Transcript Hairy root culture

Plant Cell Technology
Plant Parts and their main functions
Leaf (Photosynthesis)
Shoot (Mechanical support,
Transport of food)
Root ( Water and mineral
supply)
The Architecture of Plants
Structure of Plant Cell
Organelles Specific to Plant Cells
The characteristics of plant, animal and
microbial cultures
Characteristics
Microbial cells
Characteristics
Plant cell suspensions
Animal cell suspensions
Size
2-10µm
10-20 µm
5-100 µm
Individual cells
Often
Aggregates up to 2mm
generally form
Often, also many require
a surface for growth
Growth Rate
Rapid, doubling times
of 1-2 hrs
Slow, doubling time of
2-5 days
Slow, doubling time
12-20 hrs
Shear stress
sensitivity
Not sensitive
Sensitive and tolerant
sensitive
Aeration requirements
High
Low
low
Cultivation time
2-10 d
2-4 weeks
3-7 d
Product accumulation
Often extracellular
Mostly intracellular
Often extracellular
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Plants are obvious source for food, fiber and
fuel. Besides these plants are a source of
diverse array of chemicals as flavors,
fragrances, natural pigments, pesticides and
pharmaceuticals
( Plants derived
secondary metabolites) thus plants are
invariably the integral part of human life.
Plant Cell Culture
Plant Part
(Leaf, Shoot, Root, Embryo)
Callus culture
(Solid/Semi solid media)
Suspension culture
(Liquid media)
Bioreactor
Development of Callus Culture:Any plant part which contain the highest amount of
desired compound is taken and kept on a defined
media which contains all the nutrients required for
plant cell growth and particular growth hormones
and incubated under certain physical conditions of
temp, light/dark period etc. under these conditions
the organized plant part is converted into an
unorganized growth and forms callus. Thus callus
is unorganized growth of plant cells in vitro on a
culture medium. This callus produces the same
chemical compounds which are produced by the
mature intact plant.
Development of Suspension Culture and
Scale-up:Callus is transferred in liquid media and various
culture parameters are optimized to enhance the
yield of desired compound. For scale-up
suspension culture is grown in Bioreactor and
large-scale production of plant derived secondary
metabolite is facilitated.
Advantages of producing compounds from
Plant Cell Culture
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Control of supply of product independent of availability of
plant itself and climatic, geographical and governmental
restrictions etc.
High growth and turnover rate as compared to natural plant.
Reduction in time and space requirement for the production
of desired chemicals.
Strain improvement with programs analogous to those used
for microbial system.
Applications of Plant Cell Culture
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Production of plant derived chemicals
Development of transgenic plants
Mass multiplication of desirable genotype of
plants (Micropropagation)
Production of pathogen free plants
Compounds which are commercialized
from Plant Cell Culture Technology
Compound
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Shikonin `
Ginseng
Taxol
Vincristine &
Vinblastin
Berberin
Plant
Lithospermum
erythrorhizon
Panax ginseng
Use
Pigment
Health tonic
Taxus bacctaAnti-Cancer
Drug
C. roseus
Anti-Cancer
Drug
Coptis japonica
Anti-malarial
Callus culture of some commercially
important plants
Podophyllum
hexandrum
Azadirachta
indica
Linum album
Protocol for establishment of plant cell suspension
cultures
Various steps involved in cell culture
Setric impeller
Batch cultivation
Callus culture
Germinated seedling
Batch cultivation with fluorescence probe
Suspension culture
Seeds of P. hexandrum
Continuous cultivation
with cell retention
What bioreactor is?
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A vessel, made up of glass or steel, in
which plant cells are cultivated under
controlled environment to obtain a
desired product
Basic parts of bioreactor
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A culture vessel
Associate supply and environmental
systems
Measurement and control systems
Bioreactors for cultivation of plant cells
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Stirred tank bioreactor
Air-lift bioreactor
Rotating drum bioreactor
Spin filter bioreactor
Stirred tank bioreactor
Air-Lift Reactors
Spin Filter Bioreactor
Plant Tissue CultureDifferent Approaches for Production
of Secondary Metabolites
Plant Cell
Suspension Culture
(Plant cell suspension cultures are generated by
transferring the callus tissue in liquid media)
Tissue Culture
Hairy Root Culture
(Hairy root cultures are obtained by infection
of Agrobacterium rhizogenes, a gram negative
soil bacterium)
Induction of Hairy Roots by
Agrobacterium rhizogenes
Wounded plant cells
Transfer of
Ti/Ri Plasmind
in plant cell
Signal Molecules
Recognition by
Agrobacterium
Attachemnt of
Agrobacterium With plant cells
/rhizogenes
Transfer of Ri plasmid to
wounded plant cells
Co-Cultivation
Integration of Ri plasmid
into plant genome
Hairy Root Induction
Advantages of Hairy Root Culture Over
Plant Cell Suspension Culture
Fast
growth
Low doubling time
Genetic and biochemical stability
Growth
in hormone free media.
