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Agricultural Applications of Biotechnology

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Title: Agricultural Applications of Biotechnology


1
Agricultural Applications of Biotechnology
2
What is agricultural biotechnology?
  • Agricultural biotechnology is a range of tools,
    including traditional breeding techniques, that
    alter living organisms, or parts of organisms, to
    make or modify products improve plants or
    animals or develop microorganisms for specific
    agricultural uses. Modern biotechnology today
    includes the tools of genetic engineering.

3
How is Agricultural Biotechnology being used?
  • Crop production
  • Phytoremediation
  • Improvements in agriculture not involving plants

4
  • Agricultural Biotechnology
  • Genetically engineered, pest-resistant plants
  • Foods with higher protein or vitamin content
  • Drugs developed and grown as plant products
  • Estimated to be a 7 billion market in 2008

5
What are the new products of agricultural
biotechnology?
  • Insect resistant crops commercially available,
    e.g., Bt corn, cotton, and potatoes
  • Corn rootworm resistance in 2001

6
What are the new products of agricultural
biotechnology?
  • Animal growth hormones, e.g., bST

7
What are the new products of agricultural
biotechnology?
  • Herbicide tolerant crops, e.g., Roundup Ready
    soybeans and corn and Liberty Link corn

8
What are the new products of agricultural
biotechnology?
  • Identity-preserved or specific-attribute crops
    (vaccines, higher oil or starch content,
    additional amino acids)

9
  • Advances in biotechnology may provide consumers
    with foods that are nutritionally-enriched or
    longer-lasting, or that contain lower levels of
    certain naturally occurring toxicants present in
    some food plants.

10
  • Developers are using biotechnology to try to
    reduce saturated fats in cooking oils, reduce
    allergens in foods, and increase disease-fighting
    nutrients in foods.

11
  • They are also researching ways to use genetically
    engineered crops in the production of new
    medicines, which may lead to a new plant-made
    pharmaceutical industry that could reduce the
    costs of production using a sustainable resource.

12
Benefits of Agricultural Biotechnology?
  • The application of biotechnology in agriculture
    has resulted in benefits to farmers, producers,
    and consumers. Biotechnology has helped to make
    both insect pest control and weed management
    safer and easier while safeguarding crops against
    disease.

13
  • For example, genetically engineered
    insect-resistant cotton has allowed for a
    significant reduction in the use of persistent,
    synthetic pesticides that may contaminate
    groundwater and the environment

14
  • In terms of improved weed control,
    herbicide-tolerant soybeans, cotton, and corn
    enable the use of reduced-risk herbicides that
    break down more quickly in soil and are non-toxic
    to wildlife and humans. Herbicide-tolerant crops
    are particularly compatible with no-till or
    reduced tillage agriculture systems that help
    preserve topsoil from erosion.

15
  • Agricultural biotechnology has been used to
    protect crops from devastating diseases. The
    papaya ringspot virus threatened to derail the
    Hawaiian papaya industry until papayas resistant
    to the disease were developed through genetic
    engineering..

16
  • This saved the U.S. papaya industry. Research on
    potatoes, squash, tomatoes, and other crops
    continues in a similar manner to provide
    resistance to viral diseases that otherwise are
    very difficult to control.

17
  • Biotech crops may provide enhanced quality traits
    such as increased levels of beta-carotene in rice
    to aid in reducing vitamin A deficiencies and
    improved oil compositions in canola, soybean, and
    corn. Crops with the ability to grow in salty
    soils or better withstand drought conditions are
    also in the works.

18
  • Biotech crops can make farming more profitable by
    increasing crop quality and may in some cases
    increase yields. The use of some of these crops
    can simplify work and improve safety for farmers.
    This allows farmers to spend less of their time
    managing their crops and more time on other
    profitable activities.

19
  • The tools of agricultural biotechnology have been
    invaluable for researchers in helping to
    understand the basic biology of living organisms.
    For example, scientists recently identified the
    complete genetic structure of several strains of
    Listeria and Campylobacter, the bacteria often
    responsible for major outbreaks of food-borne
    illness in people.

