"STRATEGIES USED TO"

OPTIMIZE PLANT PRODUCT

YIELD

Culture Conditions:
 The productivity of cell lines is greatly influenced by the
culture conditions of which culture medium is the most
important. Growth and production of secondary metabolites
are inversely related, both in whole plant and cell cultures.
• In cell cultures on media defined for optimum growth,
production of secondary metabolites generally occurs in
the late stationary phase when the medium gets depleted
of some important constituents.
• Growth inhibition is often associated with
cytodifferentiation & the induction of enzymes for
secondary metabolism. So dual culture system is
preferred.
Dual culture system: It involves biomass production in a
medium optimum for cell proliferation followed by transfer of
healthy cells to a different medium which is favourable for
product yield but is not good for the growth.
This strategy was used by two:
1. zenk et al for the production of indole alkaloids by
Catharanthus roseus cells.
2. Fujita et al for the production of shikonin by cell cultures
of Lithospermum erythrorhizon.
Most useful modification made in the growth medium for use in
secondary metabolite production are:
• Reduction or elimination of 2,4-D or other
phytohormones.
• Reduction of phosphate level.
• Increase in sucrose level or alteration of
carbohydrate(C)/ nitrogen(N) ratio.
Fujita et al tested 5 basal media- LS,W,B5,BL,NN for growth &
shikonin production in cell cultures of L..erythrorhizon. Out of
those LS supported best biomass production followed by B5&
shikonin was produced only in W medium.
• Stable production occurred when nitrate was present as
the sole source of N. Even ammonium substituted for
nitrate inhibited production.
• Optimum media for the production of different
metabolites by a cell line are likely to be different.
• Plant growth regulators affect growth & differentiation &
thus affect secondary metabolite production by cultured
cells. In general an increase of auxin level such as 2,4-D
which stimulates dedifferentiation & proliferation of cells
reduces the level of secondary metabolites.Therefore
auxins are added to growth medium but used at a lower
level in production medium..
 • GA3 generally inhibits the secondary metabolite
production.
• The pH of the medium is shown to enhance permeability
of the cell membrane & thus helps in the release of
intercellular alkaloids.
• Light is an important regulatory factor in the production
of alkaloids in plant cell cultures.e.g in C.roseus light
influences the serpentine accumulation higher under 15 h
photoperiod instead of 24h illumination.
• Light affected not only production but also the site of
product accumulation.Gaseous environment mainly the
availability of oxygen & carbon dioxide also plays an
important role in production of secondary metabolites by
cell cultures.

Selection of high yielding lines:

• Explants used to initiate tissue cultures are highly
heterogenous with regard to the metabolic productivity
of its constituent cells.
• Productivity of a heterogenous culture would be an
average of the productivity of its high & low yielding
cells.Selection & cloning of high yielding cells from such
cultures is therefore regarded as an effective method to
improve invitro production of secondary metabolites.
• It requires:
1. initiation of cultures from selected high yielding
genotypes.
2. then screen a large number of individual cultures for the
best producing variant.
3. from established cultures of these lines, selection can be
made for better yielding subclones.
• Since cultured cells are prone to spontaneous genetic
changes it would be necessary to make periodic
selections to maintain high productivity of the cultures.
Selections should be made under conditions which are
suitable for product formation.
• Screening for high yielding lines for colored compounds
such as shikonin, berberine & betanin is very simple.
The most colored areas of cell clumps can be easily
isolated & cultured separately. Cells with highly
fluorescent compounds may be detected under UV light
by naked eye or fluorescene microscope.
• It is important that the selected lines are reasonably
stable.Since metabolic synthesis by plant cells is highly
influenced by the physiological state of the cells, the high
yield shown by the selected lines may be due to altered
gene expression.
• For the isolation of stable lines repeated selections
should be made& only if the product of a cell line remains
stable over several cycles.

