Plant Cell Cultures Chemical Factories of Secondary Metabolites (Review)

Biotechnology Advances 20 (2002) 101 153
Plant cell cultures:

Chemical factories of secondary metabolites
S. Ramachandra Raoa,*, G.A. Ravishankarb
a
Laboratory of Biofunctional Materials, School of Materials Science, Japan Advanced Institute of Science and
Technology, 1-1, Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan
b
Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 013, India
Abstract
This review deals with the production of high-value secondary metabolites including
pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and
transgenic roots obtained through biotechnological means. Plant cell and transgenic hairy root cultures
are promising potential alternative sources for the production of high-value secondary metabolites of
industrial importance. Recent developments in transgenic research have opened up the possibility of
the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites. The
production of the pungent food additive capsaicin, the natural colour anthocyanin and the natural
flavour vanillin is described in detail. D 2002 Elsevier Science Inc. All rights reserved.
Keywords: Secondary metabolites; Pharmaceuticals; Plant cell cultures; Hairy root cultures; Biotransformation;
Immobilization
1. Introduction
For centuries, mankind is totally dependent on plants as source of carbohydrates, proteins
and fats for food and shelter. In addition, plants are a valuable source of a wide range of
secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavours, fragrances, colours, biopesticides and food additives. Over 80% of the approximately 30,000 known
natural products are of plant origin (Phillipson, 1990; Balandrin and Klocke, 1988; Fowler
* Corresponding author. Tel.: +81-761-51-1668; fax: +81-761-51-1665.

E-mail addresses: srrao@jaist.ac.jp, raovan@mailcity.com (S. Ramachandra Rao).
0734-9750/02/$ see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 7 3 4 - 9 7 5 0 ( 0 2 ) 0 0 0 0 7 - 1
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S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153
and Scragg, 1988). The number of known chemical structures is estimated to be nearly
fourfold greater than that in the microbial kingdom. In 1985, of the 3500 new chemical
structures identified, 2600 came from the higher plants. Worldwide, 121 clinically useful
prescription drugs are derived from plants (Payne et al., 1991). The surveys of plant
medicinal usage by the American public have shown an increase from just about 3% of
the population in 1991 to over 37% in 1998 (Brevoort, 1998). The US market sales of plant
medicinals have climbed to about US$3 billion per year (Glaser, 1999). Even today, 75% of
the worlds population relies on plants for traditional medicine. In the US, where chemical
synthesis dominates the pharmaceutical industry, 25% of the pharmaceuticals are based on
plant-derived chemicals (Payne et al., 1991; Farnsworth, 1985). Plants will continue to
provide novel products as well as chemical models for new drugs in the coming centuries,
because the chemistry of the majority of plant species is yet to be characterized (Cox and
Balick, 1994). The advent of chemical analyses and the characterization of molecular
structures have helped in precisely identifying these plants and correlating them with their
activity under controlled experimentation. Despite advancements in synthetic chemistry, we
Table 1
Plant-derived pharmaceuticals of importance
Product
Use
Plant species
Ajmalicine
Artemisinin
Ajmaline
Acinitine
Berberine
Camptothecin
Capsaicin
Castanospermine
Codeine
Colchicine
Digoxin
Diosgenin
Ellipticine
Emetine
Forskolin
Ginsenosides
Morphine
Podophyllotoxin
Quinine
Sanguinarine
Antihypertensive
Antimalarial
Intestinal ailment
Antitumour
Counterirritant
Glycoside inhibitor
Sedative
Antitumour
Heart stimulant
Steroidal precursor
Antitumour
Bronchial asthma
Health tonic
Sedative
Antitumour
Antimalarial
Antiplaque
Shikonin
Taxol
Vincristine
Vinblastine
Antibacterial
Anticancer
Antileukemic
Antileukemic
Cath. roseus
Artemisia annua
Ra. serpentina
Acotinum spp.
C. japonica
Camptotheca acuminata
Ca. frutescens
Castanospermum australe
P. somniferum
Colchium autumnale
Di. lanata
Dioscorea deltoidea
Orchrosia elliptica
Cephaclis ipecaccuanha
Coleus forskolii
Panax ginseng
P. somniferum
Podophyllum petalum
Cinchon. ledgeriana
Sanguinaria canadensis
P. somniferum
L. erythrorhizon
Taxus brevifolia
Cath. roseus
Cath. roseus
n/a: not available. Adapted from Ravishankar and Ramachandra Rao (2000).
Cost
(US$ per kilogram)
37,000
400
75,000
n/a
3250
432,000
750
n/a
17,000
35,000
3000
1000
240,000
1500
n/a
n/a
340,000
n/a
500
4,800
4500
600,000
2,000,000
1,000,000
103
still depend upon biological sources for a number of secondary metabolites including
pharmaceuticals (Pezzuto, 1995). Their complex structural features are difficult to synthesize.
We have listed some plant-derived pharmaceuticals in Table 1.
Elaborative pathways from basic primary metabolites, which are synthesized immediately
as a result of photosynthetic activity, produce secondary metabolites. Many of them are
unique to the plant kingdom and are not produced by microbes or animals. However, with the
advancement of transgenic research, it is possible to produce compounds and molecules,
which were also not originally synthesized in plants.
2. Need for a biotechnological approach

Biotechnology offers an opportunity to exploit the cell, tissue, organ or entire organism by
growing them in vitro and to genetically manipulate them to get desired compounds. Many
facets of biotechnological approaches can be envisaged (Table 2). Since the world population
is increasing rapidly, there is extreme pressure on the available cultivable land to produce
food and fulfill the needs. Therefore, for other uses such as production of pharmaceuticals and
chemicals from plants, the available land should be used effectively. Hence, it is appropriate
to develop modern technologies leading to plant improvement for better utilization of the land
to meet the requirements.
The development of micropropagation methods for a number of medicinal plant species
has been already reported and needs to be adopted (Naik, 1998). Cryopreservation of cells is
an area of importance in the conservation of medicinal plants. It has already been used in
many plant species. The development and adoption of plant cell culture and organ culture
methods have lead to the production of plant products on a large scale. This has been possible
Table 2
Aspects of biotechnological approaches to plant-derived secondary metabolites
1. Plant cell tissue and organ cultures
Cell culture
Shoot culture
Root culture
Scale-up of cultures
2. Transgenic plants/organisms
Metabolic engineering
Heterologous expression
Molecular forming
3. Micropropagation of medicinal plants
Endangered plants
High-yielding varieties
Metabolically engineered plants
4. Newer sources
Algae
Other photosynthetic marine forms
5. Safety considerations
104
by the combined efforts of cell biologists and chemical engineers. Chemical engineers are
currently developing improved and appropriate bioreactors for the improvement of production systems by adopting techniques of growth and metabolite production coupled with
downstream processing of the products. The improvements in molecular biological research
have given a new dimension to in vitro culture as well as for plant improvement, enhancing
the yields of the product and resulting in multiple products or producing novel products from
genetically engineered plants. Moreover, the need for safer drugs without side effects has led
to the use of natural ingredients with proven safety. These factors have laid emphasis on the
use of biotechnological methods to enhance the production of pharmaceuticals and food
additives, both in quality and quantity.
3. Plant cell culture as a source of secondary metabolites

Plant cell cultures are an attractive alternative source to whole plant for the production of
high-value secondary metabolites (Ravishankar et al., 1999; Dornenburg and Knorr, 1997;
Scragg, 1997; Alfermann and Petersen, 1995; DiCosmo and Misawa, 1995; Stockigt et al.,
1995; Endress, 1994; Ravishankar and Venkataraman, 1990) (Tables 3 and 4). Plant cells
are biosynthetically totipotent, which means that each cell in culture retains complete
genetic information and hence is able to produce the range of chemicals found in the parent
plant. The advantages of this technology over the conventional agricultural production are
as follows.
It is independent of geographical and seasonal variations and various environmental
factors.
It offers a defined production system, which ensures the continuous supply of products,
uniform quality and yield.
It is possible to produce novel compounds that are not normally found in parent plant.
Table 3
Groups of natural products that were so far isolated from tissue and suspension cultures of higher plants
Phenylpropanoids
Alkaloids
1.
2.
3.
4.
Anthocyanins
1. Acridines
Coumarins
2. Betalaines
Flavonoids
3. Quinolizidines
Hydroxycinnamoyl 4. Furonoqui nones
derivatives
5. Harringtonines
5. Isoflavonoids
6. Isoquinolines
6. Lignans
7. Indoles
7. Phenolenones
8. Purines
8. Proanthocyanidins
9. Pyridines
9. Stilbenes
10. Tropane
10. Tanins
alkaloids
Adapted from Stockigt et al. (1995).
Terpenoids
1.
2.
3.
4.
5.
Quinones
Steroids
Carotenes
1. Anthroquinones
1. Cardiac glycosides
Monoterpenes 2. Benzoquinones
2. Pregnenolone
Sesquiterpenes 3. Naphthoquinones derivatives
Diterpenes
Triterpenes
105
Table 4
Food additives from plant cell cultures
Product type
Colours
Anthocyanins
Betalaines
Crocin
Crocetins
Carotenoids
Anthraquinones
Naphthoquinones
Flavours
Vanillin
Garlic
Onion
Basmati
Citrus flavour
Cocoa flavour
Pungent food additive
Capsaicin
Sweeteners
Stevioside
Glycyrrhizin
Thaumatin
Essential oils
Mint oil
Chamomile oil
Jasmine oil
Aniseed oil
Plant species
Reference
V. vinifera
Euphorbia spp.
D. carota
Pe. frutescens
B. vulgaris
Cheno. rubrum
Crocus sativus
Gardenia jasminoides
Lycopersicon esculentum
Cinchon. ledgeriana
M. citrifolia
L. erythrorhizon
Pepin et al. (1995)

Yamamoto et al. (1982)
Rajendran et al. (1994)
Zhong and Yoshida (1995)
Klebnikov et al. (1995)
Berlin et al. (1986)
Sujata et al. (1990)
George and Ravishankar (1995)
Fosket and Radin (1983)
Robbins and Rhodes (1986)
Kieran et al. (1993)
Sim and Chang (1993)
Va. planifolia
Allium sativum
Allium cepa
Oryza sativa
Citrus spp.
Theobromo cacao
Dornenburg and Knorr (1996)

Ohsumi et al. (1993)
Collin and Masker (1988)
Suvarnalatha et al. (1994)
Cresswell (1990)
Townsley (1972)
Ca. frutescens
Ca. annuum
Lindsey and Yeoman (1984)

Johnson et al. (1990)
Stevia rebaudiana
Glycyrrhiza glabra
Thaumatococcus danielli
Swanson et al. (1992)

Hayashi et al. (1988)
van der Wel and Ledeboer (1989)
Mentha piperata
Ma. chamomilla
Jasmine officinale
Pim. anisum
Chung et al. (1994)

Kireeva et al. (1978)
Banthrope (1994)
Ernst (1989)
Adapted from Ramachandra Rao (1998).
It is independent of political interference.

