Plant tissue culture is used to produce valuable secondary metabolites. There are three main methods: cell suspension cultures, hairy root cultures, and immobilized cell cultures. Cell suspension cultures are the most common. They involve transferring plant cells or callus to liquid medium to grow as a suspension. Medium components, regulators, precursors, elicitors, and environmental factors can be manipulated to enhance metabolite production. Hairy root cultures use plant cells transformed by bacteria to produce root-like structures that also synthesize metabolites. Immobilized cell cultures entrap or affix cells to allow contact while protecting from shear stress. Each method aims to produce metabolites through optimized bioprocessing conditions at large scales.

1. CELL & TISSUE CULTURE
Productionof secondarymetabolitesbyplanttissue culture 1
Topic
PRODUCTION OF SECONDARY METABOLITES BY PLANT TISSUE CULTURE
Submitted to:
MA’AM MARIA
Submitted by:
Amna Shehzadi (BS-BT-FO48) Group leader
Farah Masood (BS-BT-F034)
Arfa Khizer (BS-BT-F020)
Hafsa Islam (BS-BT F052)
Rizwan Abbas (BS-BT-FO44)
Date of submission:
26 October, 2015
Department:
BS-Biotechnology ”5th semester”

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Productionof secondarymetabolitesbyplanttissue culture 2
PRODUCTION OF
SECONDARY
METABOLITE BY PLANT
TISSUE CULTURE

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Productionof secondarymetabolitesbyplanttissue culture 3
TABLE OF CONTEXT
Topic Page No.
1. Introduction 3
2. Advancement of plant tissueculture over conventional agricultural production 3
3. Secondary metabolite 4
4. Strategies for enhanced production of secondary metabolites in plant cell culture 4
5. Cell Material 4
a) Shear stress 4
b) Growth kinetics 5
6) MEDIUM COMPOSITION FOR SECONDARYMETABOLITE 6
7) Manipulation of Environmental factors 6
8) METHODSFOR PRODUCTION OF SECONDARYMETABOLITEBYPLANT TISSUE
CULTURE 7
1) Cell suspension culture 7
a) Mechanism /media components 8
b) Futureaspect 9
c) Case study 10
2) Hairy root culture for production of secondary metabolite 10
a) Methods for producing hairy root 11
b) Production 12
3) Immobilized cell culture 13
a) Entrap cell 13
b) Immobilized cell surface… 13
c) Advantages of immobilized cell culture 14
d) Drawback of immobilized cell culture 14
9) References 15
10) Review about Group member 16
10) ReviewaboutgroupMembers

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Productionof secondarymetabolitesbyplanttissue culture 4
PRODUCTION OF SECONDARY METABOLITE
BY PLANT TISSUE CULTURE
Introduction:
Many higher plants are source of natural products used as pharmaceutical,
agrochemical, fragrance ingredients, food addivities and pesticides. Newly plant derived
chemicals are used in production of desirable medicinal compounds from plants. But now
biotechnological approaches and specially plant tissue culture are found to have potential as a
supplement to traditional agriculture in industrial production of bioactive plant metabolites.
Cell suspension culture systems could be used for large scale culturing of plant cells from
which secondary metabolites could be extracted. The advancement of this culture is that it can
ultimately provide a continuous, reliable source of natural products.
Advancement of plant tissue culture over conventional agricultural
production:
Plant tissue culture can be defined as the in vitro manipulation of plant cells and useful for
plant propagation and in the study of plant growth regulators. It is generally required to
manipulate and regenerate transgenic plants. The most important advantage of this in vitro
grown plants is that it is independent of geographical variations, seasonal variations and also
environmental factors. It offers a defined production system, continuous supply of products
with uniform quality and yield. Novel compounds which are not generally found in the parent
plants can be produced in the in vitro grown plants through plant tissue culture. In addition,
stereo- and region- specific biotransformation of the plant cells can be performed for the
production of bioactive compounds from economical precursors. It is also independent of any
political interference. Efficient downstream recovery of products and rapidity of production
are its added advantages.