20
  • This genetic information is providing a wealth of
    opportunities that help researchers improve the
    safety of our food supply. The tools of
    biotechnology have "unlocked doors" and are also
    helping in the development of improved animal and
    plant varieties, both those produced by
    conventional means as well as those produced
    through genetic engineering

21
Generation of Transgenic Plants
22
A. Totipotency
  • Definition
  • Entire plant can be generated from a single,
    non-reproductive cell
  • Single cells can be separated from leaf, stem or
    root tissue using enzymes to digest pectin
    holding cells together (pectinase)

23
  • Clones from cuttings in tissue culture
  • Asexual reproduction of plants can occur using
    fragments of plants
  • Shoots or stems or leaves EXPLANTS

24
  • In tissue culture, cells divide from exposed cell
    ? a callus forms
  • Callus undifferentiated cluster of rapidly
    dividing cells
  • Adventitious roots often form from callus

25
  • Callus tissue regeneration
  • Callus tissue will develop if cells are grown
    with proper balance of nutrients and plant
    hormones
  • Magenta boxes or large test tubes, sterile medium
    and transfer instruments
  • Murishigee and Skoog medium (MS medium)
    Artificial medium (agarose, nutrients and
    hormones)
  • Under influence of increased cytokinin, shoots
    will differentiate
  • Transferred to increased auxins, roots will
    establish
  • Eventually transferred to soil ? entire plant
    with reproductive structures (ovules, pollen)
  • Calluses can be split into many smaller pieces
    before hormones are added to increase of plants

26
B. DNA inserted into plants ? Transgenic plant
  • Characteristics of transgenic plants
  • All cells in the plant are derived from one cell
  • All cells express the desired genetic information
  • Why make transgenic plants?
  • Genes from distantly related plant families can
    be introduced without need for breeding (some
    families of plants are incompatible)
  • To improve crop hardiness and characteristics of
    final plant product
  • Protein content
  • Ripening rate
  • Drought resistance

27
  • Procedures for generating transgenic plants
  • Microinjection
  • DNA constructs injected using fine glass pipettes
    in combination with phase contrast microscopy
  • Electroporation of protoplasts
  • Electric pulses of high field strength
  • Reversibly permeabilize cell membranes
  • Electric discharge gun Gold beads
  • Firing DNA-coated pellets using a modified .22
    caliber gun

28
  • Whiskers of silicon carbide holes punched,
    DNA introduced
  • Agrobacterium tumefaciens
  • Viral vectors
  • Cauliflower mosaic virus vectors
  • Gemini virus vectors
  • Liposome-mediated transformation of protoplasts
  • Artificial lipid vesicles Liposomes
  • Chemically-stimulated DNA uptake by protoplasts
  • Polyethylene glycol CaCl2

29
  • Many gene transfer techniques start with
    protoplasts
  • Cell wall is digested with cellulase and cells
    are separated using pectinase
  • Plant cells are maintained in suspension
  • DNA is introduced, it integrates and expression
    of desired genes is achieved
  • Electroporation
  • Microinjection

30
  • Protoplast fusion can also be used to fuse two
    different plant types together ? New Plant
    Varieties (hybrid plantlet)
  • Fused cell acquires some of the characteristic of
    both genetic backgrounds and can be regenerated
    into a plant with some traits from both parental
    plants
  • Fusigenic agents (polyethylene glycol) or
    electroporation used to fuse membranes
  • Useful if species are sexually incompatible or
    cross with difficulty

31
  • Commercially important plants that can be grown
    from single somatic (non-seed) cells
  • Asparagus
  • Cabbage
  • Citrus fruits
  • Carrots
  • Alfalfa
  • Millet
  • Tomatoes
  • Potatoes
  • Tobacco
  • More than 30 different crop plants developed with
    rDNA techniques are being tested in field studies

32
Agrobacterium tumefaciens
  • Characteristics
  • Plant parasite that causes Crown Gall Disease
  • Encodes a large (250kbp) plasmid called
    Tumor-inducing (Ti) plasmid
  • Portion of the Ti plasmid is transferred between
    bacterial cells and plant cells ? T-DNA (Tumor
    DNA)

33
  • T-DNA integrates stably into plant genome
  • T-DNA ss DNA fragment is converted to dsDNA
    fragment by plant cell
  • Then integrated into plant genome
  • 2 x 23bp direct repeats play an important role in
    the excision and integration process

34
  • Tumor formation hyperplasia
  • Hormone imbalance
  • Caused by A. tumefaciens
  • Lives in intercellular spaces of the plant
  • Plasmid contains genes responsible for the
    disease
  • Part of plasmid is inserted into plant DNA
  • Wound entry point ? 10-14 days later, tumor
    forms

35
  • Observed in many varieties of wood plants
  • Grapes
  • Roses
  • Apples
  • Cherries
  • Pecans
  • Also infects herbaceous plants
  • Daisies
  • Asters
  • Beets
  • Turnips
  • Tomatoes
  • Sunflowers

36
  • Effects on plant
  • Distortion of tissue
  • Stunted
  • Small, chlorotic leaves
  • Wilting
  • Weaker
  • More susceptible to adverse environmental
    conditions

