moreno et al.
1993b) by Catharanthus roseus suspension cultures. Similarly, phenylalanine feeding improved accumulation of
paclitaxel in Taxus cuspidata (Fett-Neto and DiCosmo
1996), and cholesterol feeding influenced the production of
conessine in Holarrhena antidysenterica (Panda et al.
1992) cell cultures. However, factors such as the timing
and concentration of the precursor should be considered
when adding it to a cell culture medium.

Permeabilization
Plant secondary metabolites formed by plant cell cultures
are usually stored in the vacuoles; therefore, extraction of
the products into the culture medium in a way that can
facilitate the ease with which the purification procedure is
conducted is highly desirable. The removal of secondary
metabolites from the vacuoles of the cell would also
decrease product inhibition and increase productivity. Many
attempts have been made to permeabilize plant cell membranes in a reversible manner with organic solvents. Organic
solvents such as isopropanol, dimethylsulfoxide (DMSO),

123

and polysaccharides (e.g., chitosan) have been used as permeabilizing agents in a number of studies (Beaumont and
Knorr 1987; Knorr and Teutonico 1986; Brodelius 1988). In
addition, Hexadecane, decanol, and dibutylphthalate are
used for taxol permeabilization in Taxus chinensis cell cultures (Wang et al. 2001). However, when various chemicals
are used as permeabilizing agents, they affect cell viability;
therefore, selection of a chemical agent with due consideration to its effect on cell growth may lead to the substantial
release of secondary metabolites. Other permeabilization
methods such as electric field stress (Dornenburg and Knorr
1993) and ultrasound (Lin et al. 2001) have also been used
for the recovery of secondary metabolites.

Immobilization
The immobilization of plant cells with suitable matrices
has been utilized to overcome problems of low shear
resistance and cell aggregation (Dornenburg and Knorr
1995). The advantages of immobilization include the following: (1) extended viability of cells in the stationary
stage, thus, enabling the maintenance of biomass over a
prolonged time period; (2) simplified downstream processing (if products are secreted); (3) high cell density
within relatively small bioreactors showing reduced cost
and risk of contamination; (4) reduced shear stress; (5)
increased product accumulation; (6) flow-through reactors
that can be used to enable greater flow rates; and (7)
minimization of fluid viscosity, which causes mixing and
aeration problems in cell suspensions (Dicosmo and Misawa 1995). There are two major methods for cell immobilization: (1) the gel entrapment method and (2) the
surface immobilization method. The most widely used
technique involves the entrapment of cells in a specific gel
or combination of gels, which are allowed to polymerize.
Calcium alginate is the most widely used matrix; agarose,
gelatin, carrageenan, and polyacrylamide have also been
used (Nilsson et al. 1983; Dornenburg and Knorr 1995;
Ramachandra Rao and Ravishankar 2002). The matrix used
for cell entrapment should be nontoxic to the cells, inexpensive, and have good polymerization activity. The first
report of immobilization for Morinda citrifolia, Digitalis
purpurea, and Catharanthus roseus cultures was by
Brodelius et al. (1979). Surface immobilization is another
method that takes advantage of the propensity of cultured
plant cells to adhere to inert surfaces immersed in a liquid.
DiCosmo et al. (1994) reviewed the work on plant cell
adsorption to surfaces and immobilization on glass fibers,
and the surface immobilization of Catharanthus roseus,
Nicotiana tabacum, and Glycine max cultured cells have
been reported for the production of metabolites (Asada and
Shuler 1989; Archambault et al. 1989).

Plant Cell Tiss Organ Cult

Several studies have shown that the dramatic effects of

immobilization on cells for secondary metabolite production include a 100-fold increase in capsaicin production
from immobilized cells of Capsicum sps. in foam and gel
(Lindsey and Yeoman 1984; Ravishankar et al. 1988) and
13- and 3.4-fold increment in methylxanthine and ajmalicine accumulation from the gel-immobilized cells of Coffea arabica and Catharanthus roseus, respectively (Asada
and Shuler 1989; Haldimann and Brodelius 1987). The
search for new biological and synthetic polymers is
ongoing, and some immobilization strategies have been
shown to increase the plant cell bioproduction of secondary
metabolites (Dornenburg 2004).

