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substances that are widely distributed in nature, occurring virtually in all organisms In higher plants these substances would be concentrated in seeds and vegetable storage organs.

There are needed for general growth and development. Primary metabolites are low value-high bulk commodity items from plants (e.g. amino acids, starch, sugars, vegetable oils, etc.)

are biosynthetically derived from primary metabolites... They are more limited in distribution being found usually in specific families. They are not necessary for growth and development, but may serve as pollination attractants, environmental adaptations, or protection.

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Establishing a plant cell culture for secondary metabolite production is a complex problem

Within a specific cultivar of Catharanthus roseus, 62% of the clones produced the desired metabolite Whereas in another only 0.3% produced the metabolite

Culture conditions must be optimized

concentrations of sugar, hormones, and vitamins light temperature

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Metabolite production is frequently higher in cell cultures

Berberine production from Coptis japonica is about 5% of dry weight after 5 years of root growth, which equals 0.17 mg/g per week. Whereas in selected cell lines it can be 13.2% of the dry weight in cell culture after 3 weeks, which is about 44 mg/g/week or about 250 times higher

Many secondary metabolites are produced in roots

Scientists have developed a form of root culture using Agrobacterium rhizogenes, the cause of hairy root disease. Cells transformed with some of the bacterias DNA, causes the cells to be more sensitive to the hormones they produce. The cells form into roots. These roots grow very fast and produce the secondary metabolites that ordinary roots produce.

Cell Suspension Cultures

Roots often secrete the metabolites into the surrounding medium, making it easy for collection. Charcoal can be added to the medium, the metabolites are absorbed by the charcoal, and this stimulates even higher production of the metabolite.
Cell culture

Cultures of single cells and small cell aggregates that proliferate and complete a growth cycle while suspended in liquid medium..

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Suspension Cell Culture :

Rapidly dividing Homogenous cells or cell aggregates Suspended in a liquid medium Cultured to produce a cell line

Single Cell Clones :

Cell lines established from single cell origin.

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Procedure for Initiation of a Cell Suspension Culture from Callus

Cell suspensions typically are initiated by inoculating the friable callus into liquid medium

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Individual cells and/or cell aggregates are maintained in suspension by agitation or aeration, which also minimizes hypoxia. When callus pieces are agitated in a liquid medium, they tend to break up After the initial passage, culture is typically filtered to eliminate large tissue masses

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Techniques of cell culture

Initiation of cell suspension culture Fragments of undifferentiated callus Liquid medium aeration -------2 - 3 g / 100 mL

Introduction of callus into suspension

Friable callus goes easily into suspension.
2,4-D Low cytokinin semi-solid medium enzymic digestion with pectinase blending

agitation -------Subculture

Suspension cell cultures screen small size clumps

Removal of large cell aggregates by sieving. Plating of single cells and small cell aggregates - only viable cells will grow and can be reintroduced into suspension.

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Medium that results in friable callus proliferation, high auxin relative to cytokinin, w/o agar Medium Effects on Tobacco Callus Morphology
0.1 mg/L kinetin 3.0 mg/L 2,4-D 2.0 mg/L IAA 3.0 mg/L 2-iP

Procedure for Initiation of a Cell Suspension Culture from Callus

Sieve (300 to 500 m) to filter suspension

Friable Callus
friable callus compact callus

1st Passage

2nd Passage

Introduction into suspension

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Single cell clones

plating culture

Bergmann (1960), Nicotiana tabacum, Phaseolus vulgaris, Single cell isolated from plant tissue materials suspension cell cultures sieve cells to obtain true single cell

single cells suspended in agar medium layer, ca. 1mm thick observe the cell growth with inverted microscope,

plating culture count the plating efficiency : single cell clones No. of colony formed / No. of cells plated

plating culture

plating culture

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Cell Suspension Culture Types and Growth Patterns

H. hookerianum: Red pigmented callus cultures

Liquid suspensions of plant cells are grown using a variety of systems that keep the medium aerated and facilitate cell separation Gyratory or reciprocating shakers or chemostatic airlift fermentation systems

Culture types Batch culture medium volume is finite throughout the culture passage, i.e. growth continues until a nutrient becomes limiting (passage), usually Carbon (small to medium-scale) Continuous culture - medium is replenished during culture, i.e. sustained growth (bioreactors (large-scale production)

I. Batch culture
Bioreactor Shaker

finite amount of medium that is not replenished and final cell mass is dependent on the quantity of the limiting nutrient

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Growth cycle phases - typical growth cycle is 3 to 6 cell doublings,

12 to 14 days in duration Lag phase - cells activate metabolism for cell proliferation, conditioning is occurring

Shakers

Glasses

Glasses for shakers

Exponential phase - cell mass gain is exponential due primarily to cell division Linear/progressive deceleration phase - linear mass gain due primarily to cell expansion Stationary phase - growth ceases

Glasses for shakers Glasses for shakers

Rotary shaker

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Growth patterns in suspension cell culture

