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Introduction
Growth characteristics of shoot culture
Biosynthetic characteristics of shoot cultures
Physiological effects of light
Bioreactor design consideration for shoot cultures
Bioreactor studies with shoot cultures
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The proliferation of shoots in vitro without
intervening roots or undifferentiated tissue
Source of plant secondary metabolite
Also for clonal propagation of horticultural plants
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Initiation and maintenance of shoot culture
◦ Initiated from a sterile germinated seedling or a whole plant by
dissecting the shoot apical meristem, or a leaf, or a stem section
onto solidified medium
◦ Proliferate by continuously forming new meristems
◦ Axillary meristem, arising at a junction of a leaf and a stem
◦ Adventitious, arising anywhere on the leaf or the stem
◦ Important hormones- cytokinins alone or with combination with
auxin
◦ Cytokinins stimulate shoots bud growth
◦ In vitro growth-heterotrophic- need exogenous supplied of carbon
source
◦ Light is usually required for normal greening and leaf
development
◦ Infection with A. tumefaciens and A. rubi gives rise to “shooty
teratomas”which xynthesis their own hormones
◦ Increase in weight over time in a batch vessel follows a sigmoidal
curve
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Genetic stability of shoot cultures
◦ Organized meristems are generally more resistant to
genetic alterations than undifferentiated cultures
◦ Axillary shoot cultures are the most stable
◦ Adventitious shoots may arise from a single cell, they are
more prone to genetic alterations
◦ They are stable even in the absence of a constant
selection pressure.
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Biosynthetic differences between shoot cultures
and undifferentiated cultures
◦ Shoots differentiation enables the expression of
several biosynthetic pathways that are expressed at
very low levels in undifferentiated cultures
◦ Technology to scale-up shoot culture is not well
established so need to confirm that shoot culture is
really necessary for secondary metabolite
◦ If can use genetic engineering to block any step in the
biosynthetic pathway in undifferentiated culture that
will be better
◦ However, sometimes the coordinate expression of
many enzymes is necesssary, or if organeele or cell
specialization is necessary for storage the genetic
manipulation perhaps not a good choice.
◦ Need for specialized cells-many secondary metabolites
are synthesized and stored in specialized cells in the
plant. Eg. essential oil are accumulated in the structurally
differentiated structure
◦ Chloroplast and development regulation-a positive
correlation between light, chlorophyll content and
secondary metabolite production in shoots culture
system-suggesting that certain steps in the pathway may
be chloroplast-associated.
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Differences between shoot culture and whole
plants
◦ Often be incorrect to assume that a shoot culture will
produce all the compounds that are present in the
leaves of a whole plant. The site of synthesis may be
different from the site of storage
◦ Factors affecting the whole plant such as maturity and
flowering also affect secondary metabolite production.
Since shoot culture does not subjected to the same
factors in vitro their secondary metabolite profile may
vary quantitatively and qualitatively.
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Issues pertaining to secondary metabolite
productivity
◦ 1.Kinetic and product accumulation
◦ design and operation of a production process requires
a knowledge of the kinetics of product formation in
relation to growth rate
◦ If the product is intracellular accumulated, and if rapid
growth and product formation occur concomitantly-the
process favoring rapid growth of tissue in the obvious
choice
◦ If product formation occurs only after growth ceases in
a batch, then rapid growth of tissue to its final
attainable density, followed by exposure to conditions
favoring secondary metabolite formation may be
considered.
◦ 2.Product secretion and in situ removal
◦ If the product of interest is growth-associated and
completely intracellular, then the shoot culture simply
provides an alternative to whole plants as a source of
material for product extraction
◦ If part of the secondary metabolite is secreted and can be
produced by nongrowing tissue, then continuous
operation with biomass reuse, much like immobilized cell
process, is possible
◦ 3. Medium optimization
◦ 4. Use of biosynthetic precursors. Has been
demonstrated in a variety of system
◦ Factors such as level, rate and timing of precursor
addition, stoichiometry be important with shoot culture,
and precursor uptake are likely
◦ 5. Elicitor treatment
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Consists of living root tissues growing in an
organized differentiated form in the absence of
other types of plant materials such as stem, leaf,
or flower tissue.
For secondary metabolite production, frequent
lack of secondary biosynthesis in suspension and
productive instability.
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Enhanced biosynthetic expression
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Genetic and productive stability
◦ Biosynthesis or accumulation is probably
associated with the presence of certain organelles
or cell types, which form only in structurally
differentiated root tissue
◦ Suspension frequently display unstable rates of
growth and product formation
◦ Root cultures offer relatively stable growth and
production for at least 1 year
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Compatibility with Immobilization
◦ Cell are maintained within the reactor for extended
biosynthesis of secreted products
◦ Compatible with “immobilized” cell because
 Cells are “self-immobilized” in that no artificial matrix
is required for cell aggregation
 Roots are frequently capable of secretion, as is
essential for immobilized operation
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Growth rates
◦ Although the growth rates are generally too slow,
with the use of “hairy roots” transformed root
culturesand/or the judicious use of auxin , as high
as suspension cultures
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Untransformed root cultures
◦ Obtained by inoculating a sterile root tip, into
liquid medium
◦ Low auxin level, until root cultures are well
established
◦ Sterile root tips obtain from
 By excising the seedling radicle from surface sterilized
and germinated seeds.
