GMO—Detecting Genetically Modified Foods

1. GMO—Detecting Genetically Modified Foods Testing Foods for Introduced Genes

2. GMO’s • Genetically modified organisms have DNA that has been modified through genetic engineering • GM foods were first put on the market in the early 1990s fueling a revolution in agriculture • Unlike organisms developed through the conventional genetic modification of selective breeding (plant breeding and animal breeding) or mutation breeding.

3. GMO’s • Organisms that have been genetically modified by the insertion of foreign genetic material. • For plants…the gene may come from another plant, or from another species, or from another kingdom. • Introduced DNA codes for a protein that gives the GMO an advantage over the wild type

4. GMO-Animals Animal products have been proposed or produced • Pig engineered to produce omega-3 fatty acids. • A breed of pigs that are able to absorb plant phosphorus more efficiently and as a consequence the phosphorus content of their manure is reduced • Sheep that express antibodies in milk

5. GMO’s - Plants • Genetically modified plant products: soybean, corn, canola, and cotton seed oil. •  Genes encode • herbicide resistance, • insect resistance, • drought tolerance, • frost tolerance, • delayed fruit ripening and other traits.

6. GMO Plants FlavrSavr tomato • the first commercially grown genetically engineered food granted a license for human consumption • Produced by the Californian company Calgene 1992 • Sold in 1994, and was only available for a few years before production ceased

7. Flavor-sav Vs Normal • More resistant to rotting and softening by adding an antisense gene which interferes with the production of the enzyme polygalacturonase (see RNA interference). • Softening makes the tomato more susceptible to being damaged by fungal infections. • Wide Variety of Tomatoes • Picked BEFORE they are ripe….still very firm • Artificially ripen with ethylene gas • Easier handling and shelf life

8. Flavr Savr • Flavr Savr tomatoes could be allowed to ripen on the vine, without compromising their shelf-life because the inserted gene slowed the softening of tomatoes • Vine-ripe fruits have better flavor and could be harvested like green tomatoes without greater damage to the tomato itself.

9. BT-Corn • Bt corn is a variant of maize, genetically altered to express the bacterial Bt toxin • Poisonous to insect pests. • In the case of corn, the pest is the European Corn Borer

10. BT-Corn • A gene from a microorganism Bacillus thuringiensis inserted into the corn genome. • The gene codes for a protein toxin that forms a crystalline product…the product is eaten & perforates the larval digestive tract. • The pores allow naturally occurring enteric bacteria such as E. coli and Enterobacter to infect the insect causing death

11. BT-Corn In 2001, 176 varieties of bt corn were voluntarily withdrawn from the list of approved varieties by the United States Environmental Protection Agency It was found to have little or no Bt expression in the ears and was not found to be effective against second generation corn borers. (Current status of Bt Corn Hybrids, 2005)

12. BT-Corn • The Bt endotoxin is not harmful to humans, other mammals, birds, fish, or beneficial insects • Concerns for effects of Bt corn on nontarget insects • A threat to monarch caterpillars? • 1999 Monarch increased by 30%, despite Bt corn accounting for 30% of all corn grown in the USA that year. • The beneficial effects of Bt corn on Monarch populations can be attributed to reduced pesticide use

13. Round-up Ready • Roundup is the brand name of a systemic, broad-spectrum herbicide • Produced by Monsanto • The active ingredient glyphosate

14. Round up Ready • Glyphosate is the most used herbicide in the USA , and Roundup is the number one selling herbicide worldwide since at least 1980. • Monsanto also produces seeds which grow into plants genetically engineered to be tolerant to glyphosate which are known as Roundup Ready crops. The genes contained in these seeds are patented.

15. Roundup Ready Crops • In 1996, genetically modified Roundup Ready soybeansresistant to Roundup became commercially available, followed by Roundup Ready corn in 1998 • Current Roundup Ready crops include soy, maize (corn), canola, sugar beet, and cotton, with wheat, and alfalfa still under development.

16. No Till Farming • Using Round-Up eliminates ALL plants…except those that are genetically modified • No need to till (plow-turn over) the fields. • Preserves the top soil “But plow-based farming in this region cultivated an unexpected yield: the loss of fertile topsoil that literally blew away in the winds”

17. Glyphosate • The main active ingredient of Roundup is a simple amino acid analogue • In the early 1970s, it was discovered that glyphosate, inhibits the activity of an enzyme necessary for making certain amino acids. • These amino acids are needed for protein synthesis in plants ITS “LIKE” an amino acid… but NOT an amino acid Stops protein synthesis!!!

18. Glyphosate inhibits EPSPS • T he enzyme is: 5-enolpyruvylshikimate- 3-phosphate synthase (abbreviated EPSPS) • Plays a key role in the biochemical pathway that makes aromatic amino acids phenylalanine, tyrosine, and tryptophan. • This enzyme is only present in plants and microorganisms, such as bacteria and fungi. • Not present in animals and humans

19. Glyphosate Resistance Not Found in plants • In spite of extensive spraying with Roundup no resistant plant species had been detected. • Monsanto—considered the economic advantage of resistant plants…… • Spray extensively with Roundup---kills weeds but not crops---if crops are resistant to glyphosate. • NO NEED TO TILL THE SOIL

20. Bacteria also make amino acids • The target of glyphosate, EPSP synthase is also present in bacteria • A search for resistant bacteria was undertaken. • The idea is that if a glyphosate-resistant enzyme from bacteria could be transferred to plants it might make the plants resistant to the herbicide. Bacteria evolve much faster than plants

21. Glyphosate Resistance Found! • The C4 strain of Agrobacterium • A species of bacteria that was found growing in the waste-fed column at a factory that made glyphosate. • The EPSP synthase enzyme from this bacterium (C4 EPSP synthase) was almost completely insensitive to glyphosate

22. Agrobacterium tumefaciens • This bacterium infects plants and injects DNA from a plasmid into plant cells • Injected DNA enters the nucleus and becomes incorporated into the plant chromsomes. • Under normal circumstances Agrobacterium tumefaciens causes gall tumors in plants

23. Roundup Ready Cloning • The C4 EPSP bacterial gene was cloned and inserted into a bacterial plant vector in order to prepare for cloning into plants. • The Monsanto C4 EPSP cloning vectors first patented September 13, 1994

24. Roundup Ready Cloning • A plasmid vector that will work in E. coli • Needs also characteristics that allow the plasmid to work in Agrobacterium tumefaciens. • Needs a promoter…..to turn on the gene in plants!

25. Roundup Ready Cloning • A plant promoter (P-35S) is inserted at the 5' end. • This promoter is the 35S promoter from cauliflower mosaic virus (CaMV). • The 3' end of the gene is modified by inserting the polyadenylation site (NOS 3') from the nopaline synthase gene of the tumor-inducing (Ti) plasmid from Agrobacterium tumefaciens. 35S Resistance 5’ aaaaaaaaaaa 3’

26. Roundup Ready Transformation • Agrobacteriumtumefaciens. infects plants and injects DNA into plant cells where it enters the nucleus and becomes incorporated into the plant chromsomes. • The recombinat DNA is transferred and no tumors are formed.

27. Roundup Ready • Roundup Ready soybean was the first crop plant produced by Monsanto. • Today, 90% of the soybean crop in the USA consists of Roundup Ready® plants. • You can't buy soybean products that don't come from genetically modified plants.

28. HT Global area of genetically engineered crops, 1996 to 2006: By trait (million hectares) Herbicide tolerance IR Insect resistance (mostly Bt) VR Resistance to virus diseases Trait HT IR (Bt) IR/HT VR/Others Total 1996 0.6 1.1 -- <0.1 1.7 1997 6.9 0.4 <0.1 <0.1 11.0 1998 19.8 7.7 0.3 <0.1 27.8 1999 28.1 8.9 2.9 <0.1 39.9 2000 32.7 8.3 3.2 <0.1 44.2 2001 40.6 7.8 4.2 <0.1 52.6 2002 44.2 10.1 4.4 <0.1 58.7 2003 49.7 12.2 5.8 <0.1 67.7 2004 58.6 15.6 6.8 <0.1 81.0 2005 63.7 16.2 10. <0.1 90.0 2006 69.9 19.0 13.1 <0.1 102.0 GMO

29. How to make a Genetically Modified Plant • Isolate gene that direct cells to make protein of interest • (From bacteria in the sewers of the chemical plant making RR) • Attach the gene to the promoter that works in plant • (Califlower mosaic virus 35S) • Insert the promoter-gene and a gene for selectable marker into plant cells • Agrobacteriumtumefaciens • Allow the genetically altered cells to grow into plants.

30. How to Detect a GMO • Isolate DNA from plant tissue and food products.   • (PCR) is used to assay for evidence of • the 35S promoter that • drives expression of the glyphosate resistance gene and many other plant transgenes.    LOOK FOR THE GENETIC DIFFERENCE??

31. 35S PROMOTER INDICATES GMO • Herbicide resistance correlates with an insertion allele – the 35S promoter – that is readily identified by electrophoresis on an agarose mini-gel. • Amplification of tubulin, a protein found in all plants, provides evidence of amplifiable DNA in the preparation, while tissue from wild-type and Roundup Ready® soy plants are positive controls for the 35S promoter.  

32. Control for PCR • Tubulin is one of several members of a small family of globular proteins • The most common members of the tubulin family are α-tubulin and β-tubulin, the proteins that make up microtubules. • Microtubules are assembled from dimers of α- and β-tubulin. • Tubulin was long thought to be specific to eukaryotes

33. Two PCR reactions are performed for each plant or food sample. •  One primer set amplifies the 35S promoter from cauliflower mosaic virus. • The presence of a 35S product is diagnostic for the presence of a transgene. • The 35S promoter is used to drive expression of the glyphosate (Roundup) resistance gene or Bt gene in edible crops.   • A second primer set amplifies a fragment of a tubulin gene and controls for the presence of plant template DNA. • Since the tubulin gene is found in all plant genomes, the presence of a tubulin product indicates amplifiable DNA in the sample isolated.   • Tubulin is a housekeeping gene

34. Results of a GMO Test

35. PCR to Detect GMO • The following primer sets were used in the experiment: • 5'-CCGACAGTGGTCCCAAAGATGGAC-3' (Forward Primer) • 5'-ATATAGAGGAAGGGTCTTGCGAAGG-3' (Reverse Primer) • 5'-GGGATCCACTTCATGCTTTCGTCC-3' (Forward Primer) • 5'-GGGAACCACATCACCACGGTACAT-3' (Reverse Primer) • PCR Characteristics • Denaturing step:  94 C 30’ • Annealing step:   60 C 30” • Extending step:   72 C 30’ • 34X.. • 35S ----162 base pairs • Tubulin-187 base pairs

36. RG DP BS AS EC AS KR FD CB AM + c TL ASca TJ TF SC DH DH DH 20001600 1000 500 20001600 1000 500 Results: May 2010 15 Samples were successfully amplified with only 4 products testing + for GMO FD= Wheat (but we had not wheat products?? S C= TF = DH= corn pops

37. No Results…….PCR’s did not work JO MS RH AC AC BIR OSC ATM DD TP +C

38. PCR for Corn Transposon/Tubulin M JP JS CC CG TL SM M ML SH DR

39. GMO – PCR Results…….Biorad/Carolina Kits M SL AB NB CW CT RH LB RB WM RP TS AH HB EB CV AL AK M MLH NF BF TW MH SB ZP + MM S35 Promoter CHIPS Cracker Veg. pepperoni Tor. Chip pretzel PC 500 Tubulin Control PSII Control PC

40. Results: • 14 Food items were tested for genetic modification using the S35 promoter from CMV as a marker • Tubulin or Photosystem II used for a negative control • 9 samples had + PCR results • 5 samples showed +results for the S35 promoter • Problem cracker and tortilla chip PCR product wrong size. Chips and pretzel don’t have + controls

41. Conclusions: • Tortilla chips, pretzels, veggie pepperoni and club crackers appear to contain food from GMO’s • Wrong size products, no tubulin make conclusion regarding pretzel and cracker suspect • Surprize—wheat products have generally not been reported to contain this genetic modification. Perhaps they also contain corn or soy products. Further testing would be necessary to confirm. • Need to optimize the procedure.

42. Acknowledgement • Funding for this project was made available by a generous grant from the Florida Farm Bureau.