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Productionof secondarymetabolitesbyplanttissue culture 5
Secondary metabolites
Secondary metabolites can be derived from primary metabolites through modifications, like
methylation, hydroxylation and glycosylation. They are classified into terpenes, phenolic and
nitrogen and/or Sulphur containing compounds. Plants produce secondary metabolites, which
are specific to an individual species, genus and are produced during specific environmental
conditions so commercially available secondary metabolites are considered high value
products.
Strategies for enhanced production of secondary metabolites in plant cell
culture:
 The different type of genes within the cell population can be screened by selecting cell
lines capable of accumulating higher level of metabolites.
 By the manipulation of medium.
 Addition of elicitor is important which induce the production and accumulation of
secondary metabolites in plant cell.
 Precursors are added whether exogenous or endogenous, that can be converted by living
system into useful compounds or secondary metabolites. For example, amino acids
have been added to suspension culture media for production of tropane alkaloids, indole
alkaloids.
 By manipulating the permeability of cell membrane secondary metabolites that often
blocked in vacuole can be secreted out to the media.it can be achieved by electric pulse,
UV, pressure.
 Immobilization of plant cells allows better cell to cell contact and the cells are also
protected from high shear stresses. These immobilized systems can effectively increase
the productivity of secondary metabolites.
CELL MATERIAL
Plant cell culture invitro show several characteristics which must be considered before
attempting to employ such material for scale up for production of metabolite. Cell size and
rigidity which may invite shear stress, slow growth, tendency to aggregate and clump
formation, competence or level of differentiation/specialization for product accumulation and
intravacuolar storage of product.
A) Shear stress
Plant cells measure 10-100 µm in diameter (30-50 µm on average) and are encased in a fairly
rigid cellulose-based wall. Suspended in 50ml liquid medium in small flask, for example, the
cells will tolerate agitation by reciprocal or gyratory shakers (100-150rpm) over any length of
time without damage to their structure and viability. When suspended as heavy biomass in
large volume bioreactors however, agitation and aeration must be controlled so as not to effect
destructive shear stress.Kurz’(1971) chemo stat with aeration and agitation by air
bubbles(figure.1) and the advanced airlift bioreactor of today have become satisfactory
solutions to reduce shear stress. An alternative approach may be selection of shear tolerant cell

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Productionof secondarymetabolitesbyplanttissue culture 6
lines, which would permit large sale culture in conventional stirred tank reactors and much
higher biomass loadings than is likely possible in any air driven design (Fowler,1987)
Chemo stat after kurz (1971)
Figure 1
B) Growth kinetics
Plant cell grown in optimal nutrient medium may divide every 22 hours. In general however,
divisions occur at much longer interval i.e.30 to 70 hours. Also some cells may cease to divide
early in the culture period and elongate or specialize to accumulate secondary metabolite
i.e.pigmnets alkaloids phenolics.the consequences of relatively slow growth are two folds; cell
suspension require long period of culture before substantial amounts of biomass and products
have accumulated and the suspensions need to be carefully protected from contamination.
Bioreactor up to 15,500 I have, however, successfully been run (Noguchi et al.1970) and over
several month.

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Productionof secondarymetabolitesbyplanttissue culture 7
MEDIUM COMPOSITION FOR SECONDARY METABOLITE
Secondary metabolic formation can be obtained by one or two stages cultures systems .in the
first stage molecules growing the cell on the standard growth medium and second stage
molecules transferring .there cells into a production medium suitable for secondary products
synthesis . In one stage culture system both growth and production steps are combined together.
Sucrose is used as carbon and energy source and leads to optimal growth rate of plant cell
cultures. Anode, galactose .glucose and raffinose have also been used for this purpose .both
nature and concentration of sugar generally effect the yield of secondary product.
The type and concentration of group regulators in cell suspension is probably one of the most
important factors influencing their potential for secondary product synthesis. For example 2,4D
can stimulates both cell division and cell expansion but it can also bring about a dramatic
suspension of secondary metabolite synthesis.
A range of hormonal concentration is use for this purpose. Nature of plant and the
concentration of secondary metabolites generally effect on the yield.
Type and concentration of growth of regulators in cell suspension is one of the most
important factors that influencing their potential for secondary product synthesis. For example
Nicotine production by tobacco cell cultures is best achieve by a to stage approach the growth
phase is supported on MS medium 10^-5 NAA and 10^-6 KIN, While the Production phase is
optimal in MS with reduce level of growth regulators, the buffering capacity of plant tissue
culture Media is often very low which causes drift in the pH medium. thus’ Biological buffers
such as N-2 Hydroxyethyl Peprazine-N Ehane-Sulphonic Acid (HEPES) or 2 Morpholineo
Ethane sulphuric Acid add the concentration of 100mm .Medium additives such as Casin
Hydrolizate can also act as like buffers An exogenous Supplement of the biosynthetic
precursor added to the culture medium that may increases the yield of Products.
Manipulation of Environmental factors
Plants cells are influence by environmental factors light ,osmotic pressure gases atmosphere
PH ,Temperature influence on secondary metabolic production in PTC ,Higher Plants also
Influence by Photo Period increase the osmatic pressure induced by the addition of Meta-
Bolizable compound to the media can influence production of secondary Meta-Bolites.
Pear fruit suspension cultures initiated senescense and produced ethylene in medium
containing mannitol a nonmetabolizable sugar alcohol.
A high concentration of carbon dioxide in atmosphere enhance the production of isobutanol,
ethyl butyrate ethanol and ethyl acetate by apple and grape cell cultures.

