GM (Genetically Modified) Plants. Background

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1 1 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 a species from another kingdom Usually offers some benefit to the plant Pest resistance Herbicide resistance Delayed fruit ripening Increased yield.

2 Identify a protein that has the potential to improve the crop. Example: the bacterium Bacillus thuringiensis (Bt) produces a protein called delta-endotoxin that is lethal to European corn borers. Bacillus thuringiensis Protein crystals and spores produced by Bacillus thuringiensis is toxic to the corn borer 2

3 Clone the gene that codes for the protein. ordinalpos=1&itool=entrezsystem2.pentrez. Sequence.Sequence_ResultsPanel.Sequenc e_rvdocsum Engineer the gene so that the plant s cells will read it and manufacture the protein. Remove the introns, add the correct promoter (docking site for RNA polymerase) and terminator. 3

4 4 Add a promoter and a terminator that causes the foreign gene to be expressed continuously. 35S promoter from the cauliflower mosaic virus (CaMV 35S). Nopaline synthase (NOS) terminator from Agrobacterium tumefaciens. One or both of these promoters are found in about 85% of all GM crops. The engineered gene is transferred into a culture of plant cells. The DNA must pass through the cell wall, plasma membrane and the nuclear envelope. Use the soil bacterium Agrobacterium tumefaciens. Inserts its DNA into a plant s genome. Electroporation Electric current creates pores in cell membranes 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

5 5 The cells are removed from the parent plant and then grown on a special medium that causes them to form a callus Callus - A clump of undifferentiated cells. 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 used in the field. This can take years since only 50% of the genome is transferred to the genetically modified crop in each cross. Takes six to fifteen years to bring a crop to market.

6 Callus Plantlets growing in soil Plantlets growing in special medium Transplantation Unprotected peanut plant Genetically engineered BT peanut plant 6

7 7 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. Arguments for. The Debate Crops are beneficial to the environment since they reduce the use of pesticides and chemicals. Improve nutritional value of food in developing countries. Preserve land by reducing stress on the land.

8 8 The Debate The future of food. _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 the herbicide. Patent infringement disputes have occurred over these GM plants when farmers try to save and replant seeds from these GM plants. Identifying GM crops ELISA (enzyme linked immunosorbent assay) Antibody-based test which identifies specific proteins such as Bt or RR (Roundup Ready Transgene) Cannot test for GM plants in general.

9 9 Identifying GM crops PCR Identifies sequences of DNA that have been inserted into GM plants This is not a specific test. Therefore, it will identify any altered GM plant. We will test for the two most common regulatory sequences. 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 the results 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.

10 10 Experiment overview Experimental group duplex PCR two target sequences are simultaneously amplified. Two pairs of primers 35S promoter 203bp NOS terminator 225bp Some food samples contain just the 35S promoter or the NOS terminator. Some foods contain both the promoter and the terminator.

11 Choose a Test Food 11

12 12 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.