1. The Science of Transgenics Phil McClean Department of Plant Science North Dakota State University The Sociological, Economic, and Ethical Impact of Transgenic Organisms Workshop Fargo, ND February 21, 2003
2. Transgenics are a Biotechnology Product How about some definitions Biotechnology - General Definition The application of technology to improve a biological organism Biotechnology - Detailed Definition The application of the technology to improve the biological function of an organism by adding genes from another organisms
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4. What is a transgenic? Transgene – the genetically engineered gene added to a species Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant is treated with Roundup) Transgenic – an organism containing a transgene introduced by technological (not breeding) methods Ex. – Roundup Ready Crops Concept Based on the Term Transgene
5. We can develop organisms that express a “novel” trait not normally found in the species Why are transgenics important? Extended shelf-life tomato (Flavr-Savr) Herbicide resistant soybean (Roundup Ready)
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10. Next Generation of Ag Biotech Products Source: Minnesota Microscopy Society Golden Rice – increased Vitamin A content (but not without controversy) transgene = three pathway enzymes Sunflower – white mold resistance transgene = oxalate oxidase from wheat
11. Turfgrass – herbicide resistance; slower growing (= reduced mowing) Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)
12. Biotechnology is Not Just on the Farm Disease Treatment Diagnostics Environmental Cleanup Human Applications
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15. Environmental Applications Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution Indicator bacteria – contamination can be detected in the environment
22. Wheat Rye Triticale X Interspecific Cross New species, but NOT biotechnology products
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24. ATTCGA ATT G GA Susceptible Normal Gene Resistant Mutant Gene Mutagenesis Treatment Mutagenesis Changes the DNA Sequence
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27. + Glyphosate X Roundup Sensitive Plants X X Without amino acids, plant dies X Shikimic acid + Phosphoenol pyruvate 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Plant EPSP synthase Aromatic amino acids
28. Bacterial EPSP synthase Shikimic acid + Phosphoenol pyruvate 3-enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids Roundup Resistant Plants + Glyphosate With amino acids, plant lives RoundUp has no effect; enzyme is resistant to herbicide
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30. -Carotene Pathway Problem in Plants IPP Geranylgeranyl diphosphate Phytoene Lycopene -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Problem: Rice lacks these enzymes Normal Vitamin A “ Deficient” Rice
31. The Golden Rice Solution Daffodil gene Single bacterial gene; performs both functions Daffodil gene -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Phytoene Lycopene -carotene (vitamin A precursor) Phytoene synthase Phytoene desaturase Lycopene-beta-cyclase ξ-carotene desaturase Vitamin A Pathway is complete and functional Golden Rice
32. Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants
37. The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics
38. Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After
Editor's Notes
Title page.
The general definition is very broad. Many individuals prefer this definition because they can claim processes such as plant breeding or mutagenesis are actually biotechnology. The detailed definition points to the fact that a foreign gene needs to be inserted for a product to be considered a biotech product. Plant breeding is not biotechnology.
Genetic engineering is the collection of techniques necessary to create a transgene. These procedures include isolating the gene-of-interest from the tens of thousands of genes found in the genome of a species. Once that gene is isolated, it is modified so it functions better in an organism. That gene is then mixed with other genes to prepare it to be introduced into another organism. This whole step develops transgenes.
Here I am pointing out the difference between the process (using a transgene) and the product (the transgenic plant).
It is important to separate the process from the product. Focus on the product, it will highlight the benefits of biotechnology.
Bt stands for Bacillus thuringiensis , a bacteria that produces a toxin that kills the insects. The gene that encodes the toxin protein was inserted into plants.
There a multiple forms of herbicide resistance protect the crop against a variety of herbicides. Glyophosate and glufosinate are the primary classes that the plants are resistant against. Virus resistance is obtained by inserting the viral coat-protein gene into the plant. When this protein is produced in the plant, the viral immune system is activated, and the plant is resistant.
These are bacterial (chymosin) and animal (bST) examples. The chymosin gene is engineered for better cheese production. The bST hormone, engineered and expressed in bacteria, increase milk production in animals. bST was the first major ag biotech product.
FlavrSavr tomatoes were derived from a variety that was easy for introduction of foreign genes, but not particularly tasteful. In England, customers wanted labels tomato paste. The french fry industry did not want transgenic potatoes. StarLink was approved for animal but not human consumption. The corn contaminated the human corn market
Golden rice is enriched for vitamin A. The controversy surrounds the actual utility of the product. Some data suggest the child would have to significantly increase their rice intact of rice for vitamin deficiencies to be alleviated. Because of this knowledge the biotech industry is being accused of promoting a consumer-friendly product only for publicity purposes. The white mold disease is associated with elevated levels of oxalic acid. Resistance is provided by inserting a gene whose product breaks down oxalic acid.