Efficient downstream recovery and product.
Rapidity of production.
In addition, plant cell can perform stereo- and regiospecific biotransformations for the
production of novel compounds from cheap precursors.
There are a number of plant cell cultures producing a higher amount of secondary
metabolites than in intact plants (Table 5). However, there are still problems in the
production of metabolites by cell cultures resulting from the instability of cell lines, low
yields, slow growth and scale-up problems (Ravishankar and Venkataraman, 1993).
106
Table 5
High yields of secondary products
Product
Plant species
Yield (% D.W.)
Reference
Rosmarinic acid
Rosmarinic acid
Anthroquinones
Shikonin
Berberine
Jatrorhizine
Anthocyanins
Berberine
Diosgenin
Sanguinarine
Serpentine
Sa. officinalis
Col. blumei
M. citrifolia
L. erythrorhizon
Th. minus
Berberis wilsonae
Pe. frutescens
C. japonica
Diosc. deltoidea
P. somniferum
Cath. roseus
36.0
21.4
18.0
12.4
10.6
10.0
8.9
7.5
3.8
2.5
2.2
Hippolyte et al. (1992)

Ulbrich et al. (1985)
Zenk et al. (1975)
Fujita (1988)
Kobayashi et al. (1988)
Breuling et al. (1985)
Zhong et al. (1994)
Matsubara et al. (1989)
Sahai and Knuth (1985)
Park et al. (1992)
Zenk et al. (1977)
Adapted from Ravishankar and Ramachandra Rao (2000).
Several strategies have been adopted for the enhancement of metabolites (Table 6).
There has been tremendous success in the production of high-value secondary metabolites such as shikonin from cell cultures of Lithospermum erythrorhizon, berberine from
Coptis japonica and sanguinarine from Papaver somniferum. These are produced at
industrial levels.
4. Strategies to increase secondary metabolite production in plant cell cultures

During the past decade, a considerable progress has been made to stimulate formation
and accumulation of secondary metabolites using plant cell cultures (Ravishankar and
Ramachandra Rao, 2000; Dixon, 1999; Ravishankar and Venkataraman, 1993; Buitelaar
and Tramper, 1992; Fowler and Stafford, 1992; Payne et al., 1991). The adopted
strategies for enhancing the secondary metabolites of plant cell cultures have been
described in detail.
Table 6
Strategies to enhance production of secondary metabolites in plant cell cultures
1. Obtaining efficient cell lines for growth
2. Screening of high-growth cell line to produce metabolites of interest
a. Mutation of cells
b. Amenability to media alterations for higher yields
3. Immobilization of cells to enhance yields of extracellular metabolites and to facilitate biotransformations
4. Use of elicitors to enhance productivity in a short period of time
5. Permeation of metabolites to facilitate downstream processing
6. Adsorption of the metabolites to partition the products from the medium and to overcome feedback inhibition
7. Scale-up of cell cultures in suitable bioreactors
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4.1. Screening and selection of highly productive cell lines

Strain improvement begins with the choice of a parent plant with high contents of the
desired products for callus induction to obtain high-producing cell lines. Then, a screening of
the heterogeneous population for variant cell clones containing the highest levels of desired
product was employed.
The heterogeneity in the biochemical activity existing within a population of cells has been
exploited to obtain highly productive cell lines (Ogino et al., 1978). Selection can be easily
achieved if the product of interest is a pigment. For example, in cultures of L. erythrorhizon,
extensive screening of a number of clones resulted in a 1320-fold increase in shikonin
production (Fujita et al., 1984). Enhanced anthocyanin production by clonal selection and
visual screening has been reported in Euphorbia milli and Daucus carota (Yamamoto et al.,
1982; Dougall, 1980). Other techniques such as HPLC and RIA were also used to screen for
high-yielding cell lines (Matsumoto et al., 1980; Zenk, 1978).
Mutation strategies have also been employed in order to obtain overproducing cell lines.
The use of selective agents has been employed as an alternative approach to select highyielding cell lines (Rhodes et al., 1988). In this method, a large population of cells is exposed
to a toxic (or cytotoxic) inhibitor or environmental stress and only cells that are able to resist
the selection procedures will grow. p-Fluorophenylalanine (PFP), an analogue of phenylalanine, was extensively used to select high-yielding cell lines with respect to phenolics
(Berlin, 1980). Increased capsaicin and rosmarinic acid in PFP cell lines of Capsicum
annuum and Anchusa were reported (Salgado-Garciglia and Ochoa-Alejo, 1990; Quesnell
and Ellis, 1989). Other selective agents such as 5-methyltryptophan, glyphosate and biotin
have also been used to select high-yielding cell lines (Amrhein et al., 1985; Wataneba et al.,
1982; Widholm, 1974).
4.2. Manipulation of nutrients to improve yield
Manipulation of the culture environment must be effective in increasing the product
accumulation. The expression of many secondary metabolite pathways is easily altered by
external factors such as nutrient levels, stress factors, light and growth regulators. Many of the
constituents of plant cell culture media are important determinants of growth and accumulation of secondary metabolites (Stafford et al., 1986; Misawa, 1985).
4.2.1. Sugar levels
Plant cell cultures are usually grown heterotrophically using simple sugars as carbon
source and inorganic supply of other nutrients. The level of sucrose has been shown to affect
the productivity of secondary metabolite-accumulating cultures. Thus, sucrose concentrations of 2.5% (w/v) and 7.5% (w/v) in Coleus blumei media brought about rosmarinic acid
yields of 0.8 and 3.3 g/l, respectively (Misawa, 1985). For indole alkaloid accumulation in
cell culture as of Catharanthus roseus, 8% (w/v) sucrose was found to be optimal in the
tested concentration range of 4 12% (w/v) (Knobloch and Berlin, 1980). Yields of
benzophenanthridine alkaloids from suspension cultures of Eschscholtzia californica were
108
increased 10-fold to around 150 mg/l by increasing the sucrose concentration to 8% (w/v)
(Berlin et al., 1983). The osmotic stress created by sucrose alone and with other osmotic
agents were found to regulate anthocyanin production in Vitis vinifera cell suspension
cultures (Do and Cormier, 1990). A dual role of sucrose as carbon source and osmotic agent
was observed in Solanum melongena (Mukherjee et al., 1991). However, higher concentrations of sucrose at 5% (w/v) reduced the anthocyanin production in cell suspension
cultures of Aralia cordata, where 3% (w/v) favoured the anthocyanin accumulation
(Sakamoto et al., 1993).
4.2.2. Nitrate levels
Nitrogen concentration was found to affect the level of proteinaceous or amino acid
products in cell suspension cultures. The plant tissue culture medium such as MS, LS or B5
has both nitrate and ammonium as sources of nitrogen. However, the ratio of the ammonium/
nitratenitrogen and overall levels of total nitrogen have been shown to markedly affect the
production of secondary plant products. For example, reduced levels of NH4 + and increased
levels of NO3 promoted the production of shikonin and betacyanins, whereas higher ratios
of NH4 + /NO3 increased the production of berberine and ubiquinone (Bohm and Rink,
1988; Nakagawa et al., 1984; Fujita et al., 1981; Ikeda et al., 1977). Reduced levels of total
nitrogen improved the production of capsaicin in Capsicum frutescens, anthraquinones in
Morinda citrifolia and anthocyanins in Vitis species (Yamakawa et al., 1983; Yeoman et al.,
1980; Zenk et al., 1975). However, complete elimination of nitrate in cultures of Chrysanthemum cinerariaefolium induced twofold increases in pyrethrin accumulation in the
second phase of culture (Rajasekaran et al., 1991).
4.2.3. Phosphate levels
The phosphate concentration in the medium can have a major effect on the production of
secondary metabolites in plant cell cultures. Higher levels of phosphate were found to
enhance the cell growth, where it had negative influence on secondary product accumulation.
Sasse et al. (1982) have given a number of examples to show that a medium limited in
phosphate either induces or stimulates both the product and the levels of key enzymes leading
to the product. Thus, reduced phosphate levels induced the production of ajmalicine and
phenolics in Cath. roseus, of caffeoyl putrescines in Nicotiana tabacum and of harman
alkaloids in Peganum harmala. Similar results were obtained in a separate study for the
production of betacyanins in callus cultures of Beta vulgaris (Bohm and Rink, 1988). In
contrast, increased phosphate was shown to stimulate synthesis of digitoxin in Digitalis
purpurea and of betacyanin in Chenopodium rubrum and Phytolacca americana (Bohm and
Rink, 1988; Hagimori et al., 1982a,b).
4.2.4. Growth regulators
Growth regulator concentration is often a crucial factor in secondary product accumulation
(DiCosmo and Towers, 1984; Deus and Zenk, 1982). The type and concentration of auxin or
cytokinin or the auxin/cytokinin ratio alters dramatically both the growth and the product
formation in cultured plant cells (Mantell and Smith, 1984). The growth regulator 2,4-
109
dichlorophenoxyacetic acid (2,4-D) has been shown to inhibit the production of secondary
metabolites in a large number of cases. In such cases, elimination of 2,4-D or replacement of
2,4-D by naphthalene acetic acid (NAA) or indole acetic acid (IAA) has been shown to
enhance the production of anthocyanins in suspensions of Populus and D. carota, of
betacyanins in suspensions of Portulaca, of nicotine in suspensions of N. tabacum, of
shikonin in suspensions of L. erythrorhizon and of anthraquinones in M. citrifolia (Rajendran
et al., 1992; Seitz and Hinderer, 1988; Tabata, 1988; Sahai and Shuler, 1984; Bohm and Rink,
1988; Zenk et al., 1975). However, stimulation by 2,4-D has been observed in carotenoid
biosynthesis in suspensions of D. carota (Mok et al., 1976) and in anthocyanin production in
callus cultures of Oxalis linearis (Meyer and van Staden, 1995).
Cytokinins have different effects depending on the type of metabolite and species
concerned. Thus, kinetin stimulated the production of anthocyanin in Haplopappus gracilus
but inhibited the formation of anthocyanins in Populus cell cultures (Seitz and Hinderer, 1988;
Mok et al., 1976). Gibberellic acid and abscisic acid are reported to suppress production of
anthocyanins in a number of cultures (Bohm and Rink, 1988; Seitz and Hinderer, 1988).
4.2.5. Precursor feeding
Precursor feeding has been an obvious and popular approach to increase secondary
metabolite production in plant cell cultures. The concept is based upon the idea that any
compound, which is an intermediate, in or at the beginning of a secondary metabolite
biosynthetic route, stands a good chance of increasing the yield of the final product. Attempts
to induce or increase the production of plant secondary metabolites, by supplying precursor or
intermediate compounds, have been effective in many cases. The addition of phenylalanine as
a precursor led to improvement in rosmarinic acid yield in Col. blumei cell cultures (Ibrahim,
1987). Addition of phenylalanine to Salvia officinalis suspension cultures stimulated the
production of rosmarinic acid and decreased the production time as well (Ellis and Towers,
1970). Phenylalanine is also the precursor of the N-benzoylphenylisoserine side chain of
taxol, and supplementation of Taxus cuspidata cultures with phenylalanine resulted in
increased yields of taxol (Fett-Neto et al., 1994, 1993). Use of the distant precursor
phenylalanine and a near precursor such as isocapric acid resulted in enhanced capsaicin
content in cell cultures of Ca. frutescens (Lindsey and Yeoman, 1985; Yeoman et al., 1980).
Feeding of ferulic acid to cultures of Vanilla planifolia resulted in increase in vanillin
accumulation (Romagnoli and Knorr, 1988). Similarly, anthocyanin synthesis in carrot
cultures was restored by the addition of a dihydroquarcetin (naringen). Furthermore, addition
of leucine led to enhancement of volatile monoterpenes a- and b-pinine in cultures of Perilla
frutescens, whereas addition of geraniol to rose cell cultures led to accumulation of nerol and
citronellol (Mulder-Krieger et al., 1988).
4.3. Optimizing the culture environment
Culture environmental conditions such as light, temperature, medium pH and oxygen
have been examined for their effect upon secondary metabolite accumulation in many types
of cultures.
110
4.3.1. Temperature
A temperature range of 1725 C is normally used for the induction of callus tissues and
growth of cultured cells. However, each plant species may favour a different temperature.
Toivonen et al. (1992) found that lowering the cultivation temperature increased the total
fatty acid content per cell in dry weight. When the temperature was maintained at 19 C,
biotransformation of digitoxin to digoxin is favoured, whereas 32 C favours purpureaglycoside A formation in Digitalis lanata cell cultures (Kreis and Reinhard, 1992). Ikeda et al.
(1977) observed a higher yield of ubiquinone in tobacco cell cultures at 32 C when
compared to either 24 or 28 C. Courtois and Guren (1980) reported a 12-fold higher
production of crude alkaloids in cell cultures of Cath. roseus at 16 C as compared to the
normal 27 C.
4.3.2. Illumination
Accumulation of anthocyanin was strongly stimulated by light in cell cultures of D. carota
and Vitis hybrids (Seitz and Hinderer, 1988). Illumination was found to affect the composition
of sesquiterpenes in callus cultures of Marticaria chamomilla (Mulder-Krieger et al., 1988).
Exclusion of light in callus cultures of Citrus limon prompted the accumulation of
monoterpenes (Mulder-Krieger et al., 1988).
4.3.3. Medium pH
The medium pH is usually adjusted between 5 and 6 before autoclaving and extremes of
pH are avoided. The concentration of hydrogen ions in the medium changes during the
development of the culture. The medium pH decreases during ammonia assimilation and
increases during nitrate uptake (McDonald and Jackman, 1989). Photoautotrophic cell
suspension cultures of Cheno. rubrum showed that the increase in the external pH from
4.5 to 6.3 increased the cytosolic pH by 3.0 units and the vacuolar pH by about 1.3 units
(Husemann et al., 1992).
4.3.4. Agitation and aeration
The importance of aeration and agitation is crucial for large-scale production. Kreis and
Reinhard (1989) described that the influence of dissolved oxygen levels of 50% allowed an
alkaloid yield of around 3-g/l culture after 20 days of growth in an airlift bioreactor. Higher
aeration rates produced a dramatic decrease in alkaloid productivity. It is evident that airlift
and stirred tank bioreactors can allow similar secondary product levels in cultured plant
cells. However, in stirred tank vessels, the characteristics of the stirrer may be critical
(Kreis and Reinhard, 1989).
Ambid and Fallot (1981) investigated the effect of the composition of the gaseous
environment on production of volatiles by fruit suspension cultures. They reported that
the addition of carbon dioxide stimulated the synthesis of monoterpenes by Muscat grape
suspensions and induced the formation of linalool. Kobayashi et al. (1991) reported that
the use of carbon dioxide at the 2% level was critical to prevent cell browning and to
sustain berberine production in suspension cultures of Thalictrum minus in bubble
column reactors.
111
4.4. Elicitation
Plants produce secondary metabolites in nature as a defense mechanism against attack by
pathogens. Plants have been found to elicit the same response as the pathogen itself when
challenged by compounds of pathogenic origin (elicitors). Elicitors are signals triggering
the formation of secondary metabolites. Secondary pathways are activated in response to
stress. Biotic and abiotic elicitors are used to stimulate secondary metabolite product
formation in plant cell cultures, thereby reducing the process time to attain high product
concentrations and increased culture volumes (Barz et al., 1988; Eilert, 1987; DiCosmo and
Tallevi, 1985). Elicitors of fungal, bacterial and yeast origin, viz. polysaccharides,
glycoproteins, inactivated enzymes, purified curdlan, xanthan and chitosan, and salts of
heavy metals were reported for the production of various secondary metabolites (Ramachandra Rao et al., 1996a,b; Rajendran et al., 1994; Guo et al., 1992; Furze et al., 1991;
Johnson et al., 1991; Robbins et al., 1991; DiCosmo et al., 1987; Funk et al., 1987).
Treatment of P. somniferum cell suspensions with a homogenate of Botrytis mycelium
resulted in a remarkable accumulation of sanguinarine of up to 3% of the cell dry weight
(Constabel, 1990). The treatment of root cultures of Datura stramonium with copper and
cadmium salts has been found to induce the rapid accumulation of high levels of
sesquiterpenoid defensive compounds (Furze et al., 1991).
4.5. Permeabilization
In most cases, the products formed by plant cell cultures are stored in the vacuoles. In
order to release the products from vacuoles of plant cells, two membrane barriers plasma
membrane and tonoplast have to be penetrated. Cell permeabilization depends on the
formation of pores in one or more of the membrane systems of the plant cell, enabling the
passage of various molecules into and out of the cell. The permeability of the cells can be
monitored by measuring the activity of enzymes of the primary metabolism, viz.
hexokinase, glucose 6-phosphate dehydrogenase, isocitrate dehydrogenase, malic and
citrate synthetase (Brodelius, 1988b). Attempts have been made to permeabilize the plant
cells transiently, to maintain the cell viability and to have short time periods of increased
mass transfer of substrate and metabolites to and from the cell (Parr et al., 1987; Brodelius
and Nilsson, 1983). A wide variety of permeabilizing agents are used to enhance the
accessibility of enzymes or to provoke release of intracellular stored product (Berlin et al.,
1989; Knorr et al., 1985; Parr et al., 1984; Felix, 1982). Organic solvents such as
isopropanol, dimethylsulfoxide (DMSO) and polysaccharides like chitosan have been used
as permeabilizing agents (Van Uden et al., 1990; Beaumont and Knorr, 1987; Knorr and
Teutonico, 1986). Other permeabilization methods include ultrasonication, electroporation
and ionophoretic release, in which the cells are subjected to a low current in a specially
designed device (Brodelius et al., 1988). In addition, using high electric field pulses and
ultrahigh pressure (Dornenburg and Knorr, 1993) has been reported for the recovery of
secondary metabolites.
112
4.6. In situ product removal