Selective adsorption of metabolites or two-phase

systems
It has been suggested that, in some instances, the low
accumulation of secondary metabolites in cell cultures may
be due to feedback inhibition, enzymatic or nonenzymatic
degradation of the product in the medium, or volatility of
the compounds produced. In such cases, it is necessary to
develop a separation technique that can concentrate the
desired product. For in situ product separation of plant cell
cultures, liquidsolid culture systems (i.e., two-phase
systems) for plant cells consisting of an aqueous-nutrient
phase and solid-polar adsorbents have been preferred
because many plant cells are expected to be polar and,
therefore, bind weakly in the lipophilic phase of the liquid
liquid systems. The removal and sequestering of a product
in a nonbiological compartment may increase total production of secondary compounds (Beiderbeck and Knoop
1988). Polycarboxylic ester resin, neutral polymeric resin
XAD-7, can absorb berberine, a secondary metabolite from
immobilized (alginate trapped) Thalictrum rugosum cells
(Choi 1992). Some advantages of adsorbents include their
use in bioreactor operations and allowance for the easy
separation of adsorbents from cells for the repeated use of
both the cells and adsorbents (Choi 1992; Nigam et al.
1990).
Activated charcoal, RP-8 (lipophilic carrier)zeolite,
XAD-2, XAD-4, XAD-7 (XAD is a neutral resin and ion
exchanger), polyethylene glycol, b-cyclodextrin, polydimethylsiloxane, and wofatite have been tested and successfully used for the separation of secondary metabolites
in cell suspension cultures of several systems (Dornenburg
and Knorr 1995). Amberlite XAD-7 was found to efficiently facilitate the adsorption and overproduction of taxol
from suspension cultures of Taxus (Kwon et al. 1998),
anthraquinones from suspension cultures of Rubia akane
(Shim et al. 1999), and triptolide from adventitious root
cultures of Tripterygium wilfordii (Miao et al. 2013).

Biotransformation
Biotransformation involves regioselective and stereospecific chemical transformations that are catalyzed by biological systems, entrapped enzymes, or permeabilized cells
(Giri et al. 2001; Banerjee et al. 2012). Biotransformation
is another strategy that can be utilized for the production of
high-value metabolites utilizing plant cell and organ cultures. The reaction carried out by such cultures involves
hydroxylation, glycosylation, glucosylation, oxidoreduction, hydrogenation, hydrolysis, methylations, acetylations,
isomerization, and esterification of various substrates (Giri
et al. 2001).
While plant cell cultures possess the biochemical
potential for the high-rate production of specific secondary
metabolites, accumulation of the desired products can be
unsuccessful at times for any number of metabolic reasons.
However, such cultures may retain the ability to transform
exogenous substrates into the products of interest. Chemical compounds, which can undergo biotransformations
mediated by plant enzymes, are variable in nature (e.g.,
aromatic, steroid, alkaloid, coumarin, terpenoid, and lignin). It is not necessary for the compounds to be natural
intermediaries of plant metabolism, and a substrate may be
of synthetic origin. Plant cell cultures and enzymes have
the potential to transform inexpensive and plentiful substances such as industrial byproducts into rare and expensive products. For example, podophyllotoxin, a precursor
of a semi-synthetic anticancer drug, is generally extracted
from its source plant, Podophyllum species. Kutney (1993)
demonstrated that a cell line of Podophyllum peltatum,
active in the biosynthesis of podophyllotoxin, was able to
maintain repeated biotransformation of butanolide to the
podophyllotoxin analogue. Ramachandra Rao and Ravishankar (2000) used freely suspended and immobilized
cells of Capsicum frutescens for conversion of protocatechuic aldehyde and caffeic acids to vanillin and capsaicin. Moreover, Li et al. (2005) used ginseng cultured
cells and roots for the bioconversion of paeonol into its
glycosides, which have radical scavenging effects.

Organ cultures as sources of secondary metabolites

The production of secondary metabolites by cell suspension culture is not always guaranteed; thus, organ culture
methods (e.g., root, embryo, and shoot culture methods)
have been developed for various plant species as alternatives for use in the production of secondary metabolites
(Giri and Narasu 2000; Baque et al. 2012; Murthy et al.
2008a; Verpoorte et al. 2002). Shoot cultures have been
established for many medicinal plants, which have been
shown to accumulate secondary metabolites to a greater

