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PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Dining with DNA Lecture 8 6th November, 2006 Genetic Engineering: Is it the next Magic Pill? Global Context Population ... – PowerPoint PPT presentation

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Title: Prйsentation PowerPoint


1
(No Transcript)
2
Genetic Engineering Is it the next Magic Pill?
3
Global Context
  • Population growth
  • Finite resources
  • (land and water)
  • Access to food
  • Technology options to increase productivity
  • Agricultural and food policy options

4
Population Growth
  • Increasing by 80 million people per year
  • 95 of the increase in developing countries
  • 1.3 billion people live in absolute poverty
    currently

5
Demand for Food
  • Projected increase in global demand between 1993
    and 2020
  • Cereals 41
  • Meats 63
  • Roots and Tubers 40
  • Feed vs Food
  • Cereals for animal feed will double
  • Cereals for human consumption will increase by 47

6
Food Availability
  • Per capita availability of food will increase 7
    by 2020
  • Global income growth projected to increase 2.7
    per year
  • Availability does not imply access

7
Options to Meet Demand
  • Increase in cultivated land lt 20 contribution
  • Productivity increase Cereal yields expected to
    decrease to 1.5 from 2.3
  • Water stress Demand for water to increase by
    2.4 per year
  • Technology?

8
Technology Options
  • Integrated Pest Management
  • Improved cultural practices
  • Improved food preservation techniques
  • Identifying new genetic sources
  • Genetic engineering

9
What is Genetic Engineering?
10
Definitions
  • Any food that has been made from genetically
    altered plants or animals
  • Genetic alteration Altering gene/s using
    recombinant technique
  • Gene Small segment of DNA that codes for a
    specific protein
  • Recombinant technique (rDNA) Methods used to
    alter gene/s

11
Is Genetic Engineering Different from Traditional
Breeding?
  • No!
  • Traditional breeding also involves gene transfer
    but thousands of genes, good and bad, are moved

12
Plant Breeding
13
Hybridization or Cross-breeding
1000 Genes in Hybrid
1000 Genes in Line B
1000 Genes in Line A
14
DOMESTICATION OF MODERN DAY CROPS
TEOSINTE TO MAIZE
15
Some domestics and their (never domesticated)
close relatives
16
Is Genetic Engineering Different from Traditional
Breeding?
  • Yes!
  • Specific gene/s from any source can be
    introduced and is faster

17
How are GMOs Created
18
Some history...
  • In 1953 Francis Crick and James Watson published
    their discovery of the structure of DNA, which
    led to scientists being able to splice genes from
    one organism and insert them into the DNA of
    another.
  • In 1973 Stanley Cohen and Herbert Boyer created
    the first successful recombinant DNA organism.
  • In 1980 U.S. Supreme Court ruled that genetically
    altered life forms can be patented in the case of
    Diamond vs. Chakrabarty. This decision allowed an
    oil eating organism to be patented by Exxon Oil
    Company.

19
GMO Timeline
  • 1986 Federal Coordinated Framework for
    regulating biotech
  • 1993 FDA approves rBGH
  • 1994 First biotech food approved (Flavr Savr
    tomato)
  • 1996 First GM corn seed is sold GM crops enter
    the food supply

20
Non-US GMO Timeline
  • 1996 Mad cow disease linked to human brain
    disease
  • 1997 European consumers protest US shipments
    Monsanto targeted
  • 1999 Activists get violent Secretary Glickman
    is pummeled in Italy Monsanto PR campaign
    backfires in the EU Brazil, Australia and China
    threaten ban Monarch butterfly scare
  • 2000 Starlink corn crisis

21
World Political Timeline
  • 2001 Application for GM fish is submitted to
    FDA EU says labeling will be mandatory, trade
    war lingers Mexican maize contamination
    reported Monsanto abandons New Leaf potato
  • 2002 Prodigene episode
  • 2003 SubSaharan African nations reject US food
    aid with GM corn US sues EU in WTO
  • 2004 New EU rules go in effect Monsanto
    shelves GM wheat Glofish released unregulated

22
GMO Foods - VERY controversial!
http//www.teachersdomain.org/9-12/sci/life/gen/lp
_bioengfood/index.html http//www.teachersdomain.o
rg/resources/tdc02/sci/life/gen/breeding/index.htm
l
23
Example of genetically modified foods?
  • Also called genetically modified organisms (GMO).
  • Involves the insertion of DNA from one organism
    into another OR modification of an organisms DNA
    in order to achieve a desired trait.

4
5
A strawberry resistant to frost


Arctic fish DNA
strawberry
24
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)

25
A. Totipotency
  • Clones from cuttings in tissue culture
  • Asexual reproduction of plants can occur using
    fragments of plants
  • Shoots or stems or leaves EXPLANTS
  • In tissue culture, cells divide from exposed cell
    ? a callus forms
  • Callus undifferentiated cluster of rapidly
    dividing cells
  • Adventitious roots often form from callus

26
A. Totipotency
  • Callus tissue regeneration
  • Callus tissue will develop if cells are grown
    with proper balance of nutrients and plant
    hormones
  • Magenta boxes, 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

27
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..

28
B. DNA inserted into plants ? Transgenic plant
  • 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

29
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30
B. DNA inserted into plants ? Transgenic plant
  • 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

31
B. DNA inserted into plants ? Transgenic plant
  • 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

32
B. DNA inserted into plants ? Transgenic plant
  • US 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

33
C. 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)
  • 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

34
C. Agrobacterium tumefaciens
35
C. Agrobacterium tumefaciens
  • 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

36
C. Agrobacterium tumefaciens
  • 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

37
C. Agrobacterium tumefaciens
38
II. Examples of Crop Improvement Measures
39
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)

40
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

41
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 caterpillars
  • Cotton bollworm caterpillars
  • Tobacco hornworm caterpillars
  • Gypsy moth larvae
  • Sprayed onto plants but will wash off

42

C. Resistance to biological agents
  • Monsanto Chemical Company 1991 Trials
  • BT ? into cotton plants using A. tumefaciens
    vector
  • Cottton bollworms ? protection in 6 loctions, 5
    different states, consistent results
  • First crops 1996
  • Corn
  • Cotton
  • Seed potatoes
  • Soybean
  • Others

43
C. Resistance to biological agents
  • 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 ? caterpillar
    resistance
  • BT-resistant strains are beginning to emerge in
    some caterpillars

44
C. Resistance to biological agents
  • 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

45
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

46
Resistance to herbicides
  • Bromoxynil
  • bromine-based herbicide
  • Bromoxynil resistant cotton
  • Concern over movement of resistance genes into
    weeds ? making compounds useless

47
Examples
48
Examples
49
Specific Examples
50
Specific Examples
51
Specific Examples
52
III. Bioengineered foods
53
A. US-FDA Regulations
  • 1978 transgenic bacteria produce human insulin
  • 1983 First transgenic plant produced (tobacco
    with kanamycin resistance gene from bacteria)
  • May 26, 1992
  • US-FDA declares no special regulation for
    genetically modified food compared to foods
    generated by conventional means
  • No special testing
  • No mandatory labeling
  • 1994 FDA approves release of first transgenic
    crop Flavr Savr tomatoes that show delayed fruit
    softening

54
Dining with DNA
55
Impact on Humans Ecosystem
  • Unwanted transfer of genes
  • Loss of diversity
  • Safety
  • Allergic reactions
  • Toxicity
  • Antibiotic resistance

56
Environmental Issues 2 Opposing Views
but
but
but
57
Dining with DNA
58
Consumer Perception
I am required by law to tell you that everything
you ordered today may be harmful to your health.
59
Creating a balance
  • So are GM foods a good or bad thing?
  • It depend on each individual case.
  • Consumers, the government and scientists should
    be responsible for weighing the benefits against
    the costs.

Improved Nutrition Resistance to disease Reduced
use of chemicals
Environmental risks Health risks Economic risks
60
Interesting sites
www.enn.com www.propanefl.com/ images/corn.jpg www
.columbia.edu/cu/ opg/images/dna.jpg www.arctictra
vel.com/ GJOA/haven.html www.foodsubs.com/
Fruitber.html www2.utmb.edu/scccb/mouse/
images/microinjection.jpg ss.jircas.affrc.go.jp/en
gpage/ jarq/32-4/hagio/fig4.htm www.enn.com Transg
enic pollen harms monarch larvae (Nature, Vol
399, No 6733, p 214, May 1999) GM corn
poses little threat to monarch (Nature
Biotechnology, Vol 17, p 1154, Dec 1999)
www.vme.net/dvm/ARNHA/ monarch.html http//www.cs
a.com/hottopics/gmfood/overview.html www.greenpeac
e.org www.biotechknowledge.monsanto.com http//www
.inspection.gc.ca/english/ppc/biotech/labeti/respo
nse.shtml
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