A low accumulation of secondary compounds in cell cultures in a number of cases
may not be due to a lack of key biosynthetic enzymes but rather due to feedback
inhibition, enzymatic or nonenzymatic degradation of the product in the medium or
volatility of substances produced. In such cases, it should be possible to increase the net
production by the addition of an artificial site for product accumulation, for example, by
use of second solid or liquid phase introduced into the aqueous medium. Such threephase systems accumulate traces of secondary metabolites from the culture medium.
The use of in situ product removal of metabolites has a number of key potential
advantages beyond promoting secretion. The removal and sequestering of the product in
a nonbiological compartment may increase its total production (Beiderbeck and
Knoop, 1988).
Robbins and Rhodes (1986) reported that addition of amberlite XAD-7 resin stimulated
the production of anthraquinones by 15 times compared to a medium without adsorbent.
Addition of charcoal led to 2060-fold improvements in yields of coniferyl aldehyde in
Ma. chamomilla suspensions (Beiderbeck and Knoop, 1987). The addition of Miglyol or
silica gel RP8 stimulated ethanol production in cell cultures of Pimpinella anisum
(Mulder-Krieger et al., 1988). The addition of XAD-4 increased the vanilla flavour
production in Vanilla fragrans cell suspension cultures (Knuth and Sahai, 1991).
4.7. -Cyclodextrins
Many plant cell cultures hardly convert precursors in the presence of organic phases,
often as a result of dramatic decrease of cell viability. Therefore, the enzymatic activities
are reduced in these systems. A new approach to solve this problem of bioconversion of
water-insoluble precursors is to combine the advantages of apolar systems (higher
solubility if the substrate) and aqueous systems (maintenance of cell viability) by carrying
out bioconversions in the presence of clathering agents such as cyclodextrins. Cyclodextrins have the ability to form stable inclusion complexes with natural spices and
flavour substances in their cyclodextrin cavity (Haggin, 1992). Cyclodextrins may be
modified through substituting various functional compounds on the primary or secondary
phase of the molecule. The chemically modified cyclodextrins are more water soluble
than native cyclodextrins (Eastburn and Tao, 1994; Szejtli, 1988). Through complexation,
the physical properties of ligands are changed, including their solubility in aqueous media
(Qi and Hedges, 1995; Van Uden et al., 1994; Szejtli, 1986, 1982). The hydroxylation of
b-estradiol to 4-hydroxy and 2-hydroxy b-estradiol was enhanced in the presence of
b-cyclodextrin in Mucuna pruriens cell cultures (Woerdenbag et al., 1990a) and multistep
conversion of coniferyl alcohol into podophyllotoxin was demonstrated in Podophyllum
hexandrum cell suspension cultures (Woerdenbag et al., 1990b). Cyclodextrin complexation of flavours provides protection against the damaging factors of the environment (Qi
and Hedges, 1995).
113
5. Organ cultures as a source of pharmaceuticals

Since production of secondary metabolites is generally higher in differentiated tissues,
there are attempts to cultivate shoot cultures and root cultures for the production of
medicinally important compounds. These organ cultures are relatively more stable (Roja,
1994). There are a number of medicinal plants whose shoot cultures have been studied for
metabolites (Table 7). Similarly, root cultures are valuable sources of medicinal compounds
(Table 8). Root systems of higher plants generally exhibit slower growth and are difficult to
harvest. Hence, alternative methods need to be found for root-derived compounds. Until now,
however, there is no commercial process as an alternative for root-derived compounds, except
in case of utilizing hairy root culture systems, which has been described in this review.
6. Hairy root cultures for pharmaceuticals

The ability of Agrobacterium rhizogenes to induce hairy roots in a range of host plants has
lead to studies on it as a source of root-derived pharmaceuticals (Flores et al., 1999). Tepfer
(1990) summarized 116 plants belonging to 30 dicotyledonous families wherein hairy roots
have been induced. Hairy roots are induced by transfer of T-DNA from the plasmid of Agr.
rhizogenes (Ambros et al., 1986; Petit et al., 1983) to host tissue, resulting in root formation
by virtue of auxin synthesis genes coded by bacterial DNA. The Ri plasmid of Agr.
rhizogenes also elicits the synthesis of opines such as agropine or mannopine. The transformed nature of the roots is genetically checked by opine detection or Southern analysis.
The interest in hairy roots is mainly due to their ability to grow fast without needing an
external supply of auxins. Many times, they do not need incubation under light. They are
fairly well stable in metabolite yield due to their genetic stability. Because of these
Table 7
Shoot cultures of medicinal plants
Plant species
Product
Reference
Artemi. annua
Atro. belladona
Begonia spp.
Cath. roseus
Cinchona spp.
Artemesinin
Atropine
Vindoline
Vinblastine
Quinine
Cardenolides
Cardenolides
Essential oils
Kutkin
Steviosides
Withanolides
Indirubin
Alkaloids
Park et al. (1989)