123

Plant Cell Tiss Organ Cult

extent than that by natural plants. For example, shoot

cultures were established in Bacopa monnieri for the production of bacoside A, and regenerated shoots resulted in a
3-fold increase in bacoside A when compare to field-grown
plants (Praveen et al. 2009). Similarly, the shoots of
Nothapodytes nimmoniana regenerated in semisolid and
liquid media yielded higher amounts of camptothecin when
compared to their mother plants (Dandin and Murthy
2012). Hairy roots obtained by Agrobacterium rhizogenesmediated transformation, which can grow without or with
the supplementation of a growth regulator, exhibited higher
growth rates than cell suspension cultures (Giri and Narasu
2000). In addition, hairy root cultures are efficient producers of secondary metabolites; for example, the production of the terpenoid compound withanolide A is more
than adequate in the hairy root cultures of W. somnifera
(Murthy et al. 2008b). The hairy roots showed high production capacity, and withanolide A was present in
amounts that were 2.7-fold higher than those observed in
nontransformed roots. Natural adventitious roots have been
induced in many medicinal plants via flask scale to bioreactor cultivation for the production of various bioactive
compounds (Baque et al. 2012; Murthy et al. 2008a). For
example, adventitious root cultures of Morinda citrifolia
grown in bioreactors showed a several fold increment in
anthraquinone content when compared to field-grown or
greenhouse-grown plants (Baque et al. 2012).

Scale-up of plant cell and organ cultures

Plant cells have a unique set of characteristics, including
relatively unstable productivity, high shear sensitivity,
slow growth rate, and low oxygen requirements. A wide
variety of bioreactor designs have been tested and used for
plant cell cultures. Stirred tank reactors, bubble column
reactors, airlift reactors, and ebb and flood reactors used in
the cultivation of plant cells are simply extension microbial
culture systems with little modifications. The worlds
largest plant cell culture facility was established in Germany (up to 75,000 L) and was designed using the stirred
tank model (Georgiev et al. 2009). Centrifugal impeller
bioreactors, based on the principles of a centrifugal pump,
have been developed by Wang and Zhong (1996) for use in
culturing plant cells with high shear sensitivity. A successful scale-up of Azadirachta indica suspension cultures
was developed in stirred tank reactors equipped with a
centrifugal impeller for the production of azadirachtin
(Prakash and Srivastava 2007). Mechanically driven wave
reactors have been recently developed for high shearstress sensitive plant cells by Eibl and Eibl (2002), and the
absence of air bubbles and wall growth as well as reduced
foaming appears to make these reactors suitable for

123

cultivating plant cell and organ cultures (Eibl et al. 2010).

Another reactor, termed the slug bubble reactor, is
comprised of a vertical, flexible plastic cylinder that
achieves aeration for plant cells, which are high-stress
sensitive, via the generation of large cylindrical bubbles
that move from the bottom to the top of the reactors
(Terrier et al. 2007). Another reactor, named the undertow
reactor, was developed by Terrier et al. (2007); it creates a
waver/undertow motion that ensures mixing and bubblefree aeration of a culture. The system is placed on a horizontal surface and equipped, on one side, with a moveable
platform that rises and falls, which creates waves followed
by an undertow. The wave and undertow reactors may be
suitable for the cultivation of high shear-stress sensitive
cell lines (Georgiev et al. 2009).
Airlift bioreactors appear to be ideal for some plant cell
cultures that are not highly shear sensitive (Dornenburg
and Knorr 1995). Further, airlift bioreactors that spread the
air from the base of the reactor though a sparger are suitable for the cultivation of hairy and adventitious roots of
various medicinal plants; thus, scale-up and pilot-scale
cultivation is possible with the use of these reactors
(Fig. 1a). For example, with the administration of 7 g L-1
of inoculum in 1,000-L airlift bioreactors, 40.5 kg of
Echinacea purpurea adventitious root biomass can be
produced after 50 days of culture (Wu et al. 2007b). The
accumulation of 5 mg g-1 of DW of chlorogenic acid,
22 mg g-1 of DW of chichoric acid, and 4 mg g-1 of DW
of caftaric acid was achieved with adventitious roots grown
in 1,000-L bioreactors. Similarly, an inoculation of 500 g
of fresh weight of ginseng adventitious roots into 500-L
balloon-type bubble bioreactors yielded 74.8 kg of root
biomass at 8 weeks of culture. The saponin content
obtained in the small-scale (20 L) as well as pilot-scale
(500 L) bioreactors was 1 % based on DW (Choi et al.
2000). Such experimental results have led to the establishment of pilot-scale bioreactors (up to 10,000 L; Fig. 1b,
c) for the production of ginseng adventitious root biomass
for commercial exploitation by CBN biotech, Cheongju,
Republic of Korea. Ginseng adventitious root biomass
(Fig. 1d) is regularly produced, and various ginseng-based
products such as ginseng extract, ginseng wine, ginseng
soap, and ginseng capsules are currently available to
consumers.

Economic feasibility of the production of secondary

metabolites by cell and organ cultures
The economic feasibility of secondary metabolite production from cell and organ cultures varies with the plant
species, type of cultures employed (for large-scale cultivation), the type of bioreactor(s) used, the method/mode of

Plant Cell Tiss Organ Cult

Fig. 1 Pilot-scale bioreactors. a 500-L airlift bioreactor, b 10,000-L airlift bioreactor, c ginseng adventitious root biomass, d harvested ginseng
adventitious root biomass

operation, biomass yield, and value of the end product.

