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