Elicitation:
• In nature plant cells synthesize a number of compounds
in response to chemical or microbial attacks.
• Increased production of secondary metabolites in media
supporting poor growth of cells is also regarded as stress
response.
• A number of biotic like fungal extracts)&
abiotic( inorganic & organic chemicals,UV radiation)
factors have been tested as elicitor & shown to improve
the production of secondary metabolites in plant cell &
organ culture.
• For example- addition of conidia of Verticillium dahliae to
cell cultures of gossypium increased the yield of gossypol
from 5-10 g/l to a remarkable level of 500mg/l within 5
days.
• Asada & shuler noted a synergistic effect of elicitation
with a fungal homogenate, immobilization of cells with
Ca-alginate & alkaloid adsorption with a neutral
polycarboxylic ester. Cultures subjected to a combination
of the three treatments showed 45 fold higher ajmalicine
in the medium than in control.
• Simple organic and inorganic molecules can also induce
product accumulation in cultured cells.Smith observed
enhanced accumulation of catharanthine in cells of
C.roseus in response to NaCl,KCl,sorbitol individually.
• Addition of vanadyl sulphate to cell suspension cultures
of C.roseus resulted in the production of catharanthine,
serpentine and tryptamine.Effect was concentration
dependent.
• At lower conc.(25mg/l)- catharanthine& ajmalicine.
• At higher conc.(50-70mg/l)-tryptamine accumulation
occurred.
• The time of application of elicitor is critical for the yield
of secondary metabolites by cultured cells. Most of the
cultures respond to an elicitor only during the growth
phase.
• Elicitor treatment after a culture has already started to
accumulate the inducible compound does not enhance or
accelerate its production.

Immobilization of cells:
• One of the major problems in commercialization of a
cell culture based process for secondary metabolites
production is the production cost due to :
1. slow growth of plant cells
2. genetic instability of the selected lines
3. low shear resistance of cells
4. intracellular accumulation of product.
• Some of these problems can be reduced by
immobilized cell cultures. In this technique cells are
confined within a reactor system, preventing their
entry into the mobile phase which carries the
substrate and the products.
• Brodelius et al first reported the immobilization of
C.roseus & Daucus carota cells by entrapping them in
alginate beads.
• Immobilization is only relevant where the production
process involves two stages:
1. in the first stage conditions are optimized for
biomass production by suspension culture.
2. in the second stage conditions are optimized for
the product formation by immobilized cells
showing little or no growth.
• The potential advantages of immobilized cell cultures
are:
1. it may enable prolonged use of biomass.
2. by immobilization of cells the cell density in a
bioreactor can be increased 2-4 times that in
suspension cultures and this enables the use of
small reactors,reducing the cost of
medium,equipment installation and downstream
processing.
3. the entrapped cells are protected against shear
forces& a simple bioreactor can be used.
 4. it separates the cells from the medium and
therefore if the product is extracellular it can
simplify downstream processing.
5. it uncouples growth & product formation which
allows product optimization without affecting
growth.
6. the non-dividing immobilized cells are less prone
to genetic changes & therefore provide a stable
production rate.
7. it minimizes fluid viscosity which in cell
suspension cause mixing & aeration problems.
8. it promotes secondary metabolite secretion in
some cases.
• A wide range of bioreactors have been designed to
culture immobilized cells. Best design to use depends
on the method of immobilization.
• Entrapment of cells in gel or behind semi permeable
membrane is the most popular method used.
• Polymers used to entrap plant cells are alginate, agar,
agarose & carrageenan. Of these alginate has been
widely used to immobilize as it can be polymerized at
room temperature using calcium ions.
• Immobilization of cells on the surface of inert support
such as fiberglass mats& unwoven short fibre polyester
has also been examined for invitro production of
secondary metabolites.

• Binding of cells to the immobilizing support is regarded

as a two-step process.
1. cells are spontaneously attracted to the support
surface due to vanderwaal’s force aided by
entrapment of cells in pores or other irregularities
on the surface of the support.
2. the cells appear to secrete a mucilaginous
substance which firmly cements them to the
support surface.
• Surface immobilization promotes the natural tendency of
plant cells to aggregate which may improve the synthesis
& accumulation of secondary metabolites.
• A special advantage of this method over the other
methods of immobilization of cells is the absence of any
physical restriction to mass transfer between the culture
medium & the biomass surface.
 • Some reports on phytochemical production by
immobilized cells are- the accumulation of serpentine by
C.roseus & anthraquinones by Morinda citrifolia were
enhanced in the immobilized state when compared with
cell suspensions.
• Limitations of immobilized cell system are:
1. it is normally limited to systems where production is
decoupled from cell growth
2. the initial biomass must be produced in suspension
cultures.
3. secretion of products into the external medium is
imperative.
4. when secretion occurs there may be a problem of
extracellular degradation of product.
5. when gel entrapment is used the gel matrix
introduces an additional diffusion barrier.