Benjamin et al. (1987)
Takayama and Misawa (1981)
Staba and Chung (1981)
Krueger et al. (1982)
Hirata et al. (1987)
Lui and Staba (1979)
Hagimori et al. (1982a,b)
Charlwood and Moustou (1988)
Upadhyay et al. (1989)
Akita et al. (1994)
Heble (1985)
Shim et al. (1998)
Konishi et al. (1998)
Di. lanata
Di. purpurea
Pelargonium tomentosum
Picrprrhiza kurroa
Ste. rebaudiana
Withania somniferum
Polygonum tinctorium
Decentra pergrina
114
Table 8
Root cultures of medicinal plants
Plant species
Product
Reference
B. vulgaris
Bidens alba
Calystegia sepium
Coreopsis tinctoria
Hyoscamus albus
Hyoscamus muticus
Hemidesmus indicus
Artemisia absynthium
Polygonum tinctorium
Betalaines
Polyacetylenes
Tropane alkaloids
Phenylpropanoids
Hyoscyamine
Hyoscyamine
2-hydroxy-4-methoxybenzaldehyde
Volatile oils
Indigo, Indirubin
Hamill et al. (1986)

Norton and Towers (1986)
Jung and Tepfer (1987)
Thron et al. (1989)
Hashimoto and Yamada (1986)
Flores et al. (1987)
Sreekumar et al. (1998)
Kennedy et al. (1993)
Shim et al. (1998)
Adapter from Payne et al. (1991).
advantages, many of the root-derived plant products once not considered feasible for
production by cell culture are being reinvestigated for production using the hairy root culture
technology. A number of such examples are given in Table 9. Several factors influence the
yield of secondary metabolites of pharmaceutical interest in hairy root cultures. These are
nutrients, elicitors and biotransformations of precursors to products and genetic manipulation
through the Ri plasmid of Agr. rhizogenes.
Several hairy roots have been put to scale-up studies in bioreactors. However, due to their
structural features and metabolite localization characteristics, they need different type of
reactors than the ones used for plant cell cultures. Hairy roots need anchorage for growth
Table 9
Hairy root cultures producing pharmaceutical products of interest
Plant species
Product
Reference
Bidens spp.
Cinchon. ledgeriana
Cichorium intybus
Datura spp.
Cassia spp.
Duboisia leichhardtii
Echinacea purpurea
Glycyrrhiza uralensis
Hy. albus
Panax ginseng
Salvia miltorrhiza
Artemi. absynthium
L. erythrorhizon
Ra. serpentina
Rubia cordifolia
Gl. glabra
Panax ginseng
Hy. muticus
Polyacetylenes
Quinolene alkaloids
Esculetin
Tropane
Anthroquinones
Tropane alkaloids
Alkaloids
Glycyrrhizin
Alkaloids
Saponin
Diterpenes
Volatile oil
Shikonin
Ajmaline, serpentine
Anthroquinones
Isoprenylated flavonoids
Ginsenoside
Hyoscyamine
McKinely et al. (1993)

Hamill et al. (1987)
Bais et al. (1999)
Rhodes (1989)
Ko et al. (1988)
Mano et al. (1989)
Trypsteen et al. (1991)
Ko et al. (1989)
Shimomura et al. (1991)
Yoshikawa and Furuya (1987)
Hu and Alfermann (1993)
Kennedy et al. (1993)
Shimomura et al. (1986)
Benjamin et al. (1994)
Shin and Kim (1996)
Asada et al. (1998)
Kunshi et al. (1998)
Sevon et al. (1998)
115
since they are highly branched (Fig. 1). Various configurations of hairy root bioreactors are
given in Fig. 2. Certain hairy roots are tolerant to submerged cultivations. Taya et al. (1989)
found that stirred tank reactors are poor for cultivation of B. vulgaris. In our experience, hairy
roots of B. vulgaris do not produce betalaines when submerged totally. In shake flask cultures, they proliferate fast and produce betalaines after achieving a stationary phase of growth.
Air-sparging without impeller-driven agitation may also be helpful as in the case of hairy
roots of Nicotiana rustica (Rodriguez-Mendiola et al., 1991). We have found that use of airsparging system is suitable for chicory hairy roots for esculin and esculetins production (Bais
et al., 1999). Therefore, any single design of reactor will not be suitable for all hairy roots.
Most remarkable developments of scale-up in large vessels have been in the cultivation of
Panax ginseng hairy root biomass in a 20-ton cultivation tank (Scheidegger, 1990).
Hairy roots also produce valuable compounds by biotransformations (Table 10). Recently,
an innovative approach of coculture of hairy roots of Atropa belladona and shooty teratomas
of scopolamine-rich Duboisia plants was done, wherein hyoscyamine released by the former
was bioconverted to scopolamine by the latter tissues (Subroto et al., 1996). However, these
systems need to be studied carefully within parameters of uptake mechanisms, biosynthetic
potential of the tissue and, if possible, the extracellular release of the final product. Thus,
hairy root technology will be useful in not only producing root-derived compounds but also
to produce novel compounds by biotransformations.
The ability to engineer the genomic DNA of hairy root through tailormade Ri plasmid will
be a tremendous use in producing novel compounds. Using this systems, it may be possible to
Fig. 1. Highly branched hairy root cultures of Cichorium intybus.
116
Fig. 2. Configuration of hairy root bioreactors.
produce more than one product with a single type of hairy root. There already are attempts in
this direction as exemplified by the introduction of cDNA of ornithine decarboxylase (ODC)
117
Table 10
Biotransformations using hairy root cultures for production of pharmaceuticals
Plant species
Substrate
Product
Reference
Hy. niger
Hyoscyamine
Scopolamine
Cinchon. ledgeriana
Nicotiana spp.
Tryptophan
Lysine, cadaverine
Quinine
Nicotine
Putrescine, agmatine
Putrescine
Spermidine
Cadaverine
2-phenylpropanoic acid
Digitoxigenin
Hyoscyamine
Hyoscyamine
18b-glycyrrhetinic acid
Butylated hydroxytoluene
Phenolic acid
Anabasine
Scopolamine
Hyoscyamine
Hashimoto and Yamada

(1983)
Hay et al. (1986)
Walton and Belshaw
(1988)
Walton et al. (1988)
Yoshioka et al. (1989)
Duboisia myoporoides
Panax ginseng
Panax ginseng
Atro. belladona
Dubo. leichhardtii
Panax ginseng
Bidens sulphureus
Panax ginseng
Sugar esters
Digitoxin
Scopolamine
Scopolamine
Glucosides
Stilbene quinones
Glycosylated phenolic
compounds
Furuya et al. (1989)

Kawaguchi et al. (1990)
Subroto et al. (1996)
Subroto et al. (1996)
Asada et al. (1993)
Flores et al. (1994)
Ushiyama and Furuya
(1989)
from yeast into N. rustica. ODC enhances the formation of alkaloid nicotine since it is the
first step of pyrrolidine alkaloid biosynthesis. Hyoscyamine 6b-hydroxylase (H6H) enzyme
that catalyzes epoxidative conversion of hyoscyamine to scopolamine is an important step in
the biogenetic pathway (Yamada and Hashimoto, 1988), which has been induced in Atro.
belladona by the H6H gene from Hyoscyamus niger. The hairy roots of Atro. belladona
produced high amounts of scopolamine (Hashimoto et al., 1993). Such approaches would
enhance the utility of hairy roots to produce novel compounds. Most importantly, these
compounds are produced at shorter periods of time than in intact plants since the growth cycle
in vitro is shorter than in vivo conditions.
7. Production of foreign proteins in transgenic plants

Recently, transgenic plants are considered to be economically competitive production
systems for the manufacture of variety of foreign proteins. These include recombinant
antibodies and antibody fragments (James and Lee, 2001; Sharp and Doran, 2001; Doran,
2000; Smith and Glick, 2000; Wongsamuth and Doran, 1997; Tavladoraki et al., 1993; Hein
et al., 1991; Hiatt et al., 1989), enzymes such as b-glucuronidase (Kurata et al., 1998) and
invertase (Verdelhan des Molles et al., 1999) and proteins of therapeutic value such as human
interleukin (IL)-2 and IL-4 (Magnuson et al., 1998), ribosome-inactivating proteins (Remi
shih et al., 1998; Francisco et al., 1997), ricin (Sehnke and Ferl, 1999) and human
a1-antitrypsin (Terashima et al., 1999a,b). The most commonly used host species for protein
synthesis in suspension cultures is tobacco (Sharp and Doran, 1999; Liu and Lee, 1999;
Kurata et al., 1998; Wongsamuth and Doran, 1997), although rice cell suspension cultures
118
have also been used (Terashima et al., 1999a,b). Hiatt et al. (1989) estimated that plantderived antibodies are likely to cost less than the ones derived from hybridoma cells and that
plant cell media are composed of simple sugars and salts less complex than mammalian
media. Consequently, purification of secreted protein is simpler and more economical. Plant
cell-derived proteins are likely to be safer than those derived from other systems, since plant
cell pathogens are not harmful to humans. The expression of these heterologous proteins in
plants has an advantage over their expression in the microbial system, since full-length
antibodies have been produced in plants (De Wilde et al., 1996). On the other hand,
Escherichia coli produced only a monovalent Fab fragment, the largest antibody found in
a microbe (Smith, 1996).
Hairy roots of tobacco (N. tabacum) were used to produce full-length murine IgG1
monoclonal antibody and the maximum production of 18 mg/l was reported in 21-day grown
shake flasks, and up to 14% of the antibody was secreted into the medium. The decline in
antibody production was found after 14 days of cultivation and mainly due to its degradation.
Antibody production by transgenic hairy roots has a negligible effect on growth compared
with hairy roots of wild-type tobacco (Wongsamuth and Doran, 1997). The loss of protein
due to product instability during plant cell culture and subsequent purification significantly
influences the product yields due to occurrence of unfavourable environmental conditions.
Protein stability following secretion improved with the addition of appropriate chemical
stabilizers. Increased recovery of a mouse monoclonal antibody heavy chain by the addition
of DMSO, gelatin and polyvinylpyrrolidone (Magnuson et al., 1998; Wongsamuth and
Doran, 1997) was reported. The other new protein stabilizer, bacitracin, enhanced the cell
growth (Sharp and Doran, 1999) and inhibit the degradation of plant-derived proteins
(Bateman et al., 1997).
Full-length antibodies were first expressed by Hiatt et al. (1989). They have expressed a
monoclonal antibody 6D4 from a mouse hybridoma cell line in tobacco. The k and g
chains of the IgG molecule was expressed with or without mammalian leader sequence.
The transgenic lines upon crossing produced plants with expression of both the chains.
However, the plants without leader sequence did not show assembled gk complexes and
hence lacked functional characteristics. The use of plant-derived endoplasmic reticulum
(ER) targeting sequence has been studied by During et al. (1990), with demonstration of
accumulation of antibody in ER of transgenic tobacco. De Wilde et al. (1996) found that
both full-length IgG molecules and Fab fragments accumulated in the intercellular spaces of
mesophyll cells in leaves of the transgenic Arabidopsis thaliana. Their study with
immunolocalization confirms the production of full-length antibody and its secretion by
plant cells. Moreover, in plants, single cells are able to assemble secretory antibodies,
whereas two different cell types are required in mammals. Ma et al. (1995) have obtained
transgenic N. tabacum that expressed a murine monoclonal antibody k chain, a hybrid
IgAIgG heavy chain, a murine joining in chain and a rabbit secretory component. These
chains were assembled into functional, high-molecular-weight secretory immunoglobulins
that recognized the native Streptococcal antigen I/II, a surface adhesion molecule. Plant
cells also possess the requisite mechanism for the assembly and expression of other
complex recombinant protein molecules. The ability to produce monoclonal antibodies in
119
plants also led to the opportunity to develop an inexpensive method of mucosal