 Shoot apical meristem can be excised, grown on solid
medium and induced to root
 Redifferentiated rootlets from callus or suspension
aggregates can be used to initiate a root culture
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Agrobacterium rhizogenes and hairy roots
◦ Hairy roots disease is a root tumorous condition
associated with Agrobacterium rhizogenes
◦ The capacity for tumor induction is encoded by
large virulence plasmids Ri “root inducing”.
◦ On infection a portion of the plasmid, the T-DNA
is more or less randomly integrated into the host
plant genome
◦ T-DNA contains genes for synthesis of the auxin
indole-3-acetic acid (IAA)-determining tumor
root growth
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Auxin-control roots growth and development
◦ Auxin of interest
 Several auxin have been used
 Indolebutyric acid (IBA) shown to be effective for use with
root cultures
 4-chloro-IAA or 5,6-dichloro-IAA may be more effective
 Different auxin give rise to the exploitable diversity in
auxin pahrmacology
◦ Respond to auxin
 Effects on elongation rate
 At low concentration stimulates rootlet elongation and at
higher concentration, elongation is inhibited
 The stimulation of lateral root formation
 At high concentrations, auxin stimulated the initiation of
lateral root primodia, total number of emerged lateral
increase
 Both are summaries in figure (text books)
 Auxin-stimulated release of ethylene
 During balanced exponential growth, the specific growth
rate (µ)m is approximately propotional to the product of
the elongation rate and the lateral root linear density.
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Mode of auxin addition
◦ Possible to improve feeding programs,
◦ Eg: short elevation of auxin to enhance lateral root
formation followed by longer period of lower levels
favoring elongation.
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Ethylene
◦ Regulation of ethylene level
 Wounding or stress stimulate ethylene level
 High auxin give rise to ethylene concentration
◦ Response to ethylene
 Ethylene stimulate root extension at low level, at high
concentration, ethylene is inhibitory to root elongation
 Exogenos ethylene may well stimulate lateral
formation in particular case
 Increase in root diameter in response to high auxin
concentration, worsen oxygen mass transfer into the
root
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Other Hormones: Cytokinin and Gibberellic
Acid (GA)
◦ Cytokinin often result in shorter, thicker roots
and ultimately slower growth rates. AT very low
concentration lateral formation may be
stimulated. Eg: 0.1 ppm Kinetin stimulated
growth and saponin production to transformed
and un-transformed root cultures.
◦ GA, stimulated root elongation. Eg: Sugimoto et
al (1988) reported growth improvements with GA
addition to untransformed root cultures of
Stephania cepharantha
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Magnetic and electrical effects on growth
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Other nutritional and environmental factors
◦ Berry et al (1974) observed an impressive 35-40
flod increase in lateral root formation in intact
onion roots with applied electric field of 15
mV/mm. However, media of lower ionic strength
are required.
◦ Different basal media on growth and secondary
metabolite production. Eg: Catharanthus roseus
transformed root cultures grew only in halfstrength B5 medium.
◦ Dicot species prefer sucrose, whereas monocots
tend to grow better with glucose
◦ Light, might be stimulator or inhibitor
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Overall batch accumulation pattern
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Localization of sites of synthesis and of
accumulation
◦ The most common pattern is one of peak
biosynthesis in late log or early stationary phase,
biosynthesis coinciding roughly with a slowing of
the growth rate.
◦ Whether products are stored in the same cells or
transported elsewhere for storage
◦ Spatial distribution of secondary product
concentration cannot, in general be taken to
reflect the localization of biosynthesis reactions
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Achieving product secretion from living
cells is critical to efficient operation of
immobilized-root perfusion systems.
A wide variety of types of compounds are
reportedly secreted from both intact roots
and root cultures.
Factors favor secretion in roots
◦ Ethylene release’
◦ Hormones such as auxin and gibberelins and low
oxygen
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Unfavorable factors
◦ High temp, increase ionic strength, increase
calcium, decrease pH and elicitor treatment
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Root line selection
◦ Choosing promising root cultures from among
many independently initiated lines.
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Production medium development
Auxin effects on secondary biosynthesis
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Other productivity enhancement techniques
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◦ Endogenously added auxin depress secondary
product formation
◦ Precursor feeding and biotransformation
◦ Biotransformation involves a one-or two step
conversion of an advanced intermediate into final
product
◦ Elicitor treatment
◦ In situ product removal. Eg with perfusion and resin
adsorption
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Packed Bed with fluid recycle
◦ Roots form a bed in the reactor portion through
which medium is forced at a desired superficial
velocity. Aeration is accomplished by air sparging in
the recycle vessel. During continuous operation,
because the recycle rate is rapid compared to
feed/effluent flow rates, the system is well mixed
and behaves like a “continuous stirred tank reactor”
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Medium and gas cycling
◦ Medium may be alternately pumped into and out of
the culture chamber either using pumps or a pump
and siphon approach
◦ Organs may also be mechanically raised out of and
lowered into the liquid medium
◦ Organs may be fixed within a rotating “drum” which
is partially filled with medium
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Spray and aerosol system
◦ Expose root masses to gas phase containing fine
droplets of liquid medium.
◦ Advantage- potential elimination of oxygen transfer
problem