Unit Plant Science Problem Area Cellular Biology and

Unit Plant Science

Problem Area Cellular Biology and Agriculture

Lesson Biotechnology

Student Learning Objectives n n n n 1. Describe biotechnology and genetic engineering. 2. Explain the differences between genetic engineering and traditional plant breed-ing. 3. Explain the steps in engineering a plant. 4. Explain how desirable genes are located. 5. Explain how selected genes are introduced into a target organism. 6. Explain how genetically engineered crops are tested. 7. Discuss the benefits and risks of biotechnology.

Terms n n n n Agronomic trial Biolistics Biotechnology DNA probes DNA sequence map Electroporation Field trials n n n n Genetic engineering Genetic linkage map Growth chamber Immunoassay Microinjection Physical marker map Plasmid

Terms cont. n n n n Protoplast Reporter gene Safety trial Transgenic organism Varietal trial Vector Viral encoding

What are biotechnology and genetic engineering? n A. Biotechnology is simply the use of living organisms to create or improve something. Today biotechnology is centered on the modification of living organisms as a result of our new understanding of genes and DNA. It includes techniques such as: 1. Genetic engineering ¨ 2. DNA analysis ¨ 3. Genetic mapping ¨ 4. Gene transfer ¨ 5. Plant tissue culture ¨ 6. Biofermentation ¨

n B. Biotechnology is being used with microbes, plants, and animals to produce beneficial products and improve species. It is being applied to many agricultural processes including: ¨ 1. Bread making ¨ 2. Beer brewing ¨ 3. Wine, cheese, and yogurt fermentation ¨ 4. Silage fermentation ¨ 5. Classical plant breeding

n n C. Genetic engineering is the manipulation of genes. It is also referred to as recombinant DNA technology. This involves moving genetic information from one organism into a different organism or replacing it in the original organism in a new combination. These changed organisms are called transgenic. A transgenic organism is one that has either new genetic information incorporated into itself or a unique recombination of its original DNA.

How is genetic engineering different from traditional plant breeding? n Genetic engineering (GE) is different from traditional plant breeding (TPB) in many ways: n A. With TPB, crosses can be made only within the same species or closely related species. This limits the genetic material breeders can work with. With GE, there are fewer limits to the genetic material a breeder can work with. Genes can be taken from any living organism including bacteria or animals and inserted into a plant.

n n n B. When plants are crossed using TPB, nearly 100, 000 genes are combined from each plant. This requires breeders to employ the technique of backcrossing, rebreeding back to one of the original parents, many times to get rid of unwanted genes and restore desired traits. With GE, a single desired gene can be inserted into a plant. C. When a cross is made using TPB, the seeds are collected and the new generation of plants must be germinated and grown before the results of the cross can be verified. Using GE, modified plants are grown in tissue culture and the change is verified. D. TPB requires up to 14 generations to produce a new plant. GE will create a new plant in as few as five generations.

What steps are involved in engineering a plant? n The creation of a transgenic organism begins with a selected gene from a donor organism and the insertion of that gene into a host organism. Eight major steps are required to complete this process. n A. A donor which contains the gene that codes for the desired trait is identified. B. DNA is removed from this organism’s cells and cut into fragments. C. Fragments of DNA are sorted by size using gel electrophoresis and grouped. The fragment containing the desired gene is then isolated. n n

n D. The targeted fragment is joined with new DNA, making it possible to move the desired gene into the host organism. n E. The altered DNA is moved into the host cells. n F. These transformed cells are grown into a complete transgenic organism. n G. This transgenic organism is grown and tested. n H. The transgenic organism is reproduced to assure that the new gene is transferred to the progeny.

What methods are used to find a specific gene within an organism? n n Creating a transgenic organism begins with locating the specific gene that codes for the desired trait. Genes are specific sequences of DNA contained among all of the DNA inside an organism’s nucleus. In most plants and animals, less than ten percent of the DNA code for genes. A scientist will use a variety of mapping techniques to find a specific gene. There are three kinds of gene maps: genetic linkage maps, physical marker maps, and DNA sequence maps.

n n n A. Genetic linkage maps show where on the chromosome a target gene may be. It will provide the general proximity. These linkages are determined by examining the frequency that different traits are inherited together. B. Physical marker maps identify the distance between a marker and the desired gene along the strand of DNA. Markers are specific molecular characteristics of the DNA molecule which can be observed. C. DNA sequence maps describe the order of the bases (ATGC) around and including the target gene on the DNA strand within the chromosome.

How are selected genes introduced into a target organism? n n A. The desired gene must be cut of the donor organism and combined with other DNA before it can be inserted into the target organism. This combination is necessary for the gene to function, replicate, and be inheritable. This recombined DNA is usually a plasmid, a self-replicating closed loop that comes from a bacterium. Once inside the host cell, the plasmid can replicate and be passed on to the next generation.

n B. The target cell must remain intact after the transfer or it will not function. These cells must not be ruptured or they will die. The tough cellulