1. What are genetically-modified organisms?
GMO’s
What are genetically-modified organisms?

2. A bit of history Agriculture & Animals
~ For over 10,000 years people have been breeding wild animals and plants.
~Always breeding for “desirable traits” -- artificial selection
Some of the genetic diversity for ear and kernel traits found in Mexican maize. Photo: CIMMYT

3. Picture 1 center picture is wild cabbage (Brassica oleracea) man selected the other crops pictured from this plant.
Top row pictures left to right – cabbage, kale, broccoli
Bottom row pictures left to right – brussel sprouts, kohlrabi and cauliflower

4. Humans have been manipulating DNA in plants and animals for millennia
However…what’s true about this form of DNA manipulation???
How does DNA change in nature???
vertical vs. horizontal

5. What is a GMO?
“Genetically Modified Organism (GMO)” an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination FYI…GM = GMO = GE (engineered)

6. Why have GM crops? Growing human population Loss of farmable land
Remediation of soil
Enrich nutrient content
Pigs that have reduced phosphates in their feces
Pigs with omega three fa

7. GE Crop Traits
Herbicide tolerance - crop can withstand herbicide applications
Insect tolerance - plant produces toxin to kill pest
Improved nutrition – plant produces a substance of nutritive value or is changed to not produce an antinutrient
Disease resistant – crop is resistant to certain disease
Stress Tolerance – crop is tolerant of stress, low nutrient levels or excess nutrients
Increased Storage – crop can be stored longer to avoid spoilage losses
Medicinal uses – crops that produce medicines or vaccines
Industrial uses – crops to make more efficient industries

8. Transgenic - a gene is moved from one non-closely related species to another
Cisgenic/intragenic - a gene is moved within the same species or a closely related species.
RNAi -  is a biological process in which RNA molecules inhibit gene expression -
Subgenic - a gene is edited to amplify, delete, insert, silence or repress the gene.
CRISPR -

9. Concerns about Genetic Modification
• Creation of super pests
• Creation of super weeds
Loss of biodiversity
Biotechnology companies control agriculture
Health concerns (allergens)

10. Most common GM traits Ht = Herbicide tolerant Bt = Insect resistance
Most common is Roundup Ready™
Bt = Insect resistance
Bt stands for Bacillus thuringiensis -- a naturally occurring soil bacterium that makes a protein toxic to insect larva—one of a limited number of tools available to organic gardeners/farmers
Scientists took the gene for this protein and engineered it into plasmids that were then introduced to plants…the plants then make the insecticide in their own cells
80% of soy crop in GMO

14. What are the US approved GM food crops?
Corn
Apples
Soy
Melon
Papaya
Flax
Canola
Plum
Potato
Tobacco
Chicory
Salmon
Rice
Approval does not necessarily mean these crops are distributed
Squash
Sugarbeet
Database of GM crops:
Tomatoes
Bolded crops are widely grown & sold in US.

15. GE Crops that have been commercialized in US
IR = insect resistant
HT = herbicide tolerant
DT = drought tolerant
VR = virus resistant
…and potato

17. GM Crops in the U.S.
Source:

18. Additional applications…
American chestnut (Castanea dentata)
Native keystone species
Nearly wiped out by the imported fungus, Cryphonectria parasitica
Killed the tree by secreting oxalic acid
Can be detoxified by an enzyme, oxalate oxidase, found in wheat
Transgenic American chestnut ‘Darling4’
Expresses a wheat oxalate oxidase gene
Exhibits intermediate blight resistance

19. Additional applications…
American chestnut (Castanea dentata) …another angle
American chestnuts with various combinations of 6 genes from Chinese chestnuts (2014)
Field tests conducted in 2016
Available in US in about 4 years if all regulations proceed smoothly
Plans to re-establish in natural range

20. Additional applications…
ALSO…developing transgenic elm seedlings to fight Dutch Elm Disease
GM hybrid poplars (field testing)
Identified other pathogens/hosts:
Butternut
White pine
Beech
Dogwood
oak

21. Additional applications…
Citrus greening disease
Millions of acres lost in US and overseas
80% of Florida’s citrus trees infected and declining
Bacterial—incubates in tree’s roots and moves up the tree, causing nutrient flows to stop
Florida’s $5.1 billion citrus industry could be a total loss
Texas A & M scientist—insert a spinach gene to fight the bacteria
Field trials of the transgenic trees have shown high degree of resistance

22. How to Modify a Crop: Identify beneficial protein
Isolate the gene that codes for the beneficial protein
Engineer the gene into a plasmid
Add a “promoter”
Tells cell to turn gene “on”
CaMV35S is the most common promoter *
Add a “terminator”
Tells cell to turn gene “off”
NOS is the most common terminator *
*NOS and/or CaMV35S are present in 85% of GM foods
Introduce plasmid construct into the cell -- cell then make the new protein
How to Modify a Crop:

