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GM (Genetically Modified)Plants

Background• Genetically modified crops (GM) have been

used since 1996 in the U.S.• GM crops contain foreign genetic material

– The DNA may be from another plant or from aspecies from another kingdom

– Usually offers some benefit to the plant• Pest resistance• Herbicide resistance• Delayed fruit ripening• Increased yield• ….

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How to Create a GMO• Identify a protein that has the potential to

improve the crop.– Example: the bacterium Bacillus thuringiensis (Bt)

produces a protein called delta-endotoxin that islethal to European corn borers.

Bacillus thuringiensis Protein crystals and sporesproduced by Bacillusthuringiensis is toxic to thecorn borer

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How to Create a GMO• Clone the gene that codes for the protein.• http://www.ncbi.nlm.nih.gov/nuccore/870929?

ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum

• Engineer the gene so that the plant’s cells willread it and manufacture the protein.– Remove the introns, add the correct promoter

(docking site for RNA polymerase) and terminator.

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How to Create a GMO• Add a promoter and a terminator that

causes the foreign gene to be expressedcontinuously.

• 35S promoter from the cauliflower mosaicvirus (CaMV 35S).

• Nopaline synthase (NOS) terminator fromAgrobacterium tumefaciens.

• One or both of these promoters are found inabout 85% of all GM crops.

How to Create a GMO

• The engineered gene is transferred intoa culture of plant cells.

• The DNA must pass through the cellwall, plasma membrane and thenuclear envelope.

– Use the soil bacterium Agrobacteriumtumefaciens.

• Inserts its DNA into a plant’s genome.– Electroporation

• Electric current creates pores in cellmembranes

– Gene gun• Shoots gold particles coated with

engineered DNA into the plants cells.• Selectable markers are inserted with

the gene– Antibiotic resistance– Green fluorescent protein

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How to Create a GMO

• The cells are removed from theparent plant and then grown on aspecial medium that causes themto form a callus

• Callus - A clump ofundifferentiated cells.

How to Create a GMO• Once transformed the cells are induced to grow

with plant hormones to grow into complete plants.• Back cross the genetically modified plants into the

most current high yielding crop strains being usedin the field.– This can take years since only 50% of the genome is

transferred to the genetically modified crop in eachcross.

• Takes six to fifteen years to bring a crop to market.

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How to Create a GMO

Callus Plantlets growing in special medium

Plantlets growing in soil Transplantation

How to Create a GMO

Unprotected peanut plant

Genetically engineered BTpeanut plant

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The Debate

• Arguments against.– Potential for “superweeds” resistant to herbicides– “superbugs” will evolve that are no longer

susceptible to toxins in pest-resistant crops.– Allergic reactions to these new proteins.– Lack of government labeling– Not enough known about research.

The Debate

• Arguments for.– Crops are beneficial to the environment

since they reduce the use of pesticides andchemicals.

– Improve nutritional value of food indeveloping countries.

– Preserve land by reducing stress on theland.

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The Debate

• The future of food.• http://www.snagfilms.com/films/title/the_future

_of_food/• Roundup Ready (RR) Transgene

– Roundup is a herbicide.– The RR transgene makes plants resistant to

roundup so that they can be sprayed with theherbicide.

– Patent infringement disputes have occurred overthese GM plants when farmers try to save andreplant seeds from these GM plants.

Identifying GM crops

• ELISA (enzyme linkedimmunosorbent assay)– Antibody-based test

which identifiesspecific proteins suchas Bt or RR (RoundupReady Transgene)

– Cannot test for GMplants in general.

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Identifying GM crops• PCR

– Identifies sequences of DNA that have beeninserted into GM plants

– This is not a specific test. Therefore, it willidentify any altered GM plant.

– We will test for the two most common regulatorysequences.

• 35S promoter and the nopaline synthase (NOS)terminator.

Experiment overview• Multiple Controls

– Was the DNA extraction successful?• Primers which amplify a 455bp region of the photosystem II

chloroplast gene.– Was your sample contaminated?

• Analyze certified non-GMO food.• If this sample gives a GMO-positive result then it indicates

contamination. Therefore you will not be able to trust theresults of your food sample.

– Did your PCR reaction work?• Control DNA that codes for the PSII protein and the GMO

promoter and terminator sequences.• If these sequences are not amplified then you cannot trust a

GMO negative result of your food sample.• Gives you reference bands for your test samples.

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Experiment overview• Experimental group

– “duplex” PCR two targetsequences aresimultaneously amplified.

• Two pairs of primers– 35S promoter

• 203bp– NOS terminator

• 225bp– Some food samples contain

just the 35S promoter or theNOS terminator.

– Some foods contain boththe promoter and theterminator.

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Choose a Test Food

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Procedure• Day 1 – Purify your DNA samples• Day 2 – Set up and run PCR reactions• Day 3 – Set up and run gels of samples• Day 4 – Analyze results and answer the

questions in your lab notebook.