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Productionof secondarymetabolitesbyplanttissue culture 8
Sometimes temperature and incubation also influences production of secondary metabolites.
The effect of temperature cycling. Day and night temperature cycles ex-perienced by plants in
natural environment as a trigger of secondary metabolism is relatively unexplored area.
METHODS FOR PRODUCTION OF SECONDARY METABOLITE BY
PLANT TISSUE CULTURE
There are three ways for the production of secondary metabolite by plant tissue culture which
are as follows:-
1. Cell suspension culture
2. Hairy root culture
3. Immobilized cell culture
About Cell suspension culture:
Compounds that we get from plants and are used as food additives, pigments, dyes,
insecticides, cosmetics and fine chemicals is known as secondary metabolites. In 1988
Balandrin and Klocke, initiate extraction and production of secondary metabolites by
application of cell and tissue culture. Plant cell culture and tissue cultures are biotechnological
processing of whole plant cultivation for the production of secondary metabolites. In order to
successfully cultivate the plant cells at large scale, several engineering parameters such as, cell
aggregation, mixing, aeration, and shear sensitivity are often observed carefully to proceed our
aim. The media components, their concentrations and the environmental factors are selected
according to our requirement for best synthesis of a desired metabolites.
1 - CELL SUSPENSION CULTURES
Mostly in production of secondary metabolite we follows Cell suspension culture which is
more suitable and convenient technique to handle.
A) Selection of suitable cell & Callus formation
The first step in plant tissue culture is to develop a callus culture from the whole plant. For
this purpose we should select a cell having ability to generate a whole plant cells. Such cells
are known as totipotent cells. An undifferentiated mass of cells on culture plate is known as
callus. A callus must be obtained from a totipotent dividing cells.
B) Development of cell suspension culture
The important factor count in culturing cell is precise, good and sterilized medium. A
suspension culture is developed by transferring small amount of a callus into liquid medium
and is maintained under suitable conditions of aeration, agitation, light, temperature and
other physical factors
C) Mechanism /media components

