ANIMAL AND PLANT CELL REACTOR TECHNOLOGY:

Growth of animal cells in culture is currently used for manufacture of - Vaccines, proteolytic enzymes, Mabs, Interferons, etc. These show substantial potential on production of lymphokines, other enzymes, growth factors, clotting factors, hormones, etc. Though r-DNA technology provides the opportunity of expressing foreign proteins in microorganisms, animal cell cultivations also competes for the same. Usually proteins synthesized in animal cells are often subjected to PTM, but this will not happen in prokaryotes. Molecules that required PTMs are better cultivated in eukaryotes.

Animal Cell Culture

Differences between procaryotes and eucaryotes

Eucaryotes Procaryotes

size 10-30 um 1-2 um

shape

spherical,

ellipsoidal

rods, ellipses,

etc.

locomotion no yes

border membrane wall

Cells are negatively charged.

• attach to positively charges surfaces

• some cells must attach to grow, others not

• examples of surfaces: sephadex, collagen

• positively charged vesicles will attach to cell surfaces and be taken into the cell

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Animal Cell Culture Technique

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Historical events - in the development of cell culture

• 1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a living system after the death of an organism.

• 1885: Roux maintained embryonic chick cells in a saline culture.

• 1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma.

• 1903: Jolly observed cell division of salamander leucocytes in vitro.

• 1907: Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method and observed the growth of nerve fibers in vitro for several weeks. He was considered by some as the father of cell culture.

• 1910: Burrows succeeded in long term cultivation of chicken embryo cell in plasma clots. He made detailed observation of mitosis.

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Contd..

• 1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth.

• 1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods.

• 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells.

• 1923: Carrel and Baker developed 'Carrel' or T-flask as the first specifically designed cell culture vessel. They employed microscopic evaluation of cells in culture.

• 1927: Carrel and Rivera produced the first viral vaccine - Vaccinia.

• 1933: Gey developed the roller tube technique

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Contd..

• 1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture.

• 1948: Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer developed a chemically defined medium, CMRL 1066.

• 1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent monolayers of cultured cells.

• 1954: Abercrombie observed contact inhibition: motility of diploid cells in monolayer culture ceases when contact is made with adjacent cells.

• 1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.

• 1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture.

• 1964: Littlefield introduced the HAT medium for cell selection.

• 1965: Ham introduced the first serum-free medium which was able to support the growth of some cells.

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Contd..

• 1965: Harris and Watkins were able to fuse human and mouse cells by the use of a

virus.

• 1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody.

• 1978: Sato established the basis for the development of serum-free media from cocktails of hormones and growth factors.

• 1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent.

• 1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use.

• 1986: Lymphoblastoid γIFN licensed.

• 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available.

• 1989: Recombinant erythropoietin in trial.

• 1990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2).

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Growth Medium

• glucose, glutamine, amino acids,

• serum: liquid extracted from blood of offspring removed from freshly-killed pregnant cows.

• proteins: cell attachment factors; metal binding proteins; protease inhibitors

• peptides: various growth factors

• hormones: stimulate growth and nutrient uptake

• nutrients

• metabolites

• minerals

: Plasma

: Interstitial fluid

: Embryo extract

Growth factors

* cell require nutrients for use as substrate, catalysts or cofactor *

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Commercial cell culture media

: Minimum Essential Media (MEM)

: Dulbecco’s Modified Eagle Media (DMEM)

: Opti-MEM

: RPMI-1630 (for suspension cell)

: RPMI-1640 (for mammalian cell)

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Metabolism

• Animal cells can synthesize glucose from pyruvate via gluconeogenesis pathway

• waste: lactate, ammonia

• at high levels, these are toxic

• challenge for high density cultures

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Cultivation of Animal Cells

1. Tissues are removed from animals and transferred to growth medium

2. Organs -> lung, kidney, etc. (cells grow attached)

3. These are primary cultures

4. Cells can be transferred to new flasks once they have grown into a monolayer

• Remove cells with a protease – trypsin, collagenase, pronase or EDTA

• Wash cells with serum containing medium (centrifuge gently)

• Resuspend in growth medium

• Plate onto a fresh flask

Differentiated mammalian cells are mortal, however, cancer cell lines are immortal.

Animal cell lines include: mammal, insect, fish, crustaceans

Insect cells are easier to grow. They grow faster and you can use a baculovirus as a vector for genetic engineering. Insect cells may not have post-translational modifications like mammalian cells.

