2 Murthy et al 2014 Secondary metabolites review murthy2014.pdf

REVIEW

Production of secondary metabolites from cell and organ cultures:strategies and approaches for biomass improvementand 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 com-

pounds 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 selec-

tion 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

controlled in the first stage of the culture process. Elicita-

tion, 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 stage-

specific strategies, it is possible to produce large amounts

of biomass with an increase in the accumulation of sec-

ondary compounds.

Keywords Bioreactor cultures � Elicitation �Immobilization � Permeabilization � Plant cell cultures �Secondary metabolites

Abbreviations

ABA Abscisic acid

BA Benzyladenine

B5 Gamborg’s 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

H. N. Murthy (&) � K.-Y. Paek (&)

Research Center for the Development of Advanced Horticultural

Technology, Chungbuk National University, Ch’ongju 361-763,

Republic of Korea

e-mail: [email protected]

K.-Y. Paek

e-mail: [email protected]

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, Ch’ongju 361-763, Republic of Korea

123

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-014-0467-7

Introduction

Secondary metabolites are a diverse group of organic

compounds that are produced by plants to facilitate inter-

action 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, col-

ors, 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 envi-

ronmental variations). Therefore, plant cells and cultures

have emerged as attractive alternatives for the production

of secondary metabolites (Ramachandra Rao and Ravi-

shankar 2002). In recent years, various strategies have been

developed for use in biomass accumulation and the syn-

thesis of secondary compounds, such as strain improve-

ment, optimization of medium and culture environments,

elicitation, nutrient and precursor feeding, permeabiliza-

tion, 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; how-

ever, 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 experi-

mental strategies used for the production of secondary

metabolites by plant cell and organ cultures, with relevant

examples.

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 baco-

side A varies among different genotypes of Bacopa mon-

nieri, ranging from 3.53 to 18.36 mg g-1 DW (Naik et al.

2012). Similarly, the amount of quinoline alkaloid cam-

ptothecin varies among different species such as Camp-

totheca acuminata, Camptotheca lowreyana, Camptotheca

yunnanensis, Ervatamia heyneana, Ophiorrhiza pumila,

Ophiorrhiza mungos, Ophiorrhiza rugosa, Nothapodytes

foetida, and Nothapodytes nimmoniana (0.03–0.4 % DW;

Ramesha et al. 2008). Accumulation of camptothecin var-

ies 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 0.33–0.77 % DW of cam-

ptothecin, 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 pro-

duction by clonal selection and visual screening has been

reported in Euphorbia millii and Daucus carota (Yamam-

oto and Mizuguchi 1982; Dougall 1980). However, selec-

tion 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

Table 1 Strategies to enhance the production of secondary metabo-

lites in plant cell and organ cultures

Stage 1

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


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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 UV–Vis 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 metab-

olites in cell suspensions and in vitro regenerated organs of

Hypericum perforatum (Pasqua et al. 2003).

Medium optimization

Several chemical and physical factors have been recog-

nized, 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 lev-

els, 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 estab-

lishing 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 0.75-

strength 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 gym-

nemic 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 nutri-

ents. 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 concen-

trations (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 (1–8 % w/v) tested in

Gymnema sylvestre cell cultures, a 3 % sucrose concen-

tration 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 accumula-

tion, whereas higher concentrations (i.e., 5 and 7 %

sucrose) favored the production of ginkgolides and bi-

lobalides (Park et al. 2004). In Bacopa monnieri shoot

cultures, a 2 % sucrose concentration was found to be the

optimal range tested (0–6 %, w/v) for biomass accumula-

tion, and the sucrose-free medium was shown to accumu-

late 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 pro-

duction 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 melon-

gena (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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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 mark-

edly 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 experi-

ment, 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 pro-

duction of gymnemic acid and withanolide A (Praveen and

Murthy 2013; Praveen et al. 2011; Praveen and Murthy

2011). Reduced nitrogen improved the production of cap-

saicin 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 phos-

phate level was shown to stimulate the synthesis of digi-

toxin 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 concen-

tration 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

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 sapo-

nins in ginseng, and kinetin stimulated the production of

anthocyanin in slender golden weed but inhibited the pro-

duction 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 hyo-

scyamine in henbane. In contrast, GA stimulated the pro-

duction 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,


123

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 (Kano-

kwaree 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 gin-

seng (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 phenylal-

anine ammonia-lyase induced via transfer to a fresh medium

decreased with increased inoculum size. Therefore, inocu-

lum 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

1–3 and 4–6 mm or unchopped) were responsible for the

lower DW and ginsenoside yield. The root inocula chopped

to 7–10 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, tempera-

ture, 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 17–25 �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 tem-

perature 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 corre-

lated 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 incu-

bated at 20/13 �C. However, total ginsenosides was opti-

mum (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 dif-

ferent environmental factors such as light intensity and

irradiance (continuous irradiance and continuous darkness)

