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Plant biotechnology: what it means and where we

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Improve pest resistance Reduce insect and virus damage to crops, ... Transgenic plant technologies also require tissue culture Transfecting target cells requires ... – PowerPoint PPT presentation

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Title: Plant biotechnology: what it means and where we


1
Plant biotechnology what it means and where
were going
XXXX
  • Dave Law
  • Department of Biology

2
Biotechnology has a long history
  • Biotechnology use of biological organisms in
    agricultural and industrial processes to make
    products valuable for humans
  • Microbial/yeast biotech has long history making
    cheese, bread, beer, wine
  • old-style plant biotech has been used for crop
    improvement for centuries
  • Select mutants for best yield and quality
    (tomatoes!)
  • Breed plants to further improve
  • desirable characteristics
  • Ultimate improved crop is maize from from
    teosinte
  • Maize starch and oil used in
  • sweeteners,
  • ethanol fermentation
  • Glues
  • Plastics
  • Pharmaceuticals
  • cosmetics

teosinte
Early cultivated maize
Modern maize
Tomato ancestor (5 g)
Beefsteak (1000 g!)
3
Modern plant biotech is reliant on tissue culture
Making new plants Start here
  • Propagating plants in vitro
  • Many important crops and ornamentals are
    micropropagated this way (asexual reproduction)
  • This technology (and facilities needed) does not
    exist in Thunder Bay
  • Plants still grown from seed
  • NorPharms requires tissue culture facilities
  • Reasons to use tissue culture
  • Virus free reproduction
  • Bananas
  • Potatoes
  • Make many identical clones
  • Ornamentals
  • House plants

4
The best reason
  • Rapidly increase biomass versus sexual
    reproduction
  • Four-season

5
Transgenic plant technologies also require tissue
culture
Start with bacterial DNA and your gene (from
bacteria, goats, fungus, maize)
  • Modern plant biotech recombinant DNA technology
  • involves gene transfer in a much more precise
    manner than traditional breeding but still
    involves manipulation of biochemistry, physiology
    and development
  • DNA recombination involves taking DNA from one
    organism and moving it to another
  • The resulting transgenic plants can be called
    genetically modified organisms (GMOs)
  • How do you move DNA around?
  • STEP 1 Get your gene
  • Locate and remove DNA of interest using
    restriction enzymes that recognize specific
    target DNA sequences
  • Place into a plasmid for amplification in a
    bacterium (E. coli)

Grow lots of bacteria, make lots of DNA!
6
Transfecting target cells requires gene gun
(biolistics) or Agrobacteria
  • STEP 2 Prepare your receiving tissue
  • Involves tissue culture techniques
  • Often use sterile young leaf segments as targets
  • STEP 3 Get your DNA into the target plant
  • Method 1 gene gun
  • Use naked DNA (linear)
  • Coat DNA onto beads (tungsten or gold)
  • Use air pressure to fire into tissue
  • Invented at Agracetus in Wisconsin

Gene gun and technique
DNA
7
Agrobacteria allow controlled DNA insertion
  • Method 2 Agrobacteria
  • Use engineered instead of wild-type A.
    tumefasciens Ti plasmid
  • Still possesses virulence genes (allow transfer
    of T-DNA to target cell) but lacks opine and PGR
    synthesis genes
  • Wounded tissue (cut) attracts Agrobacteria that
    can infiltrate through wounds into apoplasm
  • Transfers T-DNA to genome
  • Can do in high throughput in immersion culture
  • Both methods integrate their DNA randomly into
    the genome
  • Not really desirable would like to target
    transgene to appropriate segment of genome for
    expression at correct developmental stage
  • Agrobacterium transformation tends to give lower
    copy numbers better for controlling silencing
    in long term

Original T-DNA coding for PGR, opine genes removed
Agrobacteria were first isolated from crown galls
8
Selectable markers aid greatly in identifying
positive transformants
  • STEP 4 Regenerate transgenic plants
  • Transformation is not 100 efficient not every
    targeted plant cell will be transgenic!
  • Just as in bacteria, use a selectable marker to
    find positives
  • Antibiotic, herbicide resistance common
  • Transformed explants taken through a
    dedifferentiating callus stage
  • Then manipulate auxin and cytokinin ratios to
    regenerate shoots and roots
  • Thus, tissue culture is an integral part of
    making transgenic plants

9
Making and culturing plants is expensive and
time-consuming
  • Do the math
  • Large biotech companies have armies of workers
    involved in culturing plants
  • Each transformation is an event
  • Commercially usually must do multiple events
    (250 for one trait!)
  • Then grow hundreds of plants per event and screen
    for expression
  • Select highest expressors
  • Pass regulatory approval with the USDA and FDA
  • May have a commercial product in 5 years
  • Substantial investment in plant biotech
    (comparable to drug development)
  • Cant play if you dont pay

Plant tissue culture facility
10
Plant biotech applications
  • GOAL Produce plants with a variety of desirable
    traits in high yielding seed cultivars
  • This is where the money is for biotech companies!
  • A partial list of desirable (money making)
    traits
  • 1. New horticultural varieties
  • Understand the anthocyanin (pigment) biosynthetic
    pathways, can produce novel flower varieties

