Module 2: Genetic Engineering in Food Agriculture PDF

MODULE 2: GENETIC ENGINEERING IN FOOD AGRICULTURE BACKGROUND INFORMATION PART 1 Genetic Engineering For most of history, farmers had to wait several plant generations before crops had the traits they most desired. The farmers used selective breeding, the process of choosing parent plants with the b...

MODULE 2: GENETIC ENGINEERING IN FOOD AGRICULTURE BACKGROUND INFORMATION PART 1 Genetic Engineering For most of history, farmers had to wait several plant generations before crops had the traits they most desired. The farmers used selective breeding, the process of choosing parent plants with the best traits over many generations. Selective breeding resulted in dramatic genetic changes to the species. While earlier farmers had no concept of the science of genetics, selective breeding based on observable traits allowed them to use plants’ DNA to solve agricultural challenges and to improve the food supply. This approach to selecting specific traits is exemplified by the apple activity in Module 1. Although selective breeding is still widely used, there are more modern processes available to alter the genetics of microorganisms, plants, and animals. More modern techniques to alter an organism’s genetics includes mutation breeding, molecular marker-assisted breeding, genetic engineering, and genome editing. Genetic engineering (GE) refers to deliberately modifying the characteristics of an organism by altering its genetic material. GE techniques include particle bombardment, Agrobacterium-mediated transformation, and targeted genome editing (the most recent additions to the genetic engineer’s toolbox). Using GE technology, scientists can bring us improved agricultural products and practices faster than in the past. Why genetically engineer plants? Plants are genetically engineered for many of the same reasons that selective breeding is used: Better nutrition, higher crop yield (output), greater resistance to insect damage, and immunity to plant diseases. Key biotechnology events related to food agriculture 1901 Japanese biologist Shigetane Ishiwatari discovered Bacillus thuringiensis (Bt), which makes a natural pesticide, found in soil worldwide and used by farmers since the 1920s. 1919 Karoly Ereky introduced the new term biotechnology (i.e., using biological systems to create products). 1971 Paul Berg completed a landmark gene splicing experiment. 1973 Stanley Cohen and Herbert Boyer created the first modified organism using recombinant DNA (rDNA) technology. 1974 Rudolf Jaenisch and Beatrice Mintz created the first transgenic animal (a mouse). 1978 Herbert Boyer starts a new company, Genentec and produces recombinant insulin. 1983 Mary-Dell Chilton inserted an antibiotic-resistant gene into a tobacco plant creating the first GE plant. 1987 Calgene creates the FlavrSavr® tomato. 1989 Chymosin from GE microorganisms authorized as a food processing aid by FDA. 1994 FDA concludes the FlavrSavr® tomato is as safe as comparable non-GE tomatoes. 1995 EPA approves the use of a Bt toxin as a plantincorporated pesticide in a GE crop. 1998 GE virus-resistant papaya was grown commercially in Hawaii. 2012 CRISPR-Cas9 is used as a programmable RNA-guided DNA cutting tool. Selective breeding techniques involve repeatedly crossbreeding plants until the breeder identifies offspring that have inherited the genes responsible for the desired combination of traits. However, this method may also result in the inheritance of unwanted genes responsible for unwanted traits (called linkage drag), and it can result in the loss of desired traits. 2015 Genetically modified salmon is the first GE animal approved for food use in the United States. GE techniques can be used to isolate a gene or genes for the desired trait, add a gene from another organism or edit chromosomal DNA in a single plant cell, and generate a new plant with the trait from that cell. By adding one desired gene from the donor organism or by editing the gene in the chromosomal DNA of the single cell, the unwanted traits 24 HIGH SCHOOL 2017 GE apples are available for sale in the United States. 2019 FDA completes consultation of high oleic soybean oil, first food from a genome edited plant. MODULE 2: GENETIC ENGINEERING IN FOOD AGRICULTURE BACKGROUND INFORMATION from the donor’s other genes can be excluded. GE is used in conjunction with selective breeding to produce GE plant varieties that are on the market today. Advanced Content GE techniques can be used to add new DNA to code for the expression of a new protein or to suppress expression of a native plant protein. Protein suppression can be achieved though transcriptional or post-transcriptional gene silencing (PTGS). RNA interference (RNAi) is a form of PTGS that targets mRNA transcripts for cleavage, preventing their translation into protein. The two different ways to achieve a desired trait are important, because both have been used to create GE plants that are used in food today. Development of GE Tools in Bacteria Throughout the past 100 years, several developments have led to current GE methods. After early geneticists were able to identify the gene locus for specific plant traits, various methods were used to try to transfer the specific DNA sequence from one plant to another. One method was injecting the DNA from the donor plant directly into the recipient plant cell to see if it would integrate into the recipient cell’s genome. Unfortunately, the DNA was degraded, and the method was unsuccessful. It was like trying to send an envelope through the mail with only a zip code; the postal service wouldn’t know where to deliver it. Scientists eventually used bacteria to transfer new DNA to the recipient plant cell. Transformation is the changing of the cell’s genetic makeup through the addition of new DNA. The DNA can come