These fast growing hairy roots can be used as a
continuous source for the production of
valuable secondary metabolites.
Induction of hairy roots
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Hairy roots appear within one to four weeks
of infection.
In some plant species hairy roots may appear
directly at the site of inoculation.
While in others a callus will form initially and
hairy roots appear subsequently from it.
Hairy root culture
Establishment of axenic hairy root lines
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Excise the transformed roots from the explant
after it grows more than 1 cm in its length.
Transfer these excised roots to the same solidified
growth medium with antibiotic to kill the
bacterium.
After appearance of lateral branching roots may be
transferred to the liquid medium.
Established roots may be cleared of bacteria by
several passages in the medium containing 250
mg/l Cefotaxime and 250 mg/l ampicillin.
Each root growth represents a single root line .
Measurement of growth
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By direct methods
(Biomass- drain and weigh)
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By indirect methods
(Conductivity, nutrient consumption profile)
Cultivation of hairy roots in
bioreactors
The ability to exploit hairy root culture as a
source of bioactive chemicals depends on
development of suitable bioreactor
system.
Challenges in bioreactor designing
Hairy roots are complicated biocatalysts when it comes to scaling up.
The main challenges for development of bioreactor for hairy roots
are-
• Shear sensitivity of hairy root system.
• Requirement for a support matrix.
• Restriction of nutrient/oxygen delivery to the central mass of
tissue.
• Resistance to flow due to interlocked matrix because of extensive
branching of roots.
Bioreactors for hairy root cultures
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Stirred tank bioreactor
Air lift bioreactor
Bubble column bioreactor
Turbine blade bioreactor
Mist (Trickle bed) bioreactor
Rotating drum bioreactor
Spin filter bioreactor
Bioreactor designs: A comparison
Bioreactor
Designs
Advantages
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Not suitable for HR
cultures because of
wound response &
callus formation.
Successful for hairy
roots as hairy roots
require low oxygen
supply.
Less hydrodynamic
stress
Uniform flow
pattern
Low operation cost
Better top to
bottom mixing
Dead zones,
Insufficient mixing,
rupture due to
collision
Bubbles cause less
shear stress
•Absence of moving
parts
•Ease in aseptic
condition
maintenance
Dead zones,
Insufficient mixing
Stirred Tank (STR)
Airlift or Submerged
Bubble Column
Like the Air-Lift
reactor.
Shortcomings
•
Bioreactor designs: A comparison
Bioreactor
Designs
Advantages
Easy operation
•High oxygen
tranfer
•Lack of shear
•Easiness of scaling
up
•Gas composition
can be controlled
•Pressure drop is
low
Shortcomings
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Mist
Bioreactor/Trickle
bed
Minimum shear
stress
•High oxygen
tranfer ability
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Rotating drug
bioreactor
Rotating filter
allows for spent
medium removal &
fresh medium
addition.
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Spin filter
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Bioreactors for cultivation of hairy roots
S.No.
Bioreactor
configuration
Plant species
References
1
Stirred tank
D. Stramonium
Hilton et al.,
1988
2
Stirred tank
T. petula
Buitellaar,
1991
3
Stirred tank
T. foenum graecum
Rodrigues et
al., 1991
4
Turbine blade
B. vulgaris
Dilorio et al.,
1992
5
Turbine blade
C. Sepium
Dilorio et al.,
1992
6
Turbine blade
P. ginseng
Inomata et
al., 1993
Bioreactors for cultivation of hairy roots
S.No.
Bioreactor
configuration
Plant species
References
7
Airlift
N. rustica
Rhodes et
al., 1986
8
Airlift
C. roseus
Toivonen et
al., 1993
9
Airlift
L. album
Arroo et al.,
2002
10
Airlift, batch
A. belladonna
Jung and
Tepfer, 1987
11
Airlift, batch
A. rusticana
Taya et al.,
1989
12
Airlift, continuous
D. Stramonium
Hilton et al.,
1988
Bioreactors for cultivation of hairy roots
S.No.
Bioreactor
configuration
Plant species
References
13
Airlift packed column with
amberlite XAD-2
L. erythrorhizon
Shimomura
et al., 1991
14
Airlift batch followed by
continuous
N. rustica
Rhodes et
al., 1986
15
Trickle bed
B. vulgaris
Dilorio et al.,
1992
16
Trickle bed
C. tinctorius
Dilorio et al.,
1992
17
Trickle bed
D. carota
Kondo et al.,
1989
18
Trickle bed
A. annua
Weathers et
al., 2000
Bioreactors for cultivation of hairy roots
S.No.
Bioreactor
configuration
Plant species
References
19
Trickle bed
H. muticus
Mckelvery,
1992
20
Trickle bed
H. muticus
Flores and
Curtis, 1992
21
Bubble column
H. muticus
Mckelvery,
1992
22
Bubble column
S. tuberosum
Hilton and
Rhodes,
1991
23
Bubble column
L. erythrorhizon
Sim and
Chang, 1993