11
Plant biotech apps, continued
  • 2. Improve pest resistance
  • Reduce insect and virus damage to crops, increase
    yield through eliminating competing weeds
  • Significantly reduce the amount of pesticide that
    needs to be applied to crops!
  • Pesticides will not kill beneficial predatory
    insects
  • Transfer gene that inhibits digestion of starch
    (amylase inhibitor) from bean to garden pea
  • Stops attack by weevils
  • Transfer one of many Cry genes from Bacillus
    thuringiensis to plants
  • Makes protein (Bt) toxins, only small amount
    needed in plant to kill insect pests

Nontransgenic control
Weevil resistant peas
Bollgard cotton
Nontransgenic control
Nontransgenic control
Virus resistant potato
  • Transfer viral coat proteins to plants to make
    them more resistant to viral attack
  • Viral attack reduces yield
  • tobacco mosaic virus
  • Papaya ringspot virus
  • Potato X and Y viruses

Ringspot virus resistant papaya
Susceptible plants
12
Plant biotech apps, continued
  • 2. Improve pest resistance (contd)
  • Transfer mutated gene in shikimic acid pathway
    from E. coli to make resistant (Roundup Ready)
    plants
  • Glyphosate herbicides inhibit an important enzyme
    in this pathway plants need aromatic AAs to grow!

EPSP synthase
13
Plant biotech apps, continued
  • 3. Improve nutritive value of plants
  • Use metabolic engineering to insert new pathways
    into plants or improve expression of enzymes in
    existing ones
  • Most crop plants are deficient in one or more
    amino acids
  • maize is low in lysine, methionine and tryptophan
  • Improve vitamin quality of crops
  • Golden rice higher in beta-carotene, the
    precursor to vitamin A
  • 250K go blind each year due to deficiency
  • Syngenta has just released much higher expressing
    cultivar
  • Improve value of feed crops
  • Transfer a fungal enzyme (phytase) to crops to
    remove phytic acid from feed and improve
    phosphate availability

14
Plant biotech apps, continued
  • 4. Improve resistance to stress
  • e.g., salt stress
  • Express high levels of Na/H antiporter in
    vacuole membrane
  • This allows plants to grow in high Na (50X normal
    limit) because they sequester excess
  • If transporters known, can also be used to
    engineer plants to phytoremediate toxic soils

Control tomatoes at 200 mM NaCl
Transformed tomatoes at 200 mM NaCl
15
Plant biotech apps, continued
  • 5. Improve postharvest physiology of fruits and
    vegetables
  • Delay fruit ripening slow down the ethylene
    response of the ripening pathway, allowing fruit
    to be picked ripe on the vine
  • Result better flavour for consumers
  • Could be used on any climacteric fruit especially
  • Most climacteric fruit picked green, shipped to
    market and treated with ethylene before sale at
    wholesale level
  • Flavr Savr tomato blocked polygalactonurase
    synthesis by antisense degrades plant cell wall
    pectin

16
Plant biotech apps, continued
PHB biosynthetic pathway
  • 6. Grow high value compounds in plants
  • Pharmaceuticals, vaccines, other industrial
    products molecular farming (or pharming)
  • Biopolymers make biodegradable thermoplastics
    such as polyhydroxybutyrate (PHB) by metabolic
    engineering into canola seed
  • Edible vaccines minimize need for expensive
    refrigeration, distribution and adminstration
    techniques for cholera, measles, E. coli
    enterotoxin (diarrhea causing agent), hepatitis B

PHB granules in Arabidopsis mesophyll cell nucleus
17
Plant biotech apps high value proteins
Antibody structure 2 large and 2 small subunits
  • 6. Grow high value compounds in plants (contd)
  • Antibodies to disease all medicines have several
    advantages to growth in plants versus in animal
    cell bioreactors (typically with CHO cells)
  • No prions or viruses
  • Easily scalable
  • Proteins are active (eukaryotic production
    system)
  • Can store seed for years prior to purification
  • But often not properly glycosylated, FDA must
    approve each event !
  • Clinical III trials going ahead

Fab
Fc
18
Conclusions
  • Plant biotech approaches provide alternatives to
    established agricultural practices that degrade
    the environment
  • rampant pesticide, herbicide use
  • high till farming that degrades the soil
  • Will provide crops with novel uses and with
    improved nutritional profiles

No-till farming preserves beneficial fungi in the
soil
  • Improved tolerance to environmental stresses and
    higher yield will enable higher productivity
  • Part of my research at LU will look at
    limitations of plant metabolism that hold back
    yield of edible parts and transgenic proteins
  • Also will use molecular biology to make
    transgenic plants
  • In Thunder Bay all plant biotech comes back to
    the need for tissue culture facilities
  • These presently do not exist locally
  • How do we change this situation?
  • Biotech Centre initiative at Lakehead new
    science building plant tissue culture facility
  • NorPharms initiative identifies other
    possibilities interface with city, industrial
    plant growth facilities to identify high value
    crops for cultivation/harvest
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