37
  • What is naturally encoded in T-DNA?
  • Enzymes for auxin and cytokinin synthesis
  • Causing hormone imbalance ? tumor
    formation/undifferentiated callus
  • Mutants in enzymes have been characterized
  • Opine synthesis genes (e.g. octopine or nopaline)
  • Carbon and nitrogen source for A. tumefaciens
    growth
  • Insertion genes
  • Virulence (vir) genes
  • Allow excision and integration into plant genome

38
  • How is T-DNA modified to allow genes of interest
    to be inserted?
  • In vitro modification of Ti plasmid
  • T-DNA tumor causing genes are deleted and
    replaced with desirable genes (under proper
    regulatory control)
  • Insertion genes are retained (vir genes)
  • Selectable marker gene added to track plant cells
    successfully rendered transgenic antibiotic
    resistance gene ? geneticin (G418) or
    hygromycin
  • Ti plasmid is reintroduced into A. tumefaciens
  • A. tumefaciens is co-cultured with plant leaf
    disks under hormone conditions favoring callus
    development (undifferentiated)
  • Antibacterial agents (e.g. chloramphenicol) added
    to kill A. tumefaciens
  • G418 or hygromycin added to kill non-transgenic
    plant cells
  • Surviving cells transgenic plant cells

39
  • Techniques to transform plant cells by A.
    tumefaciens
  • Wounding and direct inoculation
  • Inoculation of explants in vitro
  • Transformation of leaf-disks
  • Co-cultivation of Agrobacterium with protoplasts

40
Examples of Crop Improvement Measures
41
A. Nitrogen fixation
  • To enable plants to fix atmospheric N2 so that it
    can be converted into NH3, NO3-, and NO2- ?
    providing a nitrogen source for nucleic acid and
    amino acid synthesis
  • Thereby eliminating need to fertilize crops with
    nitrogen
  • Exploit N2 fixation metabolic machinery of
    bacteria and fungi
  • Some live freely in soil and water
  • Some live in symbiosis
  • Rhizobium spp. live in symbiosis with leguminous
    species of plants in root nodules (e.g. soy,
    peas, beans, alfalfa, clover)

42
B. Frost Resistance
  • Ice-minus bacteria
  • Ice nucleation on plant surfaces caused by
    bacteria that aid in protein-water coalescence ?
    forming ice crystals _at_ 0oC (320F)
  • Ice-minus Pseudomonas syringae
  • Modified by removing genes responsible for
    crystal formation
  • Sprayed onto plants
  • Displaces wild type strains
  • Protected to 23oF
  • Dew freezes beyond this point
  • Extends growth season
  • First deliberate release experiment Steven
    Lindow 1987- sprayed potatoes
  • Frost Ban
  • Different strain of bacteria Julie Lindemann
    led different project 1987
  • Strawberries in California

43
C. Resistance to biological agents
  • Anti-Insect Strategy - Insecticides
  • From Bacillus thuringensis
  • Toxic crystals found during sporulation
  • Alkaline protein degrades gut wall of
    lepidopteran larvae
  • Corn borer catepillars
  • Cotton bollworm catepillars
  • Tobacco hornworm catepillars
  • Gypsy moth larvae
  • Sprayed onto plants but will wash off

44
  • Monsanto Chemical Company 1991Trials
  • BT ? into cotton plants using A. tumefaciens
    vector
  • Cotton bollworms ? protection in 6 locations, 5
    different states, consistent results
  • First crops 1996
  • Corn
  • Cotton
  • Seed potatoes
  • Soybean
  • Others

45
  • Cloned BT toxin gene into a different bacterium
    that lives harmlessly in corn plants
  • Pressure applied to introduce modified bacterium
    into seeds
  • Corn stalks protected from corn borers
  • BT in poplar and white spruce ? catepillar
    resistance
  • BT-resistant strains are beginning to emerge in
    some catepillars

46
  • Anti-Viral Strategy
  • TMV-coat protein inserted into tobacco and tomato
    plant cells using Ti plasmid
  • Viral capsids inhibit viral replication of TMV
    when infected
  • Grape fan-leaf virus (GFLV)
  • Causes yellowing and deformation of grape leaves
  • Transmitted in soil by nematodes
  • Viral capsid genes introduced into champagne
    grape vines using T plasmid
  • Resistance to virus acquired
  • Other trials using capsid proteins potato
    leaf-roll virus, cantaloupe mosaic virus, rice
    strip virus
  • Concerns that recombination events may lead to
    new plant virus strains