Herein, we present the production cost of ginseng adventitious root raw material as a case study. We compared the
bioreactor production of ginseng adventitious roots with
that of field-cultivated ginseng (Table 2). Ginsenosides are
triterpene saponins obtained from Panax ginseng (Korean
ginseng), Panax notoginseng (Chinese ginseng), or Panax
quinquefolius (American ginseng). Ginsenosides have
various pharmaceutical and nutraceutical applications.
Ginseng plants that are collected from their natural habitats
are highly expensive and scarce. Ginseng is also cultivated
in the field; however, it usually takes 57 years for the
roots to reach the harvesting stage, during which close
attention is needed as growth can be influenced by various
environmental conditions such as soil, climate, pathogens,
and pests. The average yield of Korean ginseng (Panax
ginseng C. A. Meyer) roots from field cultivation in the
Republic of Korea is 523 kg per 0.1 ha, and the cost of
production was estimated at 35 US$ (Anonymous 2013;
Table 2). The cost includes seedbed preparation, custom
seeding, manure, pesticides, fumigation, fertilizer, shade
cloth (ginseng is a shade-loving plant and should be grown

Table 2 The cost of production of ginseng adventitious roots compared with field cultivated ginseng
Item

Field cultivated
ginseng

Adventitious roots
obtained from
bioreactor cultivation

Yield (kg/0.1 ha)

523a

30,000b

35

47

Production cost (US $/kg)

a

After 5 years of field cultivation (fresh root biomass). Data from

2012 Ginseng statistical yearbook, Ministry of Agriculture, Food and
Rural Affairs, Republic of Korea

Ginseng adventitious roots were cultured in four 10,000 l bioreactors for 45 days and bioreactors were operated for 78 cycles per
year

under 7080 % shade; the cost of the cloth used for

shading will vary depending on the material used), and
labor. The biomass yield of ginseng adventitious roots
cultivated in four 10,000-L bioreactors for 45 days and
operated for 78 cycles in 1 year (established by CBN
Biotech, Cheongju, Republic of Korea) is *30,000 kg per
year (30 t). The production cost of ginseng adventitious
roots is US$ 47 kg-1. The cost analysis is as follows: 13,

123

Plant Cell Tiss Organ Cult

26, 6, 11, and 44 % for chemicals, labor, electricity/gas/

water, operation, and depreciation of the machinery,
respectively. The quantum of biomass produced by bioreactor cultivation shows that there is ample scope for the
commercial application of the plant cell and organ cultures
for the production of secondary metabolites.

Conclusion
Plant cell and organ cultures are promising techniques for
the production of valuable secondary metabolites, which
have pharmaceutical, nutraceutical, and industrial importance. The recent developments in plant tissue culture
techniques and bioprocessing have shown promising
results for notably improving biomass growth and productivity. The optimization of medium ingredients and
culture environmental factors are the basic approaches that
should be ascertained with regard to individual plant species in the first stage (i.e., at the flask level) of the culture
process. Various other parameters such as inoculum density, agitation/aeration, elicitation, nutrient feeding, precursor feeding, permeabilization, and immobilization
should also be investigated in small-scale bioreactor cultures. Care should be taken when selecting the bioreactor
type and application of bioprocess parameters during this
initial stage. Adoption of organ culture techniques and a
scale-up process can lead to significant enhancement in the
productivity of secondary metabolites. Proper understanding and rigorous analysis of these parameters will pave the
way toward successful commercialization of plant cell
bioprocesses. Cost-effectiveness is the major bottleneck for
industrial production of plant secondary metabolites and
some of the criteria which contribute in reducing the production costs are (1) the detailed understanding of the
regulatory mechanisms which control the onset and the flux
of the pathways; (2) use of metabolic engineering techniques for the improvement of cellular activities by
manipulating regulatory enzymes; (3) the designing of low
cost bioreactors with minimum control systems; and (4) the
improvement in bioprocess techniques for continuous
accumulation and release of metabolites. These benchmarks should be focused on to provide momentum for
research in obtaining economically competitive yields.
Acknowledgments This study was supported by a grant from the
Korea Healthcare Technology R&D project, Ministry of Health and
Welfare, Republic of Korea (Grant No. A103017). Dr. H. N. Murthy
is thankful to the Ministry of Education, Science, and Technology,
Republic of Korea for the award of Brainpool Fellowship (131S-4-30523); this study was also supported by the Ministry of Science, ICT
and Planning (MSIP).

123

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