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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!
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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
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