47
  • Anti-Bacterial Strategies
  • Resistance to Xanthomonas oryzae (rice wilting)
  • Conferred by cloning resistance genes from wild
    rice strains
  • Anti-Worm Strategies (Animal pest)
  • Nematode resistance gene from wild beet plants
  • To protect sugar beet

48
Resistance to herbicides
  • Glyphosate resistance
  • Glyphosate Roundup, Tumbleweed Systemic
    herbicide
  • Glyphosate inhibits EPSP synthase
    (S-enolpyruvlshikimate-3 phosphate involved in
    chloroplast amino acid synthesis)
  • Escherichia coli EPSP synthase mutant form ?
    less sensitive to glyphosate
  • Cloned via Ti plasmid into soybeans, tobacco,
    petunias
  • Increased crop yields of crops treated with
    herbicides

49
  • Bromoxynil
  • bromine-based herbicide
  • Bromoxynil resistant cotton
  • Concern over movement of resistance genes into
    weeds ? making compounds useless

50
Bioengineered foods
51
Flavr-Savr tomato
  • Rot-Resistant Tomato
  • Calgen, Inc.
  • Anti-sense gene ? complementary to
    polygalacturonase (PG)
  • PG pectinase ? accelerates plant decay/rotting

52
Laurate canola oil
  • Canola plant modified with thioesterase gene
    obtained from California bay laurel tree
  • Enzyme produces lauric acid (up to 40 in oil
    from genetically modified (GM) canola seeds)
  • Low saturated fat content
  • Heat tolerant
  • Does not break down
  • Excellent for high temperature cooking processes

53
Biopharming
54
What is Biopharming?
  • Drug production in genetically modified plants
  • Tobacco
  • Alfalfa
  • Potatoes
  • Corn
  • Soybeans
  • Wheat
  • Rice
  • Oilseed rape
  • Ethiopian mustard

55
  • Drugs Biopharmaceuticals
  • Drugs synthesized organically
  • Many drugs are made naturally in plants
  • Aspirin (originally isolated from willow bark)
  • Vincristine and vinblastine (periwinkle)
  • Taxol (Pacific yew)
  • Digitalis (foxglove)
  • Recombinant DNA techniques enable many more drugs
    to be made artificially in plants
  • Human proteins in plants xenogenic proteins

56
Why Farm for Pharmaceuticals in Plants?
  • Cheaper than producing pharmaceutical proteins in
    cell culture
  • Could reduce the cost of medicine
  • Example
  • Newest factories producing GM proteins in
    mammalian cell culture costs 100 million/300
    kg, costing 1000/g
  • Biopharming producing GM proteins in plants costs
    10 million capital investment/300 kg, costing
    200/g (according to Monsantos Integrated
    Protein Technologies)

57
  • However, costs of extracting and purifying
    biopharmaceuticals can be high and processing
    strategies need to be improved

58
  • Fewer complications than producing proteins in
    animals (e.g. cell culture or milk from pharm
    animals)
  • Possible transmission of animal viruses
    zoonoses
  • Plant viruses cannot infect animals
  • Plants do not serve as hosts for infectious
    agents such as HIV, HepB, prions
  • Ethical considerations (animal welfare concerns)

59
  • Plants effectively transcribe, translate and
    assemble proteins derived from eukaryotic sources
  • Improved quality of life

60
  • Produce beneficial pharmaceuticals in tobacco
    rather than cigarettes
  • If we can actually find a medical use for
    tobacco that saves lives, what a turnaround for
    the much-maligned tobacco plant.
  • Christopher Cook, CEO of ToBio

61
  • Tobacco is favored for many reasons
  • Easy to genetically engineer (Agrobacterium-mediat
    ed transformation)
  • Excellent biomass producer
  • 1 million seeds can be isolated from a single
    plant (scale-up benefits)
  • Number one cash crop in Virginia

62
Examples of Biopharmaceuticals
  • Hepatitis B and other subunit vaccines
  • Urokinase (clot dissolving drug)
  • Human serum albumin (liver cirrhosis treatment)
  • Hemoglobin
  • Human erythropoietin
  • Glucocerebrosidase (Gauchers disease)

63
  • Blood coagulants
  • Proteases (e.g. trypsin)
  • Protease inhibitors (e.g. aprotinin - used by
    surgeons)
  • Growth promoters
  • HIV viral coat protein (HIV therapy)
  • Nutraceuticals (Vitamin A and E, amino acids)

64
  • Neurologically active agents (human enkephalins)
  • Protein based sweetener (Brazzein)
  • Avidin
  • Beta-glucoronidase
  • Indirect thrombin inhibitor (Hirudin
    anticoagulant originally isolated from the leech
    Hirudo medicinalis)
  • Human epidermal growth factor
  • Human interferon-alpha (Hepatitis B and C
    treatment)

65
  • Bacterial enterotoxins
  • Human insulin
  • Norwalk virus capsid protein
  • Natural plastic (plastic-like polymers)
    (Biopol)
  • Human GM-CSF
  • Human alpha-1 antitrypsin (cystic fibrosis/liver
    treatment)
  • Angiotensin-1-converting enzyme (hypertension)

66
  • Edible Vaccines Ongoing Research Areas
  • Hepatitis B
  • Dental caries - Anti-tooth decay Ab (CaroRxTM)
    (anti-Streptococcus mutans)
  • Autoimmune diabetes
  • Cholera
  • Rabies
  • HIV
  • Rhinovirus
  • Foot and Mouth
  • Enteritis virus
  • Malaria
  • Influenza
  • Cancer

67
EHEC Edible Vaccine
  • Foodborne Pathogen
  • Vaccine exists, cost prohibitive delivery
  • Plant-based vaccination can be cost effective
  • Improved safety of the food supply
  • Safety evaluation of the vaccine protein

68
  • Dr. Carole Cramer while at Virginia Tech
  • Engineered rDNA so that protein is only expressed
    when the tobacco leaves are cut
  • Drug is only produced when plant is wounded
  • Currently chief scientific officer of CropTech
  • Developing 20 human proteins
  • Including human protein C (blood clotting
    regulator)
  • Lysosomal enzyme - glucocerebrosidase
  • Tobacco plants produce proteins after leaves are
    shredded
  • Clinical trials must be initiated, and approval
    by FDA still lie ahead

69
  • Planet Biotechnology
  • Clinical trials involving anti-tooth decay
    antibody
  • Monoclonal antibody that binds to bacteria
    (Viridans Streptococci) associated with tooth
    decay
  • Interferes with adhesion of bacteria to tooth
    enamel

70
Potentially Harmful Effects
  • Contamination by pesticides
  • Co-purification of plant chemicals (e.g.
    nicotine)
  • Different glycosylation in plants versus animals
  • Interference with normal function of protein in
    animals
  • Stimulation of hypersensitivity reactions in
    animals (allergies)
  • Research is underway to engineer tobacco to
    synthesize human-compatible glycans

71
Environmental Risks
  • Pharmaceutical products may inadvertently be
    introduced into the general food supply
  • Cross-pollination
  • Pollen from a drug-containing crop fertilizes a
    neighboring related crop (or wild relatives) used
    for animal consumption
  • Wind
  • Insects

72
  • Consumption of GM plant by insects ? Food chain
  • Accumulation in birds extinction? (e.g. DDT
    and bald eagle)
  • Deleterious effects on non-target organisms
    (NTOs)
  • NTOs organisms in the environment that are
    affected by the product unintentionally
  • Insects, arthropods
  • Risk to NTOs
  • Depends on recombinant protein involved
  • Risk assessment carried out case-by-case

73
  • Misrouting of crops seeds during processing
  • Alteration in soil microbes
  • Leaching of drug into the soil from the roots
    (soil contamination)

74
General Risk Assessment
  • Most biopharmaceuticals are proteins which have
    little biological activity (e.g. monoclonal
    antibodies, subunit vaccines)j
  • Digestible, little hazard of toxicity
  • Some biopharmaceuticals may be toxic in higher
    doses (e.g. anticoagulants, hormones, enzymes)
  • Persistence in environment (lipophillic)

75
Management Strategies
  • Inducible genes post harvest
  • Product activation post purification
  • Terminator technology to prevent pollen
    development
  • Government permits for field trials of
    drug-producing plants
  • Double distance between crops to prevent
    cross-pollination Buffer zones

76
  • Regulate planting of drug-containing crops
    indefinitely
  • Secluded or enclosed fields
  • Transgene tracking tools
  • Marker proteins to label specific
    biopharmaceutical plants

77
Rhizosecretion
  • Soil contamination has already been observed in
    GM plants producing the Bacillus thuringensis
    toxin (Bt)
  • Biologically active Bt isolated 9 months after
    transgenic plant was harvested

78
  • Taking advantage of rhizosecretion
  • Roots of transgenic plants are submerged in
    hydroponic solutions
  • Continuous secretion of recombinant proteins
  • Economical alternative to downstream processing
    and chemical extraction of active compounds
  • Attractive but what about consequences and
    regulation?

79
  • http//www.isaaa.org/resources/publications/briefs
    /xx/pptslides/Brief43slides.pdf
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