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Other Cells for Cultivation

• Skin, epithelium cell, eye, kidney, liver, ovary

• glands, bone, nerve

• connective tissue, Skeletal muscle

• tooth primmordia, bone marrow, lymphocyte

• From young animals

• Cell cloning, Separation, Hybrids,

Large scale production

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Commonly used cell lines

• Chinese Hamster Ovary cells: CHO cells

• HeLa cells

• mouse kidney cells

Commonly used medium

• nutrients + 5-20% serum ($100-$500 per liter)

Problems with serum

• cost

• virus – safety issues

• extra-cellular proteins

• lot-to-lot variation

• availability

• foaming

Book: serum-free media contains insulin, transferrin, fibronectin, other protein components

Serum-free media can also be protein-free

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Hybridoma Cells

• antibody-producing lymphocytes fused with cancer cells – myeloma

• lymphocytes grow slowly and are mortal, hybridoma cells are immortal and produce antibodies

Production of antibody fragments by fungi and bacteria

See Nyyssonen and Eini; Bio/Technology 1993 vol 11(5) p. 591.

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Tissue culture

• Organ culture

: Maintain original structure and ability to differentiation

• Primary explant culture

: 1-3 mm in size

• Cell culture

: from single cell

: Lack cell-cell interaction and differentiation

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Growth Characteristics of Transformed cells

• Transformed cells possess some common characteristics

but are not equivalent to cancer cells

• Grown as Multilayer instead of Monolayer

• Can grow in Suspension

• Long life span

• Less serum growth factor requirement

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Types of cell culture

• Primary cell culture

• Secondary cell culture

• Diploid cell line

• Continuos cell line (or Establish cell line)

*** Grown as Monolayer or in Suspension ***

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Monolayer culture

• Requires substrate or solid surface

for attachment and growth

: Anchorage dependent of growth

• Contact inhibition of movement: Monolayer

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Anchorage dependent of growth

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Suspension Culture

• Mostly hemopoietic cells and a few others

• Specialized culture medium may be required

• Cells with high metabolic activities can be obtain

• Cells that are not able to grow in suspension

: Those can be attached onto the surface

of carrier particles and grow

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Growth Requirement

• Metabolites and intermediates from other cells

• Population dependent growth requirement

: Diploid cells require a higher number to start with

• To create a suitable condition, cells can be grown in

“microenvironment” in capillary tube

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Stationary cultivation

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Culture flask

Culture Plate 3/9/2012 28

Large scale cultivation

Roller Bottle

Culture dish Stack culture chamber

Culture bottle

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Medium Constituents

* Balance salt solution : Phosphate buffer, Mg2+, Ca2+

* Inorganic ions and trace elements

: for membrane potential and osmotic pressure

: buffer

: Monovalent- and Divalent-cation

* Energy source : glucose, glutamine

* Amino acid : metabolism and biological synthesis

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Culture Medium Sterilization

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Role of Serum

• Buffer, Chelator, Carrier proteins

• Bind to toxin

• Protease inhibitor

• Promotes attachment of cell to substratum

• Source of Intermediate metabolites,

hormone and growth factor

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• culture vessel * Glass or Plastics

* Polystyrene (gamma ray treated)

* Polyvinyl chloride

* Polycarbonate

*Polytetrafluoresthulene

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• Microcarrier

* Polystyrene (gamma ray treated)

* Sephadex

* Polyacrylamide

Growth phase of Cells in culture

* Lag phase

: adapt to new environment; repair cell membrane damage

* Log phase

: exponential growth: 90-100% of cells are dividing

* Plateau or Stationary phase

: cell growth ~ 0-10%

: Contact inhibition of movement

: Density limitation of growth 3/9/2012 34

Advantages of using cell culture:

• Can be observed microscopically

• Genetic homogeneity

• Environment

(pH, Temp., osmotic pressure, O2 and CO2 tension)

• Rapid

• Requires less amount of material

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Disadvantage of using cell culture:

• Problem of contamination

• May be expensive than using,

and may not represent the condition in, intact animal

• Chromosome instability

Primary cell culture and Establishment of cell line

• Preparation of cell suspension from intact tissue

1. Single cell preparation

: use mechanical, Chemical, and/or enzymatic method

2. Disaggregate or dissociate cell

: cutting, homogenizing, rotary shaker, vortex,

pipette, teasing

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** Enzymes used **

• Trypsin (crude)

: from cattle and pig’s pancrease

: contain Chymotrypsin, elastase, ribonuclease,

deoxyribonuclease and amylase

• Collagenase

: for connective tissue

• Pronase

: for fibroblast

• Elastase

: for fibroblast protein

• Deoxyribonuclease

: for DNA

** Enzymes used **

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** Chelating agents used **

• Ethylene diamine tetraacetic acid (EDTA) or Versene

: bind permanently to Ca2+ and Mg2+

that maintain the cellular matrix

: prevent cell aggregation

: It’s better to use in combination with Trypsin

• Sodium citrate

• Source of tissue

: Young animals e.g. kidney (Monkey, Dog, Rabbit), Chick embryo

: Old animal tissue contains a large amount of connective tissue

Common Cell lines used for animal cell cultivation

• BHK-21 : 1961

: from bay hamster kidney

: FMD and Rabies vaccine for animal use

• CHO-K1: 1957

: from Chinese hamster ovary

: use in recombinant DNA technology

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• HeLa : 1952

: from Henrietta Lach; cancer tissue

: harbors HPV type 18 genome

• Vero : 1962

: from African green monkey kidney

: preparation of Poliovirus vaccine

Contamination sources of animal cells:

