Plant Parts and their main functions Leaf (Photosynthesis) Shoot (Mechanical support, Transport of food) Root ( Water and mineral supply)

Plant Cell Technology Plant Cell Technology

The Architecture of Plants

Structure of Plant Cell

Organelles Specific to Plant Cells

The characteristics of plant, animal and microbial cultures 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

Plants are obvious source for food, fiber and fuel 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.

(Leaf, Shoot, Root, Embryo) (Solid/Semi solid media) 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

Various steps involved in cell culture Setric impeller Batch cultivation Callus culture Germinated seedling Batch cultivation with fluorescence probe Suspension culture Continuous cultivation with cell retention Seeds of P. hexandrum

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 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 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 Advantages Shortcomings Stirred Tank (STR) - Not suitable for HR cultures because of wound response & callus formation. Airlift or Submerged 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 Bubble Column Like the Air-Lift reactor. Bubbles cause less shear stress Absence of moving parts Ease in aseptic condition maintenance Insufficient mixing

Bioreactor designs: A comparison Advantages Shortcomings 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.

Bioreactors for cultivation of hairy roots S.No. Bioreactor configuration Plant species References 1 Stirred tank D. Stramonium Hilton et al., 1988 2 T. petula Buitellaar, 1991 3 T. foenum graecum Rodrigues et al., 1991 4 Turbine blade B. vulgaris Dilorio et al., 1992 5 C. Sepium 6 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 C. roseus Toivonen et al., 1993 9 L. album Arroo et al., 2002 10 Airlift, batch A. belladonna Jung and Tepfer, 1987 11 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 C. tinctorius 17 D. carota Kondo et al., 1989 18 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 Flores and Curtis, 1992 21 Bubble column 22 S. tuberosum Hilton and Rhodes, 1991 23 L. erythrorhizon Sim and Chang, 1993