immunoprotection against genital herpes (Zetlin et al., 1998). The expression of the
production of a full-length functional antibody in algal forms has also been reported
(Conrad and Fiedler, 1994). Smith (1996) states that the folding of full-size immunoglobulins in algae is possibly due to its less reducing cytoplasm. This cytoplasm contains
chaperonins, which facilitate folding.
Ma et al. (1994) have studied the prospects of using plant-produced antibodies in passive
immunization against the cell surface antigen of Streptomyces mutans, which cause tooth
decay. The fact that plant-derived antibodies have been approved for clinical trials (Ma and
Hein, 1995) is an encouraging development. However, R&D of large-scale antibody
production in plants should be pursued by developing systems of higher expression using
easy and efficient downstream processing and the large-scale cultivation of transgenic plants
in controlled greenhouses to drive this exciting research area into the realm of application.
The storage of antibodies for extended periods can be made possible by expressing them in
seeds (Fiedler and Conrad, 1995).
7.1. Vaccines
The ability to express genes encoding the antigens of bacterial and viral pathogens in
plants, with retention of immunogenic properties, is bound to revolutionize vaccine
production. The concept of edible vaccine stemmed from the landmark discovery made by
Mason et al. (1992). Plant systems will be a choice, which may be relatively less expensive
for production as well as storage of antigens.
Strategies for candidate vaccine production are of two types: (1) stable genomic
integration with foreign DNA introduced either by Agrobacterium T-DNA vectors or by
microprojectile bombardment and (2) transient expression using viral vectors (Mason and
Arntzen, 1995). The first system affords stable transformation, which may be heritable.
Furthermore, the expression could be targeted to specific loci using appropriate promoters.
The second system involves the use of viral vectors for transient expression in plants, a
potentially useful means of producing high levels of recombinant antigens. Foreign protein
expression could be achieved either by foreign gene transcription using a subgenomic
promoter or the fusion of foreign proteins to peptides, which contain the capsid protein
that normally coats the virus (Mason and Arntzen, 1995). High levels of a-trichosanthin,
an antiviral protein in transformed plants (Kumagai et al., 1993), have been obtained by
the foreign gene transcription method. Turpen et al. (1995) have developed a method of
expressing the coat promoter of the tobacco mosaic virus (TMV) using peptides of
epitopes derived from malarial sporozoites. The antigenicity measured by ELISA and
Western blot demonstrate that the recombinant coat protein recognized appropriate
monoclonal, antimalarial antibodies. Similarly, Cowpea mosaic virus has also been used
for the expression of foreign peptides such as antigens from the rabies virus and HIV-1
(Yusibov et al., 1997). Stable genomic transformation encoding foreign antigens has been
demonstrated in case of hepatitis B surface antigens as well as E. coli and Vibrio cholerae
(Thanavalla et al., 1995).
120
One can produce edible vaccines by cloning the genes of fruit plants and expressing the
vaccines in the edible parts of the fruit. However, it is necessary to address the problem
such as oral tolerance (Mahon et al., 1998) and the standardization of dosage requirement
for the scale of protection. These topics need to be studied along with ecological
considerations on a case-by-case basis to get approval for each vaccine production and
administration to target groups.
8. Immobilization of plant cells for the production of secondary metabolites

Improvement in the secondary metabolite production of cell cultures is often associated
with the organization and differentiation of plant cells. The concept of organization and
differentiation led to the use of immobilization technology, which has long been used for
microbes and enzymes. Immobilization is defined as a technique, which confines a
catalytically active enzyme or cells on a fixed support and prevents its entry into liquid
phase (Yeoman et al., 1990; Yeoman, 1987; Fowler, 1986; Lindsey and Yeoman, 1985,
1983a,b). Immobilized plant cells have been used for single and multistep biotransformations of precursors to desired products as well as for the de novo biosynthesis of
secondary metabolites.
Ever since, Brodelius et al. (1979) described the entrapment of viable cells of Cath. roseus,
M. citrifolia and Di. lanata in calcium alginate gel and this technique has received much
attention. Extensive reviews on various aspects of plant cell immobilization are available
(Williams and Mavituna, 1992; Payne et al., 1991; Scragg, 1991; Hulst and Tramper, 1989;
Brodelius 1988a; Hall et al., 1988; Lindsey and Yeoman, 1987).
Immobilization of plant cells has distinct advantages as biocatalyst over the immobilized
enzyme system. It is necessary to provide the immobilized enzyme with proper pH, the flow
of reaction mixture temperature and as supply of cofactors. Immobilized enzymes are
generally applied to single-step reactions. Furthermore, there will be loss in activity during
isolation of enzymes from the organism. The advantage of immobilized enzyme is the high
rate of activity. In contrast to immobilized enzymes, immobilized cells have distinct
advantages: (a) it can carry out multienzyme operations; (b) by selecting highly biosynthetic
cells, catalytic activity can be enhanced; (c) there is no need to provide cofactors since cells
themselves produce them and (d) immobilized cells can be easily handled as compared to
immobilized enzymes. Thus, immobilized cells are gaining much importance as biocatalysts.
The following prerequisites are essential to adopt immobilization for secondary metabolite
production (Payne et al., 1991).
The product of interest should not be strictly growth associated.
Growth of cells should be suppressed to prevent disintegration of the immobilization
matrix, which may lead to disruption of the process.
Immobilized cells should maintain prolonged viability and biosynthetic capacity with high
rates of sustainable secondary metabolite production.
The product should leach out of the cells and beads into the medium.
121
The most widely used technique involves the entrapment of cells, in some kind of gel or
combination of gels, which are allowed to polymerize around them (Novais, 1988).
Calcium alginate is the most widely used matrix. Besides this, other gels such as agar,
agarose, gelatin, carrageenan and polyacrylamide have also been used (Nilsson et al.,
1983). However, gels of alginate are most widely used because of their simplicity and
relatively lack of toxicity. The other alternative supports are polyurethane foam and hollowfibre membranes.
Table 11 gives a number of examples of the systems of immobilization, which have been
used with plant cells together with the associated plant species and their products.
Table 11
Immobilized plant cell systems used for production of secondary metabolites
Plant species
Substrate/precursor Product
Immobilization method Reference
Bioconversions
Cath. roseus
Di. lanata
D. carota
N. tabacum
P. somniferum
Cathenamine
Digitoxin
Digitoxigenin
Keto esters
Codeinone
Ajmalicine
Digoxin
Periplogenin
Hydroxy esters
Codeine
Agarose
Alginate
Alginate
Alginate
Polyurethane foam
L-Tyrosine
Ferulic acid,
vanillylamine
L-DOPA
Vanillin,
capsaicin
Alginate
Alginate
Felix et al. (1981)

Brodelius et al. (1979)
Jones and Veliky (1981)
Naoshima and Akakabe (1989)
Furuya et al. (1984)
Furusaki et al. (1988)
Wichers et al. (1983)
Ajmalicine
Alginate, agarose
Capsaicin
Polyurethane foam
Brodelius and Nilsson (1980)

Lindsey and Yeoman (1983b)
Caffeine
Caffeoyl
putrescine
Membrane
Alginate
Lang et al. (1990)

Berlin et al. (1989)
Capsaicin
Ajmalicine
Phenolics
Pigments
Diosgenin
Berberine
Thiophenes
Anthraquinones
Polyurethane foam
Alginate, agarose
Hollow fibres
Polyurethane foam
Polyurethane foam
Alginate
Alginate
Alginate
Johnson (1993)
Brodelius and Nilsson (1980)
Prenosil and Pedersen (1983)
Ishida (1988)
Kobayashi et al. (1987)
Ketel et al. (1987)
Muc. pruriens
Capsicum spp.
Synthesis from precursors

Cath. roseus
Tryptamine,
secologanin
Ca. frutescens
Isocapric acid,
vanillylamine,
valine and
ferulic acid
Coffea arabica
N. tabacum
Theobromine
Phenylalanine
De novo synthesis
Ca. frutescens
Cath. roseus
Glycine max
Lavandula vera
Diosc. deltoidea
Th. minus
Tagetes patula
M. citrifolia
122
Table 12
Dramatic effects of immobilization on secondary metabolite production in plant cell cultures
Plant species
Product
Fold change
Ca. frutescens
Ca. frutescens
Tag. patula
Coffea arabica
Cath. roseus
Cath. roseus
Capsaicin
Capsaicin
Thiophenes
Methylxanthin
Ajamlicine
Total alkaloids
Muc. pruriens
L-DOPA
Salvia miltiorrhiza
Cryptotanshinone
> 100 (I)

> 100 (I)
Ca 20 (I)
13 (I)
3.5 (I)
No de novo
synthesis
No de novo
synthesis
2.5 (D)
Type of
immobilization
Reference
Foam
Gel
Natural glass
Gel
Gel
Membrane

Ravishankar et al. (1988)
Hulst and Tramper (1989)
Haldimann and Brodelius (1987)
Asada and Shuler (1989)
Payne et al. (1988)
Gel
Wichers et al. (1983)
Gel
Miyasaka et al. (1986)
(I): increase, (D): decrease. Adapted from Payne et al. (1991).
Immobilization can have a dramatic impact on cellular physiology and secondary product
formation. The more dramatic responses are summarized in Table 12.
9. Biotransformations for the production of secondary metabolites

Biotransformation can be defined as a process through which the functional groups of
organic compounds are modified either stereo- or regiospecifically by living cultures,
entrapped enzymes or permeabilized cells to a chemically different product.
The production of high-value food metabolites, fine chemicals and pharmaceuticals can
be achieved by biotransformations using biological catalysts in the form of enzymes and
whole cells (Ravishankar and Ramachandra Rao, 2000; Krings and Berger, 1998; Meyer
et al., 1997; Scragg, 1997; Berger, 1995; Cheetham, 1995). The range of flavour metabolites and pharmaceuticals produced by plant cell cultures through biotransformation is
shown in Table 13.
Cell suspension cultures, immobilized cells, enzyme preparations and hairy root cultures
can be applied for the production of food additives or pharmaceuticals by biotransformation
process (Table 14). There are two main reasons to choose plant cells for biotransformation
purposes. Firstly, these cells are generally able to catalyze the reactions stereospecifically,
resulting in chirally pure products. Secondly, they can perform regiospecific modifications
that are not easily carried out by chemical synthesis or by microorganisms (Hamada and
Furuya, 1996; Pras et al., 1995; Stockigt, 1993; Pras, 1992; Stepan-Sarkissan, 1991). These
reactions include reduction, oxidation, hydroxylation, acetylation, esterification, glucosylation, isomerization, methylation, demethylation, epoxidation, etc. The presence of biotransformation potential in plant cells is a necessary condition for practical application.
However, for a successful and viable process, the following prerequisites must be met
(Steck and Constabel, 1974).
123
Table 13
Biotransformation of flavour compounds by plant cell culture systems
Plant species
Substrate
Product
Reference
Citrus limon
Citrus paradisi
N. tabacum
Mentha spp.
Valencene
Valencene
Linalool
Pulegone
Menthol
Steviol
Geranial
Neral
Citronellal
Ferulic acid
Nootkatone
Nootkatone
8-hydroxylinalool
Isomenthone
Neomenthol
Stevioside
Geraniol
Nerol
Citronellol
Vanillin
Drawert et al. (1984)