23. Why use CaMV 35S and NOS?
CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus.
Used because it functions in every plant cell
NOS- Sequence for nopaline synthase terminator from soil bacterium Agrobacterium tumefacians
Used because it evolved to be recognized in most plants
About 65% of food crops use 35S promoter, by adding NOS detection, can detect about 80% of GM foods.
35S promoter drives expression of 35S RNA transcript of CaMV. 2 main transcripts of CaMV. 19S and 35S. 19S codes for proteins. 35S is reverse transcribed to make the virion DNA in the cytoplasm of the infected cell for the productions of new virus particles.
Nopaline is an amino acid derivative, derived from arginine, produced in wound sites by tumor inducing (Ti) plasmid from A.tumefaciens.Nopaline provides food for A.tumefaciens and therefore gives selective advantage to A. tumefaciens. Nopaline also induces conjugation to transfer Ti plasmid into other bacteria. NOS genes are on the T-DNA that is transfered into the plant genome by the Ti plasmid

24. Clone the gene Ti plasmid Bacillus thuringiensis
Delta endotoxin crystal
Bt gene
ori
Ti plasmid
Ti genes

25. Engineer the gene Ti plasmid GO Bt gene ori Ti genes
STOP
Engineer the gene
Bt gene
Add promoter and terminator, streamline genes by removing introns. Ti plasmid from Agrobacterium.
ori
Ti plasmid
Ti genes
Antibiotic resistance

26. Backcross GM plant into high yield crops
YYgg x yyGG
YyGg
YYgG
YygG
YYgg
Yygg
YYgg x YyGg
GM plant = yyGG
High yield plant = YYgg
Repeatedly backcross GM plant to high yield plant to reintroduce hybrid traits (genome).
YYgG
YYgg
YYGg
YYGG
YYgG x YYgG

27. How to test for GMOs Test for GMOs by PCR: Grind food
Extract DNA from sample
Test sample DNA for viable plant DNA
Test sample DNA for genetic modifications

29. Which foods yield viable plant DNA?
Very Reliable
Reliable
Less Reliable
Very Difficult / Not Possible
Fresh corn
Veggie sausages
Veggie burgers
Oil
Fresh papaya
Tortilla chips
Fried corn snacks
Salad dressing
Corn bread mix
Flavored tortilla chips
Popcorn
Cereal (eg cornflakes)
Corn meal
Puffed corn snacks
Fries
Wheat flour
Soy flour
Meatballs and burgers containing soy protein
Potato chips
Soy-based protein drinks/powders

30. Extract DNA from food

31. Chelex
Chelex resin is often used for DNA extraction in preparation for PCR by binding to cations including Mg2+, which is an essential cofactor for DNases.
Chelex protects the sample from DNases that might remain active after the boiling and could subsequently degrade the DNA, rendering it unsuitable for PCR.

32. Why these steps? Grinding food to release DNA
InstaGene chelates divalent ions (e.g. Mg2+) necessary for DNA degrading enzymes (e.g. DNases)
Only 50 μl of food transferred otherwise InstaGene is overwhelmed (~ 5 mg of original material)
Boiling releases DNA from food into the InstaGene solution
Pellet InstaGene and food debris because InstaGene inhibits PCR reaction (Taq needs Mg++)
Why these steps?
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
InstaGene

33. Set up PCR reactions

34. PCR
Developed in 1983 by Kary Mullis, Still ongoing lawsuits with regard to the Taq polymerase

35. The PCR Reaction What do you need?
What is needed for PCR?
The PCR Reaction What do you need?
Template - the DNA to be amplified
Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence
Forward
Reverse
Nucleotides - dATP, dCTP, dGTP, dTTP
Magnesium chloride - enzyme cofactor
Buffer - maintains pH & contains salt
Taq DNA polymerase – thermophillic enzyme from hot springs

36. The PCR Reaction How does it work?
Heat (94oC) to denature DNA strands
Cool (59oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA
Repeat 40 cycles

37. Analysis of Results GMO positive GMO negative 1 2 3 4 5 6 7 1 2 3 4 5
1: non-GMO food with plant primers
2: non-GMO food with GMO primers
3: Test food with plant primers 1 2 3 4 5 6 7
4: Test food with GMO primers
GMO negative
5: GMO positive template with plant primers
6: GMO positive template with GMO primers
7: PCR MW Ruler

38. Trouble shooting False Positives
Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps.
False Negatives
No DNA extracted
Possible food type or possibly primers do not work on that plant species
InstaGene matrix transferred to PCR reactions