Plant biotechnology: what it means and where we’re going

1. XXXX Plant biotechnology: what it means and where we’re going Dave Law Department of Biology

2. 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 • 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… 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 Original T-DNA coding for PGR, opine genes removed 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 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) • Can’t play if you don’t 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 Nontransgenic control Weevil resistant peas 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 Bollgard cotton 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 (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! 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 (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

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 • 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 No-till farming preserves beneficial fungi in the soil