Drawert et al. (1984)
Suga and Hirata (1990)
Aviv and Galun (1978)
Aviv et al. (1981)
Furuya (1978)
Lappin et al. (1987)
G. jasminoides
Ferulic acid
Isoeugenol
and eugenol
Acetophenone
Vanillin
Isoeugenyl b-rutinoside and
eugenyl b-glucoside
(S)-aromatic alcohol
Curcuma zedoaria
Gl. glabra
Coffea arabica
Papaver bracteatum
Germacrone
Glycyrrhitinic acid
Vanillin
Linalyl acetate
Achillea millefolium
N. tabacum
Borneol, menthol
thymol and farnesol
Carvone
Guaiane-type sesquiterpenes
Hydroxyglycoside esters
Vanillin glucosides
Linalool, a-terpineol
Geraniol
Many products
Cath. roseus
N. tabacum
Cath. roseus
Cath. roseus
Coffea arabica
Euc. perriniana
Euc. perriniana
Euc. perriniana
Cymbidium spp.
Piperitone
Pulegone
Geraniol
Glycyrrhizin
Capsaicin
Menthol
Camphor
Borneol
Menthyl acetate
Ste. rebaudiana
Lavandula angustifolia
Va. planifolia
Ca. frutescens
Eucalyptus perriniana
Dihydrocarvone
neodihydrocareol
Hydroxypiperitone
Menthone 4-hydroxymenthone
10-hydroxygeraniol
Glycyrrhetic acid
Capsaicin glucoside
Menthol 3-O-b-gentiobiosides
Camphor glucosides
( ) Borneol b-gentiobioside
Menthol
Romagnoli and Knorr

(1988)
Orihara et al. (1992)
Akakabe and Naoshima
(1993)
Sakui et al. (1992)
Hayashi et al. (1992)
Kometani et al. (1993a)
Hook et al. (1990)
Figueiredo et al. (1996)
Hirata et al. (1982)
Hamada and Furuya (1996)
Hamada and Nakata (1992)
Kometani et al. (1993b)
Orihara and Furuya (1993)
Mironowicz et al. (1987)
Adapted from Ramachandra Rao (1998).
(1) The culture must have the necessary enzymes. (2) The substrate or precursor must not
be toxic to the culture. (3) The substrate must reach the cellular compartment of the cell. (4)
The rate of product formation must be faster than its further metabolism.
9.1. Advantages of biotransformations
The advantages include the production of novel compounds, enhancement in the
productivity of desired compound and overcoming the problems associated with chemical
synthesis. Importantly, the studies on biotransformation lead to basic information to elucidate
124
Table 14
Biotransformations using plant cell cultures for production of pharmaceuticals
Plant species
Substrate
Product
Reference
P. somniferum
Di. purpurea
Di. lanata
Euc. perriniana
Podo. hexandrum
Muc. pruriens
Cath. roseus
Codeine
Digoxin
Digoxin
Taxol derivatives
Podophyllotoxin
DOPA
Vincrisitne
Wilhelm and Zenk (1997)

Alfermann et al. (1980)
Alfermann et al. (1980)
Hamada et al. (1996)
Van Uden et al. (1995)
Pras et al. (1993)
DiCosmo and Misawa (1995)
Capsaicin, vanillin
Capsaicin, vanillin
Ca. frutescens
Thebaine
Digitoxin
Digitoxin
Taxol
Coniferyl alcohol
Tyrosine
Vindoline
Vinblastine
Ferulic acid
Vanillylamine
Protocatechuic aldehyde
Caffeic acid
Digitoxin
Spirulina platensis
Ca. frutescens
Codeine
Isoeugenol
Ca. frutescens
Ca. frutescens
Digoxin,
purpureaglycoside A
Morphine
Vanillin, capsaicin
Ramachandra Rao and

Ravishankar (2000a)
Ramachandra Rao et al.
(2002)
Ramachandra Rao et al. (1999)
Ramachandra Rao and
Ravishankar (1999)
the biosynthetic pathway, and catalysis can be carried out under mild conditions, thus
reducing undesired by-products, energy, safety and costs.
10. Scale-up of plant cell cultures

Since plant cells produce unique pharmaceuticals, which can be harnessed, they need to be
produced in large-scale bioreactors. Configuration of bioreactors used for microbial cells
cannot always be utilized directly for plant cells, owing to distinctive features, which are not
favourable for plant cell cultivation. Plant cells are less stable in productivity, highly shear
sensitive, exhibit low oxygen requirements (ca. 1-mmol O2 at 10 6 cells) slow growth
(doubling time 25110 h) and often occur as cell clumps of 24-mm diameter. Different
Table 15
Bioreactor types used for plant cell cultures
Reactor type
Plant species
Reference
Stirred tank
Cath. roseus
N. tabacum
N. tabacum
M. citrifolia
L. erythrorhizon
Cath. roseus
Cath. roseus
Ruta vulgaris
Drapeau et al. (1986)

Kato et al. (1977)
Noguchi et al. (1977)
Wagner and Vogelmann (1977)
Tanaka (1987)
Morris et al. (1984)
Shuler et al. (1983)
Shuler and Hallsby (1985)
Bubble column
Airlift stirred
Aerated
Fluidized bed
Tubular membrane
Disc turbine
125
Table 16
Large-scale suspension cultures reactors for plant cell cultures
Plant species
Product
Bioreactor capacity
Reference
Cath. roseus
Col. blumei
L. erythrorhizon
N. tabacum
Serpentine
Rosmarinic acid
Shikonin
Biomass
Biomass
Saponins
Biomass
Biomass
Biomass
100 l airlift
300 l airlift
750 l agitated
20,000 l agitated
1500 l bubble column
20,000 l agitated
750 75,000 l agitated
Smart and Fowler (1981)

Rosevear (1984)
Tabata and Fujita (1985)
Kato et al. (1976)
Noguchi et al. (1977)
Ushiama et al. (1986)
Ritterhaus et al. (1990)
Panax ginseng
E. purpurea
Ra. serpentina
Panax ginseng
Adapted from Sahai (1994).
configuration of bioreactors has been adopted depending on the nature of cells. Few
representative references are made in Table 15. Several studies are available for large-scale
cultivation of plant cells (Table 16).
Cell/tissue types such as cell suspension cultures, immobilized cells and hairy roots
have been very ideal for scale-up. However, the configuration of reactors will be different
for different cell or tissue types. No one design could be recommended as a common
one. The most important contribution, however, is to produce not only the biomass but
also the metabolite in an economical manner. In the present state of art, any compound
less than US$ 1000 per kilogram is difficult to produce. Hence, it is advantageous
to utilize bioreactor technology for compounds, which are costlier and has a higher
market demand.
10.1. Scale-up of biotransformations
So far, a few reports are available on scale-up of biotransformation. In the food area, the
development of a process for making high-fructose corn syrup by immobilized glucose
isomerase was reported. In the pharmaceutical industry, the first biotransformation was the
production of modified penicillin by immobilized penicillin acylase using single-step
biotransformation (West, 1996). Biotransformation of 12b-hydroxylation of methyldigitoxin
to methyldigoxin and other metabolites such as deacetayllanatoside C by Di. lanata cell
Table 17
Biotransformation of b-methyldigitoxin to b-methyldigoxin by Di. lanata cells in 20-l reactor
Precursor
Productivity (g)
% Conversion
b-methyldigitoxin added
Unconverted b-methyldigitoxin
b-methyldigoxin formed
By-product
Yield
17.24
2.04
14.36
0.28
100
11.8
81.7
1.4
94.9
Adapted from DiCosmo and Misawa (1995).
126
cultures was reported (Kreis and Reinhard, 1992; 1990; Reinhard et al., 1989). A
biotransformation process for the production of digoxin was developed using Di. lanata
in cell suspension cultures using 20- and 300-l airlift bioreactors. The results of the study
indicated that maximum digoxin was reached in 8% (w/v) glucose medium, and about 80%
of the digoxin produced was found in the medium (Table 17). Panda et al. (1992)
demonstrated the alkaloid production by precursor feeding using stirred tank bioreactors
in plant cell cultures of Holarrhena antidysentrica.
11. Food additive production from plant cell cultures studied in the authors laboratory
11.1. Capsaicin
Capsaicin (Fig. 3) is a pungent principle of green pepper (Capsicum spp.). Capsaicin
preparations are used as a counterirritant in lumbago, neuralgia and rheumatic disorders.
Taken internally, Capsicum preparation has a tonic and carminative action especially useful
in dyspepsia. Capsicum oleoresin is added to tannin or rose gargles for pharyngitis and
relaxed sore throat. Capsaicin is administered as a bacteriostatic (Gal, 1965) or fungistatic
compound in the form of powder, tincture, liniment, plaster, ointment and medicated wool. In
the US, two commercial creams, viz. Zostrix and Axsain, are formulated with purified
capsaicin for alleviation of arthritis pain (Anonymous, 1991). Commercial production of
capsaicin by separation from Capsicum oleoresin and its subsequent purification would
involve several steps.
Capsaicin can be produced by immobilized cell cultures, which leach out to the medium,
facilitating easy separation and purification. The cultivation of the Capsicum crop takes 45
months and therefore cannot offer a continuous production procedure. In contrast, immobilized cell cultures of Capsicum offer a continuous method of producing capsaicin under in
vitro controlled conditions. There are several reports of capsaicin production in immobilized
cell cultures (Johnson et al., 1990, 1991; Ravishankar et al., 1988; Mavituna et al., 1987;
Lindsey and Yeoman, 1984). Our attempts to enhance the level of capsaicin by treating
immobilized cell cultures of Ca. frutescens with elicitors (Johnson et al., 1990) and
permeability agents (Johnson et al., 1991) or by manipulating culture conditions (Johnson,
1993) have been successful. An innovative method of capsaicin production by immobilizing
the placenta, the site of synthesis of capsaicin (Iwai et al., 1979), was reported by Johnson and
Ravishankar (1996). Capsaicin production increased several folds higher in immobilized
Fig. 3. Chemical structure of capsaicin.
127
placenta than in chilly fruit. With precursor biotransformation using coumaric acid, it
registered an 11-fold increase (Johnson and Ravishankar, 1996).
Here, we provide capsaicin production in 2-l airlift culture vessel using immobilized cells/
placenta. An airlift culture vessel of cylindrical shape was constructed with Corning glass of
the configuration given in Fig. 4.
11.2. Inputs of operation
1. 20-day-old Capsicum cells to placenta (100-g fresh weight).
2. Alginate and calcium chloride solution required to envelop 100-g cells/placenta in
beads. One litre of 2.5% (w/v) sodium alginate with cells extruded into 2 l of 0.9% (w/v)
calcium chloride dihydrate.
3. Beads washed with water were transferred into the vessel containing 1 l of MS
medium supplemented with 3% sucrose, 2-mg/l 2,4-D and 0.5-mg/l kinetin [standard
medium (SM)].
4. Airflow (2:1 mixture of CO2 + air for the initial 7 days of culture and 4:1 for the latter
7 days of production) at a rate of 4 V.V.M.
5. Incubation at 25 2 C under continuous light of 2000 lx.
6. pH adjustment to 5.8 during culture.
7. Replenishment of entire medium after 7 days with fresh medium.
8. Capsaicin recovery and analysis in 2 weeks of culture with two harvests at 7-day intervals.
Fig. 4. Schematic representation of column reactor process for capsaicin production using immobilized cell
cultures of Ca. frutescens.
128
11.3. Yield components