Turfgrass are being targeted because of how much inputs are required for their maintenance. Spider silk protein is one of the strongest known natural products, and a natural for the development of a biologically-based product.
A list of human and environmental applications of biotechnology.
These are the general classes of human applications. Pharmaceutical products will be discussed on the next slide. Disease diagnosis using the tools of biotechnology to determine if an individual is at risk for contracting a certain disease. In some cases, the result will predict a 100% probability, whereas in other cases a positive result means a significantly increased risk. Gene therapy involves the insertion of a functioning gene into a human. The gene provides a protein product that is not being produced in the individual because of a mutation in their corresponding gene.
Each of these products were developed by the pharmaceutical industry using biotechnology approaches. Most of these are engineered into bacteria, and the protein product is extract in large quantities for future sale.
Recombinant (genetically-engineered) bacteria have been developed that help clean up contaminated sites. They can also be used to determine if a specific site is contaminated with a specific pollutant.
The health and pharmaceutical industry uses biotech approaches for vaccine production. The normal bacteria in the mouth produces an acid that destroys enamel. The engineered version of the bacteria does not produce the acid. Children will be treated with the engineered version, which also has a gene that will destroy the other bacteria in the mouth, allowing it to become established.
Edible vaccines may be the most important and accepted biotech product. The principles are the same as those used for normal vaccines: a protein enters the body in some manner, and the human immune system produces antibodies against that protein. When the human is then exposed to the pathogen, the immune system is turned on and destroys the pathogen.
GMO: notice that foreign DNA is a requirement for this definition. Plant breeding products are not GMOs, as some would like to claim.
Not all biological products are GMOs as some would have us believe.
Again, here were trying to show that breeding is not biotechnology. Some would like to claim breeding is biotechnology. In that way, they can claim that we actually been using biotech products for a long time. This is a hollow argument that will backfire if pressed.
These are important definitions related to crop improvement. Not the distinction; one is a biotechnology procedure, the other is not.
These photos illustrate the use of plant breeding to produce a new crop of agronomic utility.
It is important to understand that the mutagenesis procedure does not produce a GMO. The genes within the species are changed by the treatment. A new gene is not inserted.
Notice that the mutagenic treatment changed a single base in the gene sequence. This change created a resistant plant because the gene product targeted by herbicide is not affected by the herbicide. This is a change of gene in the plant; a foreign gene was NOT involved.
BASF Clearfield line is an example of products developed by mutagenesis. Some of these herbicide resistant genes have been stacked with transgenes, so those products are also considered to be transgenics or GMOs.
The Roundup Ready technology is the most visible plant biotechnology product on the market. To better understand plant biotechnology in general, it is important to understand the development of these transgenic organisms. Roundup is a brand name herbicide manufactured by Monsanto Corp. The active ingredient in this herbicide is glyphosate. The chemical binds to the active site of the EPSP synthase enzyme. This enzyme is a key to the development of a group of amino acids called the aromatic amino acids. When this enzyme is bound by glyphosate, it can not synthesize those amino acids, and the plants die because protein synthesis is severely disrupted. Glyphosate will not bind the to a particular genetically-engineered version of EPSP synthase. Therefore RoundUp Ready crops with this altered enzyme will survive when sprayed with the herbicide.
This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as Roundup, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death.
Roundup Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive.
The second major plant biotechnology product is more recent and was developed to address the vitamin A deficiency problems prevalent throughout the world. This vitamin deficiency is very critical because it can cause blindness and affects the severity of many diseases including diarrhea and measles. This is a severe problem that affects more than 100 million children worldwide. A simple solution would be to distribute vitamins to the affected children. Unfortunately, many countries where the deficiency is chronic do not have the necessary infrastructure to deliver the vitamin tablets to the most needed. The solution that is currently being promoted is to improve the vitamin content in widely-consumed, and readily available to the consumer. Transgenic rice plants were developed that contain elevated levels of the precursor to vitamin A. This GMO is called “Golden Rice” because of its color: it is yellow rather than white. It is yellow because β -carotene, a yellow precursor to vitamin A is abundant in the seed.
Unlike the single-step RoundUp Ready pathway, the β –carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels.
In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β -carotene. It is this product that gives "Golden Rice" its characteristic yellow color.
It is now time to cover the development of transgenic crops in greater depth. The two major steps are creating a transformation cassette that contains the gene of interest, and then successfully introducing the cassette into the plant.
All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red. Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green. The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium . For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray.
This slide summarizes the steps necessary for plant transformation.
Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens . The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun. Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants.
And this slide illustrates those steps.
The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants.
What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated.