• fungal contamination

• Bacterial contamination

• Mycoplasma contamination

• Viral contamination

• Other cell line

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• Original tissue : primate virus, mycoplasma

• Biological: Serum

• Laboratory personnel : from body, aerosols

• Laboratory environment

: culture vessel cap

: humidified Incubator

: Water bath

: Insect

Animal Cell Storage:

*** Prevent genetic drift ***

: Freezing Medium

* Serum (~ 20-90%)

* Culture medium

* Cryoprotective agent : ~ 5-10% (DMSO, Glycerol)

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: Temp. decline rate 1-10oC/min

* (-20oC) Freezer

* (-70oC) Freeze : 6M-2 yr

* liquid nitrogen: Years

: cell concentration ~ 5 x 106-2 x 107 cells/ml

: % cell viability is decrease 2-3%/yr

** Slow Freeze : Quick Thaw **

Cell Thawing and culture

1. Quick thaw in 37oC water bath

2. Pipette to culture vessel

3. Slowly growth medium adding

4. Incubate for overnight

5. Refresh with new growth medium

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Subcultivation or Passage

Age of cells in culture can be determined by

* Number of subcultivation (Passage number)

* Number of population doublings

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• Kinetics of growth are similar to bacterial culture

• There is a difference between attached and suspended cultures

• Disposable bioreactors

• Cell growth measured by actual cell counts

“Hemocytometer”

Stain cells and drop on the slide – count all the white ones

Cell growth is measured in days.

Production can continue in non-growth conditions – hopefully!

Oxygen requirements: .06 - .2 x 10-12 mol O2/h/cell OUR ~ 0.1-0.6 mmol O2/l/hr

Compare to bacteria at 10 – 200 mmol/l/hr! 3/9/2012 43

Animal Cells are shear sensitive – cannot sparge reactors

• cells respond to shear with apoptosis

Fritted metal fittings create very small bubbles

Chemical (e.g. Pluronic F-68) can be added to provide shear protection

Typical kLa of suspension cultures (106 cells/ml) 5 – 25 hr-1

Bioreactor Considerations for Animal Cell Culture

Microcarriers: sephadex, etc: 70,000 cm2/liter: get ~ 107 cells/ml

• cells grow in mono – multi-layers on microcarriers

Hollow Fiber reactors

• cells grow on the outside of the tubes, nutrients pass through the tubes

• uncontrolled, unmixed environment

• get high cell concentrations (eg hybridoma demonstrated at 5 – 50 mg/ml antibody

Stirred-Tank reactors

• use pitched blade or other impeller (10 – 30 RPM for stirrers)

•Tank and bubble columns are used (especially with cells on multicarriers) 3/9/2012 44

Perfusion reactor

• simultaneous cell cultivation and product concentration and byproduct removal

• sonic separator

Products from Animal Cell Cultures

1. Immunobiologicals:

(i) monoclonal antibodies

(ii) immunobiological regulators

a) Used for diagnostic assay systems, therapeutics for biological separation systems (affinity chromatography)

b) Interferon

2. Virus Vaccines

3. Hormones: glycosylated peptides (e.g. erythropoetin)

4. Enzymes: TPA, collagenase,factor VII, factor VIII, factor X

5. Insecticides

6. Whole cells and tissue culture

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Work in Safety cabinet Class-II

: 30 min UV

: 70% ethanol for decontamination

Culture medium and Reagent

1. (1x) PBS

2. Growth medium

: 5%FCS-DMEM

3. ( 0.1%) Trypsin-Versene 3/9/2012 46

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PLANT CELL REACTOR TECHNOLOGY

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Differences between plant cells and microbes and their implications for Bioreactor design

Differences Implications for reactor design

Lower respiration rate Lower OTR required

More shear sensitive May require operation under low-shear conditions

Cells often grow as aggregates or clumps Mass transfer limitations

Degree of aggregation Optimal aggregate size for product

Volatile compounds may be important for cell metabolsim (ethylene)

May need to sparge gas mixtures

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Advantages and disadvantages of Plant cell cultivation

Advantages

• Can manipulate environment

• Can feed precursors

• Possible to select in culture

• Possible to get all cells in a culture producing.