Lag phase -- Logarithmic (log, exponential) -- Stationary phase
Stationary Progressive deceleration
Sigmoidal (S) growth :

Small batch culture

Plateform shaker : speed : stroke range : Orbital shaker 30-150 rpm

Log

Linear

Lag

Exponential

Factors affect the growth cycle : 1. Interval of subculture Subculture at log phase -->

1. Interval of subculture Subculture at stationary phase

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Long lag phase

Long growth cycle

Short

Short

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Factors :

Factors :

2. Initial cell density High initial cell density --> short lag phase few cell division

Low initial cell density --> long lag phase long exponential growth

2. Initial cell density Critical initial density -->

In general, 0.5 2.5 X 10 cells / mL 4 6 division 1 4 X 10 cells / mL

II. Continuous culture

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- better control of the culture conditions; - optimal supply of nutrients and growth regulators; - renewal of the culture atmosphere; - changing the medium during the culture period according to the developmental stage; - Filtration of the medium for exudates; contamination control; and - production of clusters of buds or somatic embryos for automated handling of the propagules.

bioreactor-culture offers many advantages, including:

Cell cultures can be grown on shakers or in fermentors

Various steps involved in cell culture

Hairy root cultures:

Definition: It is the culture produced after the infection of explants or cultures by the gram negative soil bacterium Agrobacterium rhizogenes. This processes take advantage of the naturally occurring hairy root disease in Dicotyledons.

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Characteristics of the Hairy Roots Cultures

Hairy roots are fast growing and laterally highly branched, able to grow in hormone-free medium. not susceptible to geotropism anymore. genetically stable produce high contents of secondary metabolites characteristic to the host plant. The secondary metabolite production of hairy roots is stable compared to other types of plant cell culture. The secondary metabolite production of hairy roots is highly linked to cell differentiation. some hairy roots is their ability to occasionally excrete the secondary metabolites into the growth medium

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Agrobacterium cell

Plant cell

1. Proliferate by increasing the rate of cell division (cytokine expression) and cell elongation (auxin expression) to produce the hairy roots. 2. Produce the opines which is a type of unusual amino acids (octopine, agropine,nopaline, mannopine, and cucumopine) which is used by the bacterium as a carbon, nitrogen and energy source.

Ri-plasmid

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Structure of Ri-plasmid (root inducing plasmid)

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Clone Generation
Plasmid Constructio n in E. coli Desired gene Ri Sterile Grown Plants (5 weeks) ATCC 15834 A. rhizogenes Adapt to Liquid Media (16 weeks)

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Infection (6 weeks) Selection Media (6 weeks)

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Transfer of Ti/Ri Plasmind in plant cell

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Normal Root Cultures

Hairy Root Cultures

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Roots often secrete the metabolites into the surrounding medium, making it easy for collection. Charcoal can be added to the medium, the metabolites are absorbed by the charcoal, and this stimulates even higher production of the metabolite.

model for metabolic engineering (

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Elicitor are substances that, when applied in very small concentrations, enhance the biosynthesis of specific compounds in a number of biological systems

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Elicitors of plants and microbial cells

Elicitors Physical elicitors Chemica l elicitors Injury Abiotic Biotic Complex composition Defined Composition Metal ions (lanthanum, europium, calcium, silver, cadmium), oxalate Yeast cell wall, Mycelia cell wall, Fungal spores Carbohydrates Polysaccharides Alginate LBG Pectin Chitosan Guar Gum Oligosaccharides Mannuronate Guluronate Mannan Galacturonides Proteins Lipids Glycoproteins Volatiles Peptides Proteins Glutathione Cellulase, Elicitins, Oligandrin Lipopolysaccharides Not characterized C6-C10 Reported effects on P Pc Pc, F Pc, F, B F Pc, F Pc Pc F F F Pc Pc Pc Pc Pc Pc

Carbohydrate elicitors and metabolites in plant cell cultures

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Elicitor -linked glucopyranosyl -1,4-Oligogalacuronide Chitosan Hepta- -glucoside Pectic oligomer -1,5-1,3-Glucans Chitin, alginate, guar gum, pectin Chitin -D-Glucans Chitosan Oligogalacturonoides Chitin, chitosan oligosaccarides mannan Chitosan N-Acetylchito-oligosaccaride B-glucan N-Acetylchitohexaose Culture Glycine max Glycine max N. tabacum Glycine max Citrus lemon Glycine max Morinda citrifolia Papaver sammiferum N. Tabacum Lupinus albus N. tabacum Taxus canadensis Hypericum perforatum Rheum palmatum Avena sativa Glycine max Taxus canadenis Metabolites Phytoalexins Phytoalexins Phytoalexins Phytoalexins Phytoalexins Isoflavonoids Anthraquinones Sanguinarine Disease resistance Isoflavonoid H2O2 Taxol hypericins Anthranilate Anthranilate H2O2 taxol

Abbreviations: P, plants; Pc, plant cell culture; B, bacterial cell culture; F, fungal cell culture

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