from the environment surrounding the cell via “horizontal gene transfer” or be added in a laboratory through GE methods. The laboratory method developed to combine genetic sequences that would not otherwise be found in the genome is called recombinant DNA (rDNA) technology. In 1973, Herbert Boyer and Stanley Cohen produced the first successful GE organism. Boyer had expertise using restriction endonucleases (enzymes that cut DNA at specific nucleotide sequences), and Cohen studied plasmids (small rings of DNA) in bacteria. They were able to use a restriction enzyme to cut open a plasmid loop from one bacterial species, insert a gene from a different bacterial species, and close the plasmid, which combined the genes from different bacteria into one rDNA molecule. An enzyme called ligase was used to help join the cut DNA strand. Then they transformed this rDNA plasmid into the bacterium Escherichia coli (E. coli) and showed that the bacteria could utilize the rDNA. In Boyer and Cohen’s experiment, one gene coded for tetracycline resistance and the other for kanamycin resistance. Tetracycline and kanamycin are antibiotics that kill bacteria that do not have resistance genes. It was possible to see which of the E. coli in their experiment had successfully acquired the new genes by culturing them in the presence of the antibiotics, where only the successfully transformed bacteria could grow. These experiments showed that bacterial transformation could be used to deliver the desired DNA to a useful site, just as the postal service delivers mail to the correct address. Restriction enzymes are like scissors that cut DNA at specific sequences. Some restriction enzymes leave blunt DNA ends while others leave short, single-stranded overhangs called sticky ends. Restriction Enzyme DNA G AA T T C C T T AA G GAA T T C C T T A AG Enzyme Cuts DNA DNA Fragments have Sticky Ends Ligase enzymes are like the glue or tape for connecting DNA sequences in GE, or molecular biology, procedures. Bacterial transformation still serves as the basis for a number of DNA technologies. Bacteria are used extensively in the laboratory for rDNA research. There are even some species of bacteria that go through the transformation process naturally, but most bacteria needs manipulation to become competent (able to take up the plasmid). Using the techniques from bacterial transformation, scientists have learned how to change the genome of plants, including plants that we use for food. Scientists worldwide continue to use the Boyer and Cohen techniques to improve GE tools that develop, modify, and improve consumer products, including many of the food products we eat. Nature’s Own Genetic Engineer A widely used method of transferring a transgene to a plant is to use the soil bacterium Agrobacterium tumefaciens (A. tumefaciens). This bacterium has a natural ability to enter a plant cell and insert its own DNA into a plant’s genome. A plasmid is constructed to include A. tumefaciens genes needed for transferring DNA into the recipient plant cell, the transgene of interest, and a selectible marker, such as a gene conferring antibioticresistance or herbicide tolerance. Scientists now use the bacterium’s natural behavior to insert the transgene into a plant’s genome. HIGH SCHOOL 25 MODULE 2: GENETIC ENGINEERING IN FOOD AGRICULTURE BACKGROUND INFORMATION Simplified Steps of Plasmid Development 1. Plasmid Cut with a Restriction Enzyme Selection Marker Gene Restriction Enzyme Sticky End 2. Plasmid and Desired Gene Joined Using DNA Ligase Ligase Enzyme Application of GE Tools in Plants Plants can be genetically engineered to be resistant to pests and herbicides, to increase crop yield, or to tolerate adverse weather conditions using a process similar to bacterial transformation. Plants can also be engineered to produce fruits and vegetables that have longer and more stable shelf-lives in the grocery store. These GE uses have potential trickle-down benefits from the farmer to consumers, animals, and the environment. Because plants are eukaryotic and contain a nucleus, a slightly different method than the one used for bacterial transformation is used to insert the gene of interest. For example, if scientists find a gene for enhanced drought resistance in a plant, and they want to use the gene to make another plant more drought resistant, an advantage of GE over selective breeding is that less time is required and linkage drag is avoided. The desired gene to be transferred and added to the genome of the recipient plant is often referred to as a transgene. 3. Plasmid with Desired Gene Inserted Desired Gene That was Cut with Same Restriction Enzyme as the Plasmid The technologies used to clone or synthesize genes are changing and evolving. The three major methods currently used are: • Traditional cloning – isolating DNA directly from the genome of the donor organism and inserting it into a plasmid for later use • Subcloning the gene of interest – copying the gene from an existing collection of DNA clones (“DNA library”) • De novo gene synthesis – building a gene from scratch, using single nucleotides or short oligonucleotide strands without the need for a physical template The techniques used by scientists to assemble and insert DNA pieces into the plasmid are also evolving along with the complexity of multi-gene DNA constructs. While simple restriction enzyme protocols can be used to create a single gene insert, multi-gene constructs such as those required for complex plant traits require more complex assembly strategies. Genetic Engineering What is a DNA Library? • Allows the direct transfer of one or just a few genes between either closely or distantly related organisms • Achieves crop improvement in a shorter time compared to conventional breeding • Allows plants to be modified by adding, removing, or switching off particular