5-cell cultures and secondary metabolites [compatibility mode]

7/30/2019 5-Cell Cultures and Secondary Metabolites [Compatibility Mode]

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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 ofCatharanthus

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 areproduced 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 bacteriasDNA, 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.

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.

Cultures of single cells and small cellaggregates that proliferate and complete agrowth cycle while suspended in liquid

medium..

Cell Suspension Cultures

Cell culture


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Rapidly dividing

Homogenous cells or cell aggregates

Suspended in a liquid medium

Cultured to produce a cell line

Suspension Cell Culture :Suspension Cell Culture :

Single Cell Clones :Single Cell Clones :

Cell lines established from single cell origin.

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Medium Effects on Tobacco Callus Morphology

0.1 mg/L kinetin3.0 mg/L 2,4-D

2.0 mg/L IAA3.0 mg/L 2-iP

friable callus compact callus

Medium that results in friable callus proliferation, high

auxin relative to cytokinin, w/o agar

!

Sieve (300 to500 m) to filtersuspension

2nd Passage1st PassageFriable Callus

Procedure for Initiation of a Cell SuspensionCulture from Callus

Introduction into suspension

+

Plate out

Sieve out lumps

1 2

Pick off

growing

high

producers

Initial high

density

Subculture

and sieving

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Sigmoidal (S) growth :

Lag phase -- Logarithmic (log, exponential) -- Stationary phase

Growth patterns in suspension cell cultureGrowth patterns in suspension cell culture

Lag

Log

Stationary

Exponential

Linear

Progressivedeceleration

Plateform shaker :

speed :

stroke range :

Small batch culture

30-150 rpmOrbital shaker

1. Interval of subcultureSubculture at stationary phase -->

Long growth cycle

Longlag phase

Factors affect the growth cycle :

1. Interval of subcultureSubculture at log phase -->

1. Interval of subcultureSubculture at log phase -->

Short Short


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Factors :

2. Initial cell densityHigh initial cell density -->

2. Initial cell densityHigh initial cell density --> Low initial cell density -->Low initial cell density -->

short lag phase

few cell division

long lag phase

long exponential growth

Critical initial density -->

Factors :

2. Initial cell density2. Initial cell density

In general, 0.5 2.5 X 105

cells / mL

4 6 division

1 4 X 106

cells / mL

II. Continuous culture


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bioreactor-culture offers many advantages,including:- 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 thedevelopmental stage;- Filtration of the medium for exudates; contamination control; and- production of clusters of buds or somatic embryos for automated

handling of the propagules.

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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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 tothe 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 linkedto cell differentiation.

some hairy roots is their ability to occasionally excrete thesecondary metabolites into the growth medium

Characteristics of the Hairy Roots Cultures

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1. Proliferate by increasing the rate of cell division (cytokineexpression) and cell elongation (auxin expression) toproduce the hairy roots.

2. Produce the opines which is a type of unusual aminoacids (octopine, agropine,nopaline, mannopine, andcucumopine) which is used by the bacterium as a carbon,nitrogen and energy source.

Agrobacterium cell Plant cell

Ri-plasmid


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Structure of Ri-plasmid (root inducing plasmid)

Ri-Plasmid

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,

Clone Generation

PlasmidConstruction in E. coli

ATCC 15834A. rhizogenes

Ri

SterileGrownPlants(5 weeks)

Infection

Desiredgene

(6 weeks) SelectionMedia(6 weeks)

Adapt toLiquidMedia(16weeks)

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!

Transfer of

Ti/Ri Plasmind

in plant cell

/rhizogenes

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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 compoundsin a number of biological systems

&


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Elicitors Reported

effects onPhysical

elicitors

Injury P

Abi ot ic M etal i ons ( lant han um, eur opi um, cal ci um, sil ver, cad miu m), oxal at e Pc

Chemica

l elicitors

Bi ot ic Compl ex

composition

Yeast cell wall, Mycelia cell wall, Fungal spores Pc, F

Defined

Composition

Carbohydrates Polysaccharides Alginate Pc, F, B

LBG F

Pectin Pc, F

Chitosan Pc

Guar Gum Pc

Oligosaccharides Mannuronate F

Guluronate F

Mannan F

Galacturonides Pc

Proteins Peptides Glutathione Pc

Proteins Cellulase, Elicitins, Oligandrin Pc

Lipids Lipopolysaccharides Pc

Glycoproteins Not characterized Pc

Volatiles C6-C10 Pc

Elicitors of plants and microbial cells

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

No Elicitor Culture Metabolites

1 -linked glucopyranosyl Glycine max Phytoalexins

2 -1,4-Oligogalacuronide Glycine max Phytoalexins

3 Chitosan N. tabacum Phytoalexins

4 Hepta--glucoside Glycine max Phytoalexins

5 Pectic oligomer Citrus lemon Phytoalexins

6 -1,5-1,3-Glucans Glycine max Isoflavonoids

7 Chitin, alginate, guar gum, pectin Morinda citrifolia Anthraquinones

8 Chitin Papaver sammiferum Sanguinarine

9 -D-Glucans N. Tabacum Disease resistance

Carbohydrate elicitors and metabolites in plant cell cultures

10 Chitosan Lupinus albus Isoflavonoid

11 Oligogalacturonoides N. tabacum H2O2

12 Chitin, chitosan oligosaccarides Taxus canadensis Taxol

13 mannan Hypericum perforatum hypericins

14 Chitosan Rheum palmatum Anthranilate

15 N-Acetylchito-oligosaccaride Avena sativa Anthranilate

16 B-glucan Glycine max H2O2

17 N-Acetylchitohexaose Taxus canadenis taxol

5-cell cultures and secondary metabolites [compatibility mode]

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