• Can continuously extract.

• Can retain biomass

Disadvantages

• High cost

• Contamination

• Low intrinsic production

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Cost of production

• Plant cells are slow growing.

• Full of water (90% - 95%).

• Easily contaminated.

• Shear-sensitivity means specially modified fermenters necessary

• All this puts the cost of production of dry mass to $25 per kilogram. Product only a fraction of this.

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Plant cell culture systems

Organised

• Shoot cultures.

• ‘Hairy root’ cultures

• Embryo fermentations.

Unorganised

• Callus

• Cell suspension culture

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Shoot cultures

• Under conditions of high cytokinin, a culture producing a mass of shoots may be produced by adventitious shoot formation.

• For light-associated products, may be much more high yielding.

• Sensitive to shear

• Illumination a problem for scale up

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‘Hairy root’ cultures

• ‘Hairy roots’ are produced by infecting sterile plants with a natural genetic engineer, Agrobacterium rhizogenes.

• Genes for auxin synthesis and sensitivity are engineered into plant cells leading to gravity-insensitive mass root production.

• Very useful for products produced in roots.

• Aggregration and shear sensitivity are a major problem for scale-up

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Embryo Fermentations

• Somatic Embryos may be produced profusely from leaves or zygotic embryos.

• For micropropagation, potentially phenomenally productive.

• Shear sensitivity is a problem.

• Maturation in liquid is a problem.

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Shikonin production in culture

• Shikonin production in the intact plant

• Introduction into culture

• Optimisation of production through medium manipulations

• Fermentation

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Callus

• Equimolar amounts of auxin and cytokinin stimulate cell division. Leads to a mass proliferation of an unorganised mass of cells called a callus.

• Requirement for support ensures that scale-up is limited (Ginseng saponins successfully produced in this way).

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Cell suspension culture

• When callus pieces are agitated in a liquid medium, they tend to break up.

• Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support.

• Large scale (50,000 lit.) commercial fermentations for Shikonin and Berberine.

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Introduction of callus into suspension

• ‘Friable’ callus goes easily into suspension.

– 2,4-D

– Low cytokinin

– semi-solid medium

– enzymic digestion with pectinase

– blending

• Removal of large cell aggregates by sieving.

• Plating of single cells and small cell aggregates - only viable cells will grow and can be re-introduced into suspension.

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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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Growth kinetics

1. Initial lag dependent on dilution

2. Exponential phase (dt 1-30 d)

3. Linear/deceleration phase (declining nutrients)

4. Stationary (nutrients exhausted)

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16 18 20 22

Dry

weig

ht (g

/l)

time (d)

Plant Cell Suspension typical Growth curve

1

2

3 4

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Characteristics of plant cells

• Large (10-100mM long)

• Tend to occur in aggregates

• Shear-sensitive

• Slow growing

• Easily contaminated

• Low oxygen demand (kLa of 5-20)

• Will not tolerate anaerobic conditions

• Can grow to high cell densities (>300g/l fresh weight).

• Can form very viscous solutions

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Shear and plant cells

• Oxygen demand proportional to cell density.

• Shear rate proportional to viscosity

• shear rate proportional to **power of viscosity

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Special reactors for plant cell suspension cultures

• Modified stirred tank

• Air-lift

• Air loop

• Bubble column

• Rotating drum reactor

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Modified Stirred Tank

Standard Rushton turbine Wing-Vane impeller

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Airlift systems

Bubble column Airlift (draught tube)

Poor mixing

Airloop (External Downtube)

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Rotating Drum reactor

• Like a washing machine

• Low shear

• Easy to scale-up

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Ways to increase product formation

• Select

• Start off with a producing part

• Modify media for growth and product formation.

• Feed precursors or feed intermediates (bioconversion)

• Produce ‘plant-like’ conditions (immobilisation)

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Selection

• Select at the level of the intact plant

• Select in culture

– single cell is selection unit

– possible to plate up to 1,000,000 cells on a Petri-dish.

– Progressive selection over a number of phases

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Selection Strategies

• Positive

• Negative

• Visual

• Analytical Screening

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EXAMPLES OF PLANT PRODUCTS OF POTENTIAL COMMERCIAL INTEREST

1. Pharmaceuticals - Ajmalicine, atropine, berberine, codeine, digoxin, taxol,etc 2. Food colors & Dyes - Anthocyamins, betacyanins, saffron, shikonin 3. Flavors - Vanilla, strawberry, grape, onion, garlic 4. Fragrances - Jasmine, lemon, mint, rose, sandalwood 5. Sweeteners - Miraculin, monellin, thaumitin 6. Agriculture chemicals - Alloepathic chemicals, rotenone, salannin, etc.