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

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

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

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 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

Callus culture of some commercially important plants

Podophyllum hexandrum

Azadirachta indica

Linum album

Protocol for establishment of plant cell suspension cultures

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

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

Basic parts of bioreactor A culture vessel Associate supply and

environmental systems Measurement and control systems

Bioreactors for cultivation of plant cells

Stirred tank bioreactor Air-lift bioreactor Rotating drum bioreactor Spin filter bioreactor

Stirred tank bioreactor

Air-Lift Reactors

Spin Filter Bioreactor

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)

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

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.

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.

Hairy root culture

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 .

Measurement of growth

By direct methods (Biomass- drain and weigh)

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

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 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

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

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 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

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