on cell biomass yield and anthocyanin production in cul-

tures of Melastoma malabathricum. Moderate light inten-

sity (300–600 lx) induced an increase in the accumulation

of anthocyanins; the cultures exposed to 10 days of con-

tinuous darkness exhibited the lowest pigment content,

while the cultures exposed to 10 days of continuous


123

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 Hahl-

brock 1973), anthocyanins in Centaurea cyanus (Kakega-

wa 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 ginse-

nosides in ginseng hairy root cultures and reported that

hairy root growth was stimulated by red-light-incubated

cultures when compared to dark-incubated cultures. Fluo-

rescent irradiation enhanced the accumulation of ginseno-

sides (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 pro-

duction (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

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 con-

trolled 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 advanta-

geous 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 bio-

mass 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 vol-

atile 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 cell’s metabolic activities and

may decrease nutrient availability (carbon dioxide) from

the culture broth (Georgiev et al. 2009). Carbon dioxide is


123

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 produc-

tion 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 Ravishan-

kar 2011). These varied stress conditions have been des-

ignated 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 Ravi-

shankar 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 over-

production 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 cul-

tures; 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 gin-

senosides and Rb-group ginsenosides by 5- and 5.6-fold,

respectively were noted. By following the two-step meth-

odology, Yu et al. (2002) showed that it was possible to

achieve both biomass growth and ginsenoside accumula-

tion. 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 0–20 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 a-

linolenic 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

A major disadvantage of batch processes is that a sig-

nificant 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, semi-

continuous, 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 cul-

tivation or during the batch process. Continuous cultivation

includes variants without feedback control (e.g., chemo-

stats, 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 dis-

solved 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. Sim-

ilarly, 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 mem-

branes in a reversible manner with organic solvents. Organic

solvents such as isopropanol, dimethylsulfoxide (DMSO),

and polysaccharides (e.g., chitosan) have been used as per-

meabilizing 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 cul-

tures (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 consider-

ation 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 fol-

lowing: (1) extended viability of cells in the stationary

stage, thus, enabling the maintenance of biomass over a

prolonged time period; (2) simplified downstream pro-

cessing (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 Mis-

awa 1995). There are two major methods for cell immo-

bilization: (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, inex-

pensive, 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).


123

Several studies have shown that the dramatic effects of

immobilization on cells for secondary metabolite produc-

tion 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 ajmali-

cine accumulation from the gel-immobilized cells of Cof-

fea 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, liquid–solid 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 pro-

duction 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, poly-

dimethylsiloxane, and wofatite have been tested and suc-

cessfully used for the separation of secondary metabolites

in cell suspension cultures of several systems (Dornenburg

and Knorr 1995). Amberlite XAD-7 was found to effi-

ciently 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 stereospe-

cific chemical transformations that are catalyzed by bio-

logical 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 cul-

tures. The reaction carried out by such cultures involves

hydroxylation, glycosylation, glucosylation, oxidoreduc-

tion, 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. Chemi-

cal compounds, which can undergo biotransformations

mediated by plant enzymes, are variable in nature (e.g.,

aromatic, steroid, alkaloid, coumarin, terpenoid, and lig-

nin). 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 sub-

stances such as industrial byproducts into rare and expen-

sive 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 Ravi-

shankar (2000) used freely suspended and immobilized

cells of Capsicum frutescens for conversion of proto-

catechuic aldehyde and caffeic acids to vanillin and cap-

saicin. 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 suspen-

sion 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 alterna-

tives 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

extent than that by natural plants. For example, shoot

cultures were established in Bacopa monnieri for the pro-

duction 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 rhizogenes-

mediated 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 pro-

ducers of secondary metabolites; for example, the pro-

duction 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 pro-

duction 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 biore-

actor 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 world’s

largest plant cell culture facility was established in Ger-

many (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 suc-

cessful 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 shear-

stress 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

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 bubble-

free aeration of a culture. The system is placed on a hori-

zontal 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 suit-

able 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 estab-

lishment 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 produc-

tion from cell and organ cultures varies with the plant

species, type of cultures employed (for large-scale culti-

vation), the type of bioreactor(s) used, the method/mode of


123

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

Herein, we present the production cost of ginseng adven-

titious 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 5–7 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

under 70–80 % 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 7–8 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,

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

Table 2 The cost of production of ginseng adventitious roots com-

pared with field cultivated ginseng

Item Field cultivated

ginseng

Adventitious roots

obtained from

bioreactor cultivation

Yield (kg/0.1 ha) 523a 30,000b

Production cost (US $/kg) 35 47

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 Koreab Ginseng adventitious roots were cultured in four 10,000 l biore-

actors for 45 days and bioreactors were operated for 7–8 cycles per

year


123

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

water, operation, and depreciation of the machinery,

respectively. The quantum of biomass produced by biore-

actor 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 impor-

tance. The recent developments in plant tissue culture

techniques and bioprocessing have shown promising

results for notably improving biomass growth and pro-

ductivity. The optimization of medium ingredients and

culture environmental factors are the basic approaches that

should be ascertained with regard to individual plant spe-

cies in the first stage (i.e., at the flask level) of the culture

process. Various other parameters such as inoculum den-

sity, agitation/aeration, elicitation, nutrient feeding, pre-

cursor feeding, permeabilization, and immobilization

should also be investigated in small-scale bioreactor cul-

tures. 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 understand-

ing 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 pro-

duction 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 tech-

niques 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 bench-

marks 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-4-3-

0523); this study was also supported by the Ministry of Science, ICT

and Planning (MSIP).

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