plant biotechnology: what it means and where we’re going
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XXXX. Plant biotechnology: what it means and where we’re going. Dave Law Department of Biology. Biotechnology has a long history. teosinte. Early cultivated maize. Biotechnology: use of biological organisms in agricultural and industrial processes to make products valuable for humans - PowerPoint PPT PresentationTRANSCRIPT
Plant biotechnology: what it means and where we’re going
Dave LawDepartment of Biology
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Biotechnology has a long historyteosinte
Modern maize
• 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…
Early cultivated maize
Tomato ancestor (5 g)
Beefsteak (1000 g!)
Modern plant biotech is reliant on tissue cultureMaking 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 facilitiesReasons to use tissue culture:• Virus free reproduction
– Bananas– Potatoes
• Make many identical clones– Ornamentals– House plants
The best reason…
• Rapidly increase biomass versus sexual reproduction
• Four-season
Transgenic plant technologies also require tissue culture
• 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)
Start with bacterial DNA and your gene (from bacteria, goats, fungus, maize…)
Grow lots of bacteria, make lots of DNA!
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
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
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
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)• Can’t play if you don’t pay
Plant tissue culture facility
Plant biotech applicationsGOAL: 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
Plant biotech apps, continued2. 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
– 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
Bollgard cotton
Nontransgeniccontrol
Nontransgeniccontrol
Virus resistantpotato
NontransgeniccontrolWeevil resistant peas
Ringspot virusresistant papayaSusceptible plants
Plant biotech apps, continued
EPSP synthase
2. Improve pest resistance (cont’d)– 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!
Plant biotech apps, continued3. 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
Plant biotech apps, continued4. Improve resistance to
stresse.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
Plant biotech apps, continued5. 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
Plant biotech apps, continued6. 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
PHB biosynthetic pathway
Plant biotech apps: high value proteins6. Grow high value compounds in plants
(cont’d)• 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
Antibody structure: 2 large and 2 small subunits
Conclusions
• 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 locallyHow 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
• 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