Hairy root culture:

• It is desirable to use morphologically undifferentiated
plant cells for the production of secondary metabolites
but the genetic instability of these cells has been a
serious limitation. Moreover in many cases the
production of a metabolite occurs only when the
unorganized structure is induced to undergo organogenic
or embryonic differentiation.
• Undifferentiated calli of Atropa belladonna do not
produce the tropane alkaloid hyoscyamin but the cultures
gained the ability to synthesize this compound with the
differentiation of roots.
• In nature, roots are the source of a variety of food
products & other useful chemicals. The tropane
alkaloids,atropine & hyoscyamine ,steroidal precursors &
Catharanthus alkaloids are some of the high value
compounds synthesized in roots. Therefore, root cultures
are a potentially useful system for in vitro system for
commercial production of metabolites.
• In 1934, White established continuously growing root
cultures of tomato in a medium containing minerals,
sucrose & yeast extract. subsequently, root cultures of
many dicotyledonous & monocotyledonous species were
established.
• Stable integration of a portion of root inducing plasmid
into plant genome, following infection by the pathogenic
Agrobacterium rhizogenes, induces the hairy root disease
 in which proliferation of roots occurs at the site of
infection.
• Hairy roots can also be induced in most of the
dicotyledonous plants by genetic transformation with
A.rhizogenes or certain mutant strains of A.tumefaciens.
These roots which retain the ability to synthesize natural
products as normal roots can be used to establish
continuously growing hairy root cultures.
• The hairy root cultures are characterized by a high
degree of lateral branching, a profusion of root hairs & an
absence of geotropism. Due to extensive branching the
hairy root cultures show very high growth rate in the
absence of a growth regulator.
• Another important feature of the hairy root system from
the commercial point of view is their stable & high level
production of secondary metabolites.
• The ability to exploit plant root cultures as a commercial
source of natural products depends on the development
of suitable bioreactors. The unique structure of root
cultures presents a tremendous challenge for aseptic
engineering.
• The standard tank reactor is not suitable for mass
cultivation of roots because the contact of impeller not
only damages the root tip but also induces callus
formation.
• Mostly trickle bed reactor is used for hairy root culture, in
which the medium is sprayed over the roots & permitted
to drain down through the root tissue, provided a growth
rate comparable to the control.

Biotransformation:
• A possible reason why de novo synthesis of many
secondary metabolites in tissue cultures is lacking or
unsatisfactorily low is the absence or poor expression of
one or more corresponding biosynthetic enzyme. e.g the
lack of serotonin biosynthesis by Peganum harmala cell
cultures is due to the lack of tryptamine decarboxylase.
These non- producing cell cultures were able to transform
large amounts of externally supplied tryptamine to
serotonin. Thus even non- producing cultures may be
used for specific biotransformation or as a source of
enzymes of a non- expressed pathway.
• The chemical conversion of an exogenously supplied
substance by living cell cultures, permeabilized cells or
 entrapped enzymes derived from cell cultures is reffered
to as biotransformation.
• It may be a single step (mediated by single enzyme) or a
multistep (mediated by two or more enzymes) process.
The substrate used for biotransformation may be natural
or synthetic.
• Generally single step biotransformations are more
efficient, the yield decreases with increase in the number
of steps between precursor & product.
• E.g Arbutin a skin depigmentation agent is produced by
biotransformation of hydroquinone using Catharanthus
roseus cells.
• Addition of the inexpensive precursor into the liquid
culture medium of this cell line produced arbutin
efficiently.
• The dimeric indole alkaloids vinblastine & vincristine are
high value drugs in cancer therapy. They are currently
produced commercially by extraction from C.roseus
plants but the process is not efficient because of the low
yields.
• In order to produce these anticancerous drugs more
efficirently,many scientists have tried to apply plant
tissue culture. However the suspension cultures of
C.roseus do not produce either of these alkaloids due to
their inability to produce vindoline, a precursor of these
alkaloids.
• Some of the cardiac glycosides produced by Digitalis
species have been employed in the treatment of heart
diseases.
• Biotransformation of digitoxin to digoxin using D.lanata
cells which are unable to produce cardiac glycosides de
novo is the most famous example of biotransformation.

Permeabilization of cells:
• Generally secondary metabolites accumulate
intracellularly only a few lines release the product into
the surrounding medium.
• Permeabilization of cells to allow controlled release of
products into the medium has been attempted by
various techniques:
1. dimethyl sulphoxide application
2. using media of low pH
3. sonication with continuous ultrasound
4. electric treatment
• The essential feature of permeabilization is that it
should be reversible.

Removal of secreted products:

• The secreted products released into the medium
naturally or by induced permeabilization of cells may
suffer enzymatic or non-enzymatic degradation.
Therefore it is important that the secreted metabolites
are removed from the medium.
• Amberlite XAD-7 was used to adsorb indole alkaloids &
anthraquinones from the culture medium of C.roseus &
cinchona respectively.
• The best time to add the adsorbent was 7 days after
culture initiation.
• The removal of anthraquinones from 1.2 to 17.4
mg/l/day which was more than with any chemical or
elicitor treatment.
• Activated charcoal & reverse phase silica are other solid
phases employed to increase the production of
secondary metabolites.