genes A DNA library is a collection of cloned DNA fragments that are stored in plasmids, which in turn are maintained and propagated in bacterial or yeast cells. The type of library is classified by the source of the DNA and the plasmid – referred to as a cloning vector – used to construct the library. Sources of DNA may be a single cell, a tissue, an organism, or an environmental sample containing multiple organisms. The DNA may be obtained from genomic sequences or from isolated mRNA and converted to complementary DNA (cDNA). Scientists use DNA libraries to find and study DNA encoding proteins or other functions of interest. Adapted from: Agricultural Biotechnology (A Lot More than Just GM Crops). www.isaaa.org/resources/publications/agricultural_ biotechnology/download/Agricultural_Biotechnology.pdf 26 HIGH SCHOOL MODULE 2: GENETIC ENGINEERING IN FOOD AGRICULTURE BACKGROUND INFORMATION General Plasmid Preparation Bacterial plasmids are used to store a ready supply of the gene of interest. In the case of Agrobacterium-mediated plant transformation, the plasmids are used to transfer the gene of interest to the genome of the recipient plant. To receive the gene of interest, the bacterial plasmids are treated with a restriction enzyme that is compatible with the gene. This way, the plasmid DNA will have the same sticky ends as the gene, so they will combine more easily. The gene and plasmid DNA preparations are mixed with DNA ligase to seal the sticky ends of the DNA molecules together. Scientists may also modify the bacterial plasmid using a similar process to insert one or more selectable marker genes. The selectable marker genes will be important later in the GE process when bacteria or plant cells with the gene of interest are being isolated. There are many selectable markers used to screen for bacterial, as well as plant transformants. Selectable markers include: • Auxotrophy (selects for the ability to grow on certain carbon sources) • Antibiotic resistance (selects for ability to grow in the presence of a specific antibiotic) • Herbicide tolerance (selects for ability to grow in the presence of a specific herbicide) This new bacterial plasmid is called a transformation plasmid and has the gene of interest as well as the selectable marker gene. The transformation plasmid is added to bacteria using a bacterial transformation method. Finally, the bacteria are plated onto a medium containing the selection factor that will inhibit the growth of bacteria that did not take up the plasmid. The Petri plates are incubated to encourage bacterial growth, and only the bacteria that have taken up the transformation plasmid with the selectable marker gene will grow. Bacteria without it will not grow, resulting in millions of bacteria with the gene of interest in their DNA. The next step is to transfer the gene to the plant cells. Currently, the most frequently used technique is Agrobacterium-mediated transformation. Bombardment with a gene gun is less common and typically used in cases where Agrobacterium-mediated methods don’t work. Agrobacterium is a plant pathogen that has the natural ability to transfer DNA to plant cells. GE methods use a version of the Agrobacterium plasmid that has been “disarmed”: the modified plasmid still has the ability to transfer DNA into the plant’s genome, but it’s diseasecausing genes have been removed. Agrobacterium that have been transformed with the plasmid carrying the gene of interest and selectable marker are mixed with the plant cells. The Agrobacterium enters the plant cells and inserts a segment of the plasmid DNA (containing the gene and selectable marker gene) into the plant’s genome. Once the Agrobacterium has had time to transform the plant cells, the cells are placed on medium containing: (1) An antibiotic that kills the Agrobacterium, (2) the selection factor that will inhibit growth of plant cells that did not take up the plasmid DNA, and (3) plant hormones that encourage the transformed cells to grow into new plants. After a gene has been successfully inserted into the plant’s genome, the modified plant must be able to grow and reproduce with its newly modified genome. First, the genotype of the plant must be studied so that the scientists only grow plants in which the genome has been modified correctly. When this is done, the GE plants will be grown under controlled conditions in a greenhouse and then in field trials to make sure that the new plants possess the desired new trait and show no new undesired characteristics. Food from GE Plants The first GE plant evaluated by the FDA for human consumption was the FlavrSavr® tomato. FDA concluded that the FlavrSavr® tomato was as safe as comparable non-GE tomatoes. It was brought to market in 1994, but it was not sufficiently profitable to continue production. Although there are currently no GE tomatoes on the market, other GE food crops are commercially available. Most of these GE plants were engineered to increase resistance to disease or pests, or tolerance to specific herbicides. As of 2019, there were 10 GE food crops available in the U.S. Of these, only a few GE crops in the grocery store are available as whole produce. Whole produce could include certain cultivars of apple, potato, papaya, sweet corn, and squash. Ingredients derived from GE corn, soybeans, sugar beets, and canola (such as flour, oil, starch, and sugar) are used in a wide variety of foods including cereal, corn chips, veggie burgers, and more. The 10 GE crops today are: Alfalfa, apples, canola, corn (field and sweet), cotton, papaya, potatoes, soybeans, squash, and sugar beets. Animal food: In the United States, more than 95 percent of food-producing animals consume food containing ingredients from GE crops. GE plants can also be found in food for non-food producing animals, such as cats and dogs. HIGH SCHOOL 27