The airlift culture vessel was run with immobilized cells or placenta for 2 weeks under
three different conditions using (1) SM, (2) SM + elicitor or (3) SM + precursor ( p-coumaric
acid). The results are presented below.
11.3.1. Yields in SM
1. Immobilized cells: 35 mg/100-mg fresh cells/15 days or 0.4% on dry weight basis
2. Immobilized placenta: 300 mg/100-g fresh placenta/15 days or 0.1% on dry
weight basis
11.3.2. Yields in SM+elicitor
Elicitors used were curdlan at 8-mg/l concentration for immobilized cells and Rhizopus
oligosporus mycelial extract equivalent to 2.5 g of mycelium/l of medium administered for
placental immobilized cultures.
1. Immobilized cells: 76 mg/100-g fresh cells/15 days or 0.95% on dry weight basis
weight basis
11.3.3. Yields in SM+precursor (2.5-mM coumaric acid)
1. Immobilized cells: not done
weight basis
By our method, we are able to get a maximum production of 1.15 g/l. However, increases
can be obtained using continuous cultivation with adsorption of capsaicin and recycling of
medium to minimize the cost. The plant may not be viable if it is less than 10,000-l capacity.
We have recently studied capsaicin production in bubble column and packed-bed reactor at
1-l level, which needs further scale-up and its performance to be evaluated vis-a`-vis alginate
immobilized system (Table 18).
Apart from the production of capsaicin from immobilized cell cultures of Ca. frutescens,
attempts have been made in our laboratory to produce vanilla flavour metabolites from
Capsicum-immobilized cultures upon feeding of phenylpropanoid intermediates protocatechuic aldehyde, caffeic acid (Ramachandra Rao and Ravishankar, 2000a), ferulic acid,
coniferyl aldehyde and veratraldehyde (Ramchandra Rao and Ravishankar, 1998). The
feeding of phenylpropanoid precursors not only produced the vanilla flavour metabolites
but also increased the yields of capsaicin (Ramachandra Rao, 1998; Ramachandra Rao and
Ravishankar, 2000a). Feeding of clove principle, isoeugenol, to immobilized cultures of Ca.
frutescens showed formation of vanilla flavour metabolites (Ramachandra Rao and
Ravishankar, 1999).
129
Table 18
Comparison of continuous production of capsaicin from bubble column and packed-bed reactor
Bioreactor
Productivity
Bubble column
Biomass density
Average productivity
Average specific productivity
Maximum duration of continuous operation
Total production
Packed-bed reactor
Biomass density
Average specific productivity
Maximum duration of continuous operation
Total production
Production under fungal elicitation
100 g fresh weight/l

1.7 mg/l/day
17 mg/g fresh weight/day
21 days
35 mg/l in 21 days
380 g/l
7.4 mg/l/day
19.5 mg/g fresh weight/day
28 days
207 mg/l in 28 days
330 mg/l in 28 days
Adapted from Madhusudhan (1998).
11.4. Vanillin
Vanillin (Fig. 5) is the most universally accepted aroma chemical used in processed foods,
pharmaceutical products and perfumeries (Prince and Gunson, 1994; Anonymous, 1993;
Webster, 1995). It occurs in the vanilla bean at a level of 2% dry weight, and it is associated
with many other compounds. Approximately 12,000 tons of vanillin is consumed annually,
from which only 20 tons are extracted from the vanilla beans (Dornenburg and Knorr, 1996;
Feron et al., 1996; Berger, 1995). The cost of the pure natural vanillin flavour is priced at US$
4000 per kg, while the synthetic equivalent costs about 12 US$ per kg (Krings and Berger,
1998; Berger, 1995). Other than flavour qualities, use of vanillin as antimicrobial against molds
and yeast has been described (Lopez-Malo et al., 1995; Cerrutti et al., 1997; Cerrutti and
Alzamora, 1996). Vanillin was found to be an antioxidant (Burri et al., 1989) and will be useful
in the development of health foods. Vanillin is also known for its antimutagenic activity
(Kometani et al., 1993a) and glucosylvanillin has been found to impart distinctive flavour note,
hence would be useful in tonics and syrups. Interestingly, at the 8th FAOBMB Congress,
Iekhsan et al. (1998) reported the discovery of use of vanillin as antidote for neutralizing the
toxic effect of toxin from Chinorex fleckeri (boxjellyfish). There are a number of cases of
poisoning by boxjellyfish, and often, it is fatal to individuals. The findings of Iekhsan et al.
(1998) hence provide newer utility of vanillin as a pharmaceutically important compound.
Fig. 5. Chemical structure of vanillin.
130
Production of vanillin by conventional method by cultivating Vanilla plants is tedious and

expensive. Though vanilla flavour has its own utility as flavouring, the use of vanillin
obtained through biotechnology assumes significance since there is worldwide demand for
natural compounds. In this context, vanillin and its other flavour metabolites are being
produced through biotransformation to provide alternate method of production of biovanillin
through cheaper substrates (Table 19). The increase in production of vanillin and its other
flavour metabolites has been made possible by several factors using optimization by
enhanced substrate availability mediated by cyclodextrin (Ramachandra Rao and Ravisha-
Table 19
Biotransformations leading to vanillin formation through microbial and plant cell cultures
Organism bioconverting the substrate
Ferulic acid
Aspergillus niger
Pycnoporous cinnabarinus
Pseudomonas fluorescens
Pseudomonas fluorescens AN 103
Corynebacterium glutamicum
Ca. frutescens
Spir. platensis
Haemotococcus pluvialis
Va. planifolia
Va. planifolia
Eugenol
Corynebacterium glutamicum
Pseudomomnas spp.
Arthrobacter globiformis
Spir. platensis
Isoeugenol
Enterobacter spp.
Serratia spp.
Spir. platensis
Ca. frutescens
Vanillylamine
Asper. niger
E. coli
Spir. platensis
Ca. frutescens
Coniferyl aldehyde
Ca. frutescens
Spir. platensis
Coniferyl aldehyde
Vanillyl alcohol
Ca. frutescens
Spir. platensis
Penicillum simplicissimum
Reference
Lesage-Messen et al. (1996)
Andreoni et al. (1995)
Narbad and Gasson (1998)
Labuda et al. (1993)
Ramachandra Rao (1998)
Ramachandra Rao et al. (1996a)
Usha et al. (1999)
Romagnoli and Knorr (1988)
Funk and Brodelius (1990)
Tadasa and Kayahara (1983)
Rabenhorst (1996)
Cooper (1987)
Rabenhorst (1991)
Ramachandra Rao and Ravishankar (1999)
Yoshida et al. (1997)
Sudhakar Johnson et al. (1996)
Markus et al. (1992)
Ramachandra Rao and Ravishankar (1998)
Fraaije et al. (1997)
131
nakar, 1998), elicitation of bioconversion process using microbial elicitors (Ramachandra

Rao, 1998) and in situ adsorption of products using adsorbents, viz. activated charcoal (Knuth
and Sahai 1991; Westcott et al., 1994) and amberlite XAD-4 and XAD-7 (Ramachandra Rao
and Ravishankar, 2000b), to overcome the feedback inhibition, thereby enhancing yield and
recovery. The formation of vanilla flavour metabolites from various phenylpropanoids in cell
cultures of C. frutescens is shown in Fig. 6
11.5. Anthocyanin
Anthocyanins are the most ubiquitous pigments seen in nature, widely distributed in the
pericarps of several fruits, flowers and vegetables. They are glycosylated polyhydroxy and
polymethoxy derivatives of flavylium (2-phenylbenzopyrrolium) salts belonging to the group
Fig. 6. Vanilla flavour metabolites formation from various phenylpropanoid precursors in Ca. frutescens
cell cultures.
132
Table 20
Effect of antioxidants on lipid peroxidation
Compound
IC50
Butylated hydroxyanisole
Butylated hydroxytoluene
a-tocopherol
Anthocyanin from carrot cell cultures
0.125
0.250
0.500
0.005
Adapted from Narayan et al. (1999).
of two or three portions the aglycone base, a group of sugars and a group of acyl acids. The
aglycone moiety is referred to as anthocyanidin. There are more than 15 anthocyanidins
(Francis, 1982). Anthocyanins are widely used a colourants. However, they are known to
have several pharmacological attributes such as antiinflammatory, antiulcer and woundhealing properties (Koichi and Hasashi, 1990; Vega et al., 1987). We have recently
established the antioxidant properties of anthocyanin obtained through cell culture (Narayan
et al., 1999). Culture-derived anthocyanin effectively prevented autooxidation of lipids as
well as lipid peroxidation in biological systems (Table 20). Our studies have shown high
anthocyanin production in mutated cell cultures of D. carota (Table 21). Chromatographic
analyses of anthocyanin extract showed the presence of cyanidin xylosyl galactose, cyanidin
monoglucose and cyanidin galactose in the molar ratio of 5:3:2. The chemical structure of
cyanidine-3-glucoside and delphinidin-3-glucoside is shown in Fig. 7.
Presently, the commercial source of anthocyanins is the grape peel in view of its quoted
price of US$ 12001500 per kilogram (Ilker, 1987) and estimated market of US$ 200
million. Plant cell cultures are an alternative source of anthocyanin. The possibility of
obtaining novel anthocyanin with two to three times greater stability at slightly acidic pH has
been demonstrated using D. carota cells (Vunsh et al., 1986) Research in our laboratory has
shown that anthocyanin production in carrot cultures is stimulated by indole-3-acetic acid
Table 21
Growth and production of anthocyanin in D. carota cell cultures
Parameters
Growth
Specific growth rate
Doubling time
Growth index
Maximum cell density
Dry weight/fresh weight ratio
Packed cell volume
Anthocyanin production
Average content
Specific productivity ( qp)
Total production
Adapted from Madhusudhan (1998).
Value
0.217/day
3.2 days
12.2
18.92 1.17 g/l
0.085
0.95 ml/g fresh weight
15.58% (w/w) dry mass
10.52 mg/g/day
3.1 g/l in 15 days
207 mg/l/day
133
Fig. 7. Chemical structure of anthocyanin.
(Rajendran et al., 1992). Elicitation of anthocyanin by fungal elicitor has been useful to
enhance the yields by 1.25-fold using Aspergillus flavus mycelial extract (Rajendran et al.,
1994). Similarly, its elicitation by phycocyanin was also reported (Ramachandra Rao et al.,
1996a,b). The cell aggregate size of 500850 mm was critical for maximum anthocyanin
production (Madhusudhan and Ravishankar, 1996).
The high yields obtained by several researchers have resulted in exciting possibility of
scale-up of anthocyanin production systems. In the future, anthocyanins will be used for
antioxidant effect as well as their value as colours. Developments in genetic engineering
of anthocyanin production have been elegantly reviewed by Guttason (1994). There is a
possibility of obtaining plants with flowers of different hues. This aspect may be
adopted for cell culture production. However, to date, no enhancement of hue has
been demonstrated.
12. Metabolic engineering of biochemical pathways

It is envisaged that the use of plant cell culture approach for pharmaceutically important
compounds can be made industrially applicable only upon the overproduction of metabolites
or the production of novel compounds that too with the expression of more than one
compounds of interest in a plant. This is not realized by routine cell line selection and other
parameters of growth/production media development. Therefore, there is a need to study the
metabolic pathways and its regulatory steps for overexpressing the regulatory genes for ratelimiting steps. The recent advancement in plant cell and tissue culture techniques and the
genetic transformation of plants using Agrobacterium-mediated or direct gene transfer make
possible the incorporation of foreign genes into plants producing new genotypes of desirable
traits. Thus, genetically engineered or modified plants can be used as green bioreactors for
the production of value-added chemicals (Saito et al., 1992).
The progress of genetic manipulation largely depends on (a) the ability to obtain genes
encoding desirable traits, (b) the proper alignment of the expressed gene with its endogenous
substrate and the compartmentation/transport of its product, (c) the successful transformation
and reproducible regeneration of transgenic plants, (d) the proper site of gene integration
combined with the level and patterns of gene expression and (e) the evaluation of the altered
metabolic pathways.
134
12.1. Molecular strategies

Strategies for overproduction of metabolites by genetic manipulation can be as follows.
1.
2.
3.
4.
5.
Overproduction of precursors of secondary metabolites (e.g. A).