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Productionof secondarymetabolitesbyplanttissue culture 9
Cell suspension cultures components include,
 Fresh friable fragments of callus into 50 ml of MS liquid media in 250 ml Erlenmeyer
flasks.
 Regulators includes kinetin (0.5, 1.4 or 2.3 µM) and 2, 4-D (2.2, 4.4 or 6.6 µM), and
their combinations Benzyl adenine combinations with kinetin contained NAA (0.54
µM) in one treatment.
 Macro and micro nutrients.
 Precursor feeding exogenous supply of a biosynthetic precursor to culture medium is
applied to increase the yield of the desired product.
 According to Roberts and Shuler, 1997 elicitation, is one of the most effective strategies
for improving the productivity of bioactive secondary metabolites. Elicitors are signals
triggering the formation of secondary metabolites.
 Agitation. . Suspension cultures is incubate on rotary shaker (100 rpm) for agitation
under the dark in the growth room.
 Each treatment consisted of four completely randomized replicates without sub samples
and growth assessment was performed by recording data every five days during a 30
day period for cells Fresh Weight (FW).
 Optimization of other process parameters
 Appropriate nutrients, their concentrations and environmental factors are known to
enhance the yield and productivity of metabolites in plant cell suspension cultures.
 Optimum aeration-agitation condition with respect to capacity of oxygen supply and
intensity of hydrodynamic stress effects on the plant cells.
 Control of temperature, pH and nutrient concentration inside the bioreactor.
 Control of aggregate size (which may be important to enhance secondary metabolite
production).
 Maintenance of aseptic conditions for relatively longer cultivation period.
D) Permeabilization of Plant Cells
As the culture under our
processing matures and
developed after complete
regulation and nourishment by
providing all requirement we
are able to proceed our aim.
Plant secondary metabolites
are normally produced
intracellularly which carried
out by downstream processing
of a specific product. It is,
therefore, desirable to extract
the products into the culture
medium such that the
purification procedure becomes easier. Extraction of secondary metabolites from the vacuoles
of the cells would also reduce the product inhibition and increase the productivity.

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Productionof secondarymetabolitesbyplanttissue culture 10
Selection of a specific solvent system with due consideration to its effect on cell growth may
lead to substantial release and increase in the production of secondary metabolites. Organic
material such as Dimethyl sulfoxide (DMSO) has been used in many cases, because it is known
to extract sterols from the membranes of the eukaryotic cells. Of various cells tested, only
Catharanthus roseus survived the treatment of DMSO. Taxol has recently been extracted by
various organic solvents such as hexadecane, decanol and dibutylphthalate, in the range of 5-
20% (v/v), in the culture medium of Taxus chinensis.
FUTURE PROSPECTS /Advantages
Plant cell cultivation is a suitable alternative to whole plant cultivation for the production of
desired compounds. However, due attention must be given to the biological and engineering
parameters related to the growth of and secondary metabolite production by plant cells in
suspension cultures. The inherent difficulties associated with in vitro plant cell cultivation e.g.
genetic variation of plant cell lines, sensitivity to shear stress, complex regulatory mechanism
etc. are to be properly addressed for a specific cell line.

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Case Study /Example: Production of Taxol from Taxus mairei by cell suspension cultures
In 1969, Wani used Taxol as breast cancer treatment as (FDA) food diterpene amide. Taxol is
considered as the prototype of a new class of cancer chemotherapeutic agents.
The thin bark of yew tree contains 0.001% of Taxol by dry weight basis. Due to the scarcity of
the slow growing trees and the relatively low content of Taxol they used cell suspension
technique for its large scale production.
Stages
I. Collected different tissues of Taxus mairei, a species.
II. The extracts of bark and leaf tissues were analyzed by using HPLC for the content of
Taxol and Taxol related compound to determine some facts.
III. Inoculation in Medium: Taxus mairei Callus were induced from needle and stem
explants on B5 medium supplemented with 2 mg/l 2, 4-D or NAA. Different cell lines
were established using stem and needle-derived callus.
IV. One of the cell lines, after providing culture requirements, 6 weeks of incubation,
produced 200 mg Taxol per liter cell suspension cultures.
2- HAIRY ROOT CULTURE FOR PRODUCTION OF SECONDARY
METABOLITE
A development that may revolutionize the role in vitro culture is fine chemical synthesis is the
production of fastly growing Hairy root culture that is obtained by the genetic transformation
of plant tissue by a soil bacteria called agrobacterium rizogene.
Effect of transformation
The plant infection with rhizogene causes two pieces of transfer DNA (TL, TR) contained on
the bacterial plasmid to be inserted on plant genome. Metabolism of transferred tissue cause
the hairy root phenotype. Both (TL DNA, TR DNA) has rhizogenic function.TL DNA is more
effective in determining the Hairy root culture.
Methods for producing hairy roots
a) Infection
The production of hairy root culture is extremely straight forward process. Several strains of a
rhizogenes show good virulent character. We use bacterial strain LBA which contain plasmids
pRI to induce root on dicotyledonous plants.
Transformation may be induce on aseptic pants, detach leaves, isolated protoplast.
Transformation may can be induce by co-cultivation of plant protoplast with A.rhizogenes.
Depending on the species profusion of roots may appear at the site of inuculation.callus will
form. From which roots will emerge within 1-4 week. We can obtain infection of all suitable
species.

