Plant Cell Technology. Plant Parts and their main functions Leaf (Photosynthesis) Shoot (Mechanical...
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Transcript of Plant Cell Technology. Plant Parts and their main functions Leaf (Photosynthesis) Shoot (Mechanical...
![Page 1: Plant Cell Technology. Plant Parts and their main functions Leaf (Photosynthesis) Shoot (Mechanical support, Transport of food) Root ( Water and mineral.](https://reader036.fdocuments.us/reader036/viewer/2022062518/56649ece5503460f94bda7f7/html5/thumbnails/1.jpg)
Plant Cell Technology
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Plant Parts and their main functions
Leaf (Photosynthesis)
Shoot (Mechanical support, Transport of food)
Root ( Water and mineral supply)
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The Architecture of Plants
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Structure of Plant Cell
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Organelles Specific to Plant Cells
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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.
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Plant Cell Culture
Plant Part(Leaf, Shoot, Root, Embryo)
Callus culture(Solid/Semi solid media)
Suspension culture(Liquid media)
Bioreactor
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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.
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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.
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Advantages of producing compounds from Plant Cell Culture
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.
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Applications of Plant Cell Culture
Production of plant derived chemicals Development of transgenic plants Mass multiplication of desirable genotype of
plants (Micropropagation) Production of pathogen free plants
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Compounds which are commercialized from Plant Cell Culture Technology
Compound Plant Use
Shikonin ` Lithospermum Pigment erythrorhizon
Ginseng Panax ginseng Health tonic Taxol Taxus baccta Anti-Cancer
Drug Vincristine & C. roseus Anti-Cancer Vinblastin Drug Berberin Coptis japonica Anti-malarial
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Callus culture of some commercially important plants
Podophyllum hexandrum
Azadirachta indica
Linum album
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Protocol for establishment of plant cell suspension cultures
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Seeds of P. hexandrum
Germinated seedling
Callus culture
Suspension culture
Batch cultivation
Batch cultivation with fluorescence probe
Continuous cultivation with cell retention
Setric impeller
Various steps involved in cell culture
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What bioreactor is? A vessel, made up of glass or steel,
in which plant cells are cultivated under controlled environment to obtain a desired product
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Basic parts of bioreactor A culture vessel Associate supply and
environmental systems Measurement and control systems
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Bioreactors for cultivation of plant cells
Stirred tank bioreactor Air-lift bioreactor Rotating drum bioreactor Spin filter bioreactor
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Stirred tank bioreactor
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Air-Lift Reactors
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Spin Filter Bioreactor
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Plant Tissue Culture- Different 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)
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Induction of Hairy Roots by Agrobacterium rhizogenes
Wounded plant cells
Signal Molecules Recognition by
Agrobacterium Attachemnt of Agrobacterium With plant cells
Transfer of Ri plasmid to wounded plant cells
Co-Cultivation
Integration of Ri plasmid into plant genome
Hairy Root Induction
Transfer of Ti/Ri Plasmind
in plant cell
/rhizogenes
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Advantages of Hairy Root Culture Over Plant Cell Suspension Culture
Fast growthLow doubling timeGenetic 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.
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Induction of hairy roots
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.
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Hairy root culture
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Establishment of axenic hairy root lines
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 .
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Measurement of growth
By direct methods (Biomass- drain and weigh)
By indirect methods (Conductivity, nutrient consumption
profile)
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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.
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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.
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Bioreactors for hairy root cultures
Stirred tank bioreactor Air lift bioreactor Bubble column bioreactor Turbine blade bioreactor Mist (Trickle bed) bioreactor Rotating drum bioreactor Spin filter bioreactor
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Bioreactor designs: A comparison
Bioreactor Designs Advantages Shortcomings
Stirred Tank (STR)- Not suitable for HR
cultures because of wound response & callus formation.
Airlift or SubmergedSuccessful for hairy roots as hairy roots require low oxygen supply.Less hydrodynamic stressUniform flow patternLow operation costBetter top to bottom mixing
Dead zones,Insufficient mixing, rupture due to collision
Bubble Column Like the Air-Lift reactor.
•Bubbles cause less shear stress•Absence of moving parts•Ease in aseptic condition maintenance
Dead zones,Insufficient mixing
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Bioreactor designs: A comparison
Mist Bioreactor/Trickle
bed
•Easy operation•High oxygen tranfer •Lack of shear•Easiness of scaling up•Gas composition can be controlled•Pressure drop is low
-
Rotating drug bioreactor
•Minimum shear stress•High oxygen tranfer ability
-
Spin filter
•Rotating filter allows for spent medium removal & fresh medium addition.
-
Bioreactor Designs Advantages Shortcomings
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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
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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
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Bioreactors for cultivation of hairy rootsS.No. Bioreactor
configurationPlant species Referenc
es
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
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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