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

Shoot culture

The proliferation of shoots in vitro without intervening roots or undifferentiated tissue

Source of plant secondary metabolite Also for clonal propagation of horticultural plants

Introduction

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

Growth characteristic of shoot cultures

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.

Growth characteristic of shoot cultures cont..

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.

Biosynthetic characteristics of shoot cultures

◦ 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.

Biosynthetic characteristics of shoot cultures cont….

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.

Biosynthetic characteristics of shoot cultures cont….

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.

Biosynthetic characteristics of shoot cultures cont….

◦ 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

Issues pertaining to secondary metabolite productivity..cont…

◦ 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

Issues pertaining to secondary metabolite productivitycont…

Roots Culture 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.

Advantages Enhanced biosynthetic expression

◦ Biosynthesis or accumulation is probably associated with the presence of certain organelles or cell types, which form only in structurally differentiated root tissue

Genetic and productive stability◦ Suspension frequently display unstable rates of

growth and product formation◦ Root cultures offer relatively stable growth and

production for at least 1 year

Advantages cont… 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

Advantages cont… 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

Types of root cultures 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

Types of root cultures cont… 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

Physiology: Hormones and Growth Regulator

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

Physiology: Hormones and Growth Regulator cont….

◦ 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.

Physiology: Hormones and Growth Regulator cont…. 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.

Physiology: Hormones and Growth Regulator cont….

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

Physiology: Hormones and Growth Regulator cont…. 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

Other Medium and Environmental Factors

Magnetic and electrical effects on growth◦ 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.

Other nutritional and environmental factors◦ Different basal media on growth and secondary

metabolite production. Eg: Catharanthus roseus transformed root cultures grew only in half-strength B5 medium.

◦ Dicot species prefer sucrose, whereas monocots tend to grow better with glucose

◦ Light, might be stimulator or inhibitor

Product Formation in Root Cultures Overall batch accumulation pattern

◦ 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.

Localization of sites of synthesis and of accumulation◦ 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

Product Secretion 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

Product Secretion cont… Unfavorable factors

◦ High temp, increase ionic strength, increase calcium, decrease pH and elicitor treatment

Productivity Enhancement Consideration

Root line selection◦ Choosing promising root cultures from among

many independently initiated lines. Production medium development Auxin effects on secondary biosynthesis

◦ Endogenously added auxin depress secondary product formation

Other productivity enhancement techniques◦ Precursor feeding and biotransformation◦ Biotransformation involves a one-or two step

conversion of an advanced intermediate into final product

Productivity Enhancement Consideration cont….

◦ Elicitor treatment◦ In situ product removal. Eg with perfusion and

resin adsorption

Design Approaches 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”

Design Approaches cont…. 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

Design Approaches cont…. Spray and aerosol system

◦ Expose root masses to gas phase containing fine droplets of liquid medium.

◦ Advantage- potential elimination of oxygen transfer problem