Overexpression of the gene product, which limits the specific pathway (e.g. B).
Creating a new branch for a preexisting pathway (e.g. F to C).
Down-regulation of existing reaction using antisense methods (blocking, e.g. C to E).
Manipulation of regulatory genes whose products may bind DNA and function as
transcriptional activators or repressors.
6. Selection of regulatory mutants with increased tissue-independent expression for in
vitro production of metabolites.
7. Sometimes, the tissue/organ-specific expression by using appropriate promoters is
required to target the metabolite production. This would result in metabolic energy
saving by localized production of metabolites.
Studies related to the importance of plants by metabolic engineering or transgenic
protocols are presented here, where success has been obtained for enhancement of yields.
12.2. Tryptophan decarboxylase (EC 4.1.1.28)
Tryptophan decarboxylase catalyses the decarboxylation of L-tryptophan to tryptamine.
Tryptamine is a substrate for strictosidine synthase (Stockigt and Zenk, 1977). Interestingly,
cDNA clone from Cath. roseus encoding tryptophan decarboxylase has been expressed in
tobacco plants (Songstad et al., 1991), which resulted in the increased levels of tryptamine
and tyramine as predicted. This study has been extended to metabolically engineer Brassica
napus, wherein tryptophan, which is generally bioconverted to glucosinolate (an undesirable
product of oil seed cake, which renders the product unpalatable by cattle), is diverted away by
expressing tryptophan decarboxylase gene of Cath. roseus. In such a situation, transgenic B.
napus produced low amount of glucosinolates, making the oil seed cake palatable to cattle
(Chavadej et al., 1994).
12.3. Strictosidine synthase (EC 4.3.3.2)
Strictosidine synthase catalyzes the stereospecific condensation of primary amino group
of tryptamine and aldehyde group of iridoid glycoside secologanin to form the indole
135
alkaloid strictosidine. Strictosidine synthase is of biotechnological interest in the biomimetic

synthesis of monoterpenoid indole alkaloids because the reaction product is a mixture of
the diastereomers.
The cDNA encoding strictosidine synthase was isolated from Rauwolfia serpentina and it
has been functionally expressed in E. coli, Saccharomyces cerevisiae and insect cells using a
baculovirus vector (Kutchan, 1989; Kutchan et al., 1988). Strictosidine synthase from Cath.
roseus has been expressed in tobacco (McNight et al., 1990).
12.4. H6H (EC 1.14.11.11)
The biosynthesis of scopolamine from hyoscyamine is catalyzed by H6H. This enzyme
catalyzed hydroxylation and epoxidation reactions (Robbins et al., 1994).
The cDNA encoding H6H from Hy. niger was introduced into Atropa belladona using
either Agrobacterium tumefaciens- or Agr. rhizogenes-mediated transformation. The resultant
Atropa transgenic plants contained high levels of scopolamine (Yun et al., 1992). This is a
first example of metabolic engineering to produce medicinally useful compound.
12.5. Berberine bridge enzyme (EC 1.5.3.9)
Berberine bridge enzyme catalyses the stereospecific conversion of N-methyl group
(S)-reticuline into (S)-scoulerine (Kutchan, 1995). Direct conversion of N-methyl group into
a methylene bridge moiety is not easily achievable presently by synthetic chemistry. It is of
interest to note that berberine bridge enzyme is an elicitor-induced yeast cell wall (Schumacher et al., 1987), thereby providing possibility of overproduction of berberine in cell
cultures. Moreover, cDNA encoding the berberine bridge enzyme has been isolated by
Dittrich and Kutchan (1991) in elicited cell suspensions of Es. californica (California poppy)
and expressed heterologously into insect cultures using baculovirus expression vector, which
resulted in the production of sufficient quantities.
12.6. Berbamunine synthase
Berbamunine synthase (EC 1.1.3.34) catalyzes the formation of the ether linkage between
one molecule of (R)-N-methylcoclaurine and one molecule of (S)-N-methylcoclaurine to form
the bisbenzylisoquinoline alkaloid berbamunine. The cDNA encoding berbamurine synthase
was isolated from B. stolonifera cell suspension cultures and expressed in insect culture using
a baculovirus expression vector (Kraus and Kutchan, 1995).
12.7. Polyphenol oxidase (PPO)
PPO is ubiquitous in higher plants and its regulation provides several advantages. Downregulation of PPO increases the quality attributes of crop plant-derived commodities,
whereas overexpression reduces danger from pests. It has been concluded that no phenotypic
136
effects are visible in transgenic tomato or tobacco plants with high or low PPO levels
(Steffens et al., 1994).
12.8. p-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL)
Higher plants possess the phenylpropanoid pathway for the production of lignin and some
secondary phenolic compounds. Coumaric acid and ferulic acids and their CoA esters are
intermediates in these routes. Expression of the bacterial gene for the side chain cleavage
enzyme in plants may offer the potential to generate vanillin and related flavour compounds
in a range of crops by diversion of phenylpropanoid metabolism. At IFR, attempts have been
made to produce vanillin in hairy root cultures of Dat. stramonium by expressing the HCHL
gene from Pseudomonas fluorescens AN103. The expression of the HCHL gene in Dat.
stramonium hairy root cultures caused substantial changes in the content of p-hydroxybenzoic acid in the form of glucosides and glucose esters and along with traces of glucosides of
p-hydroxybenzyl alcohol and vanillic acid. The potential of this line to biotransform added
ferulic acid to vanillin is being studied (Mitra et al., 1999).
12.9. 4-coumarate-CoA ligase (4CL)
4CL (EC 6.2.1.12) catalyses a branch-point reaction (conversion of hydroxycinnamic acids
into the corresponding CoA esters) between the central phenylpropanoid pathway and
pathways leading to various secondary metabolites such as flavonoids, lignans, lignin and
related phenylpropanoid compounds (Holton and Cornish, 1995; Douglas, 1996). Brodelius
and Xue (1997) isolated and characterized 4CL from Va. planifolia cell suspension cultures.
Based on studies using the inhibitors of 4CL (Funk and Brodelius, 1990), they concluded that
the expression of antisense mRNA for 4CL gene would redirect the flow of phenylpropanoid
precursors from lignin biosynthesis into flavour compound biosynthesis in Va. planifolia.
Thus, genetic engineering for metabolic alteration or overexpression of pathway has much
to offer. There are exciting possibilities to produce high-value, low-volume pharmaceutical
products such as the ones described here. Moreover, one can imaginatively produce novel
compounds by expression of foreign genes. The questions regarding environmental study of
transgenic plants and regulatory considerations of administration of the phytopharmaceuticals
derived from them has not been dealt since it is beyond the scope of this review.
13. Conclusion
Though plant cell cultures can produce a whole range of secondary metabolites, there are
few success stories of production at commercial scale. The prospect of production of highcost, low-volume products such as anti-HIV and anti-cancer compounds is very high, thus
putting this technology to a position of being able to make a commercial impact in a few
selected pharmaceuticals. The advancement of knowledge in phytochemistry, regulation of
secondary pathways and ability to express desired traits by transgenics is expected to drive
137
technology to produce a range of pharmaceutical and healthcare products. Vaccines and

antibodies from plants are already fast-advancing fields, with investments from multinational
companies. They aim to provide protection against diseases of all sections of society, since
the poor have been deprived of expensive immunization schedules.
Use of transgenic plants for the production of pharmaceuticals will be bound by the IPR
protection. Those who can invest in the R&D will benefit in the era of processes and patents.
Developing countries, which are still behind in recombinant DNA research, may not be able
to develop their own complete technologies. Nations rich in biodiversity and knowledge base
of their local vegetation may protect their plants and develop cultivation methods. The
technique of micropropagation by in vitro culture methods will be a handy tool, which does
not need high levels of biotechnology. The R&D of production of secondary metabolites
through biotechnology has made a beginning and is expected to provide disease-remedial or
disease-preventive molecules at affordable costs for the benefit of mankind.
Acknowledgments
The authors would like to thank the Council of Scientific and Industrial Research (CSIR),
Department of Science and Technology (DST) and the Department of Biotechnology (DBT),
Government of India, New Delhi for sponsoring the research grants on plant cell cultures for
secondary metabolites. They also like to thank B. Suresh for his help in the preparation of
this review.
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Biotechnology Advances 20 (2002) 101 153

Plant cell cultures:

* Corresponding author. Tel.: +81-761-51-1668; fax: +81-761-51-1665.

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

2. Need for a biotechnological approach

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

3. Plant cell culture as a source of secondary metabolites

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Pepin et al. (1995)

Dornenburg and Knorr (1996)

Lindsey and Yeoman (1984)

Swanson et al. (1992)

Chung et al. (1994)

Adapted from Ramachandra Rao (1998).

It is independent of political interference.

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Hippolyte et al. (1992)

Adapted from Ravishankar and Ramachandra Rao (2000).

4. Strategies to increase secondary metabolite production in plant cell cultures

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

4.1. Screening and selection of highly productive cell lines

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

4.6. In situ product removal

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

5. Organ cultures as a source of pharmaceuticals

6. Hairy root cultures for pharmaceuticals

Park et al. (1989)

Adapted from Ravishankar and Ramachandra Rao (2000).

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Hamill et al. (1986)

Adapter from Payne et al. (1991).

McKinely et al. (1993)

Adapted from Ravishankar and Ramachandra Rao (2000).

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Fig. 1. Highly branched hairy root cultures of Cichorium intybus.

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Fig. 2. Configuration of hairy root bioreactors.

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Hashimoto and Yamada

Furuya et al. (1989)

7. Production of foreign proteins in transgenic plants

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

plants also led to the opportunity to develop an inexpensive method of mucosal

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

8. Immobilization of plant cells for the production of secondary metabolites

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

Immobilization method Reference

Felix et al. (1981)

Brodelius et al. (1979)

Brodelius and Nilsson (1980)

Lang et al. (1990)

Synthesis from precursors

S. Ramachandra Rao, G.A. Ravishankar / Biotechnology Advances 20 (2002) 101153

> 100 (I)

Lindsey and Yeoman (1984)

Wichers et al. (1983)

Miyasaka et al. (1986)