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Productionof secondarymetabolitesbyplanttissue culture 12
b) Confirmation of transformation
Opine synthesis is the indication of hairy root culture transformation. Detection of T-DNA by
southern hybridization is more indication that culture root tissue is transformed.
E.g. Nicotiana rustica hairy root culture developed in laboratory did not synthesize opines but
possessed TR T-DNA.
c) Maintenance of culture
In many species subculture of great inoculum cause the formation of callus, which may grow
at the site of expense of root tissue reducing the capacity of culture. The best growth of hairy
roots tissue is obtained by inoculating a flask of medium with 3 or 4 tips of 2-3 cm length.
Medium conditioning is not necessary such root tips grow with minimum lag period.
d) Characteristic of the culture Growth and Morphology
Hairy roots have profusion of root hairs and high degree of branching resulting in high growth
rate in culture. Both the growth and rate vary between the species.
Production
• Key feature of hairy roots is the production of secondary metabolites.
• The technology of hairy root culture is applicable to all the species that can
transformed, providing the culture that can grow faster than untransformed roots
without decrease production per unit.
• The production of secondary metabolites by hairy roots is increasing. Hairy roots
contain cells in range of morphological and biochemical state with a number of
meristematic calls at root tips. Active compounds occur in increased concentration
compares to invitro culture.
• The 2nd key feature of hairy root culture is their productivity is very stable and
retain the biochemical potential of original plant. Incotrast to unorganized culture
chromosome number is that of the parent plant.
• Hairy root culture can be used for the production of vaccine. There are several
approaches for the production of vaccine in plants transient expression system based
on agrobacterium delivery of binary vectors and plant viral vector and transgenic
plant, plant cell and tissue culture. Hairy root cultures, generated from edible plants
and producing target antigens, provide a potential approach for the development of
vaccines for oral delivery.
• Hairy root culture can also be used for regeneration of whole plants and for
production of artificial seed.
• Hairy root culture can be used for phtoremediation.
• Bioreactors can be also use to culture hairy roots for secondary metabolites
production.
Reactors used for culture are of three types
 Liquid phase

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Productionof secondarymetabolitesbyplanttissue culture 13
 Gas phase
 Hybrid reactor (that are combination of both)
The only company based on Non GMO Hairy root Cultures as an industrial level is a Swiss
company called ROOTTec Bioactive AG in Witterswil. There also is a French company called
ROOT LINES TECHNOLOGY SAS which uses Genetically Modified Hairy root cultures.
3- IMMOBILIZED CELL CULTURE
a) Entrapment of cells
Immobilized of plant cells as pioneered by Brodelius et al. (1979) is technology which enables
entrapment of cell in gel of calcium alginate, polyvinyl alcohol resin or other polymer or
restrains cell on a fixed support of foam, fabric or hollow fibres. Yeoman (1987) has presented
technique, characteristics, properties and commercial potential of immobilized plant cells. The
procedure to follow in the case of entrapment in gels is as follows:
Cells (8 gFW )suspended in 100ml alginate (1.5%) are from nozzles (1mm in diameter) or the
orifice of a pipette into a 50µm Calcium chloride solution, the calcium alginate beads (ca
2mm in diameter) formed are left in the solution for 3 hours at 25⁰C in the dark, then washed
in 30ml medium. Growth or production medium is inoculated with beads containing a total 7
g cells in a bubble column bioreactor and incubated. Entrapped cells have been used repeatedly
for product formation. Using a different matrix cultured cells of Lavandula Vera have been
entrapped in synthetic resin pre-polymer which are derivatives of polyvinyl alcohol (degree of
polymerization 1700 to 1800) with photosensitive functional sites which initiate dimerization
by irradiation with visible light. Entrapped cells grew well inside gels and synthesized blue
pigment in the presence of L-
cysteine as an inducer. The
entrapped cells were superior to
calcium alginate entrapped cells
in both cell growth and
metabolite production. Since
blue pigment was released into
the medium, cells could be used
for metabolite production
several times (Nakajima et
al.1986)
b) Immobilized cell surface
The tendency of cells suspended
in liquid medium to adhere to
glass surfaces in culture vessels
can be exploited for
immobilization. Lindsey and
Yeoman (1984) found that after introduction of particles of polyurethane foam into cell
suspension of capsicum Frutescens and daucus carota, cell settled preferentially in the pores of

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Productionof secondarymetabolitesbyplanttissue culture 14
the foam. The resulting aggregates were stable and could be used in bioreactor
systems.Archambaults et al. (1990) purposed a system to be operated in an airlift fermenter.
Here, the surface consisted of sheets of non-waven short fibre polyester material formed into a
square spiral configuration on a supporting structure made of welded stainless steel rods, the
structure was placed into a. bioreactor. Catharanthus roseus inoculum attached readily and
uniformly to the immobilizing structure. All cells were retained with in the first 24 hours of
culture. In the B5 medium (gram borg et al., 1968) a constant carbohydrate consumption of
ca.2g/1/day was observed during the growth phase. The stationary or idiophase was
characterized by a much lower sugar consumption rate of ca.0.5g/1/day.
Adjustment will be required if high biomass concentration and product formation are to
attained with other cell cultures. For example with papaver somniferum cells the medium had
to be supplemented with an elicitor 9 (botrytis spp.mycelium homogenate) 7.5 days after
inoculation in order to stimulate metabolite production. After 6.5 days of culture, immobilized
cells in a 2-1 culture had released into the medium 6 mg of sanguinarine (total i.e. cells and
medium =690mg, kurz et al….1990)
Immobilization creates for cells a situation which is to imitate membership in a tissue of a
whole plant.as such, cells are expected to cease to grow and accumulate metabolites. The
technical advantage of this system clearly is superior metabolite formation compared to cell
suspension and media exchange without loss of cells.
Development of cell immobilization conceivably leads to continuous culture and extreme
reduction of upstream costs due to saving of biomass were not growth control and product
release major stumbling blocks.immobilzed cells would also be the perfect catalyst for
biotransformation
Advantages of immobilized cell culture
• Cells are protected from the shearing forces normally found in bioreactors and can
be grown in high densities.
• Usually, immobilized cells are able to synthesized chemical over longer periods
than in suspension so that continuous, rather than batch production becomes a
possibility.
• Immobilization also keeps cells away from the interface of the medium with air
where coagulation, frothing and sometimes deactivation can occur
Drawback of immobilized cell culture
One of the greatest drawback of immobilized cell systems are heterogeneous cultures is the
possible occurrence of mass transport limitation inside the beads or biofilms (i.e. oxygen,
carbon dioxide and substrates). Due to mass transport limitation immobilized cell systems are
heterogeneous culture with respect to gradient. As a result, control and measurement of
important process parameters is difficult. Furthermore, methodical model require a complex
structure taking in to account substrate and product concentration profiles throughout the
immobilized particle.suprisingly, the nutrient gradient (oxygen and substrate) in immobilized
systems are often mentioned as an advantage of immobilized systems. These limitation would
enhance secondary metabolism although no evidence has been presented to explain the result.

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REFERENCES
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16. Plant Cell Culture Secondary MetabolismToward Industrial ApplicationBy Frank
DiCosmo, Masanaru Misaw
17. Plant Tissue Culture: Practices and New Experimental Protocols By B. N.
Sathyanarayana
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19. Introduction to Plant Biotechnology By H. S. Chawla

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Productionof secondarymetabolitesbyplanttissue culture 17
REVIEW ABOUT GROUP MEMBERS
NAMES WORK DONE IN ASSIGNMENT
ARFA KHIZER Introduction of secondary metabolite by
plant tissue culture and compare plant tissue
culture over conventional agricultural
production and strategies.
FARAH MASOOD Media composition for secondary metabolite
formation and manipulation of environment
factors.
RIZWAN ABBAS Cell suspension culture for production of
secondary metabolite. Mechanism, future
aspect and case study.
HAFSA ISLAM Hairy root culture for production of
secondary metabolite ,Method and
Production
AMNA SHEHZADI Cell material and immobilized cell culture
for production of secondary metabolite.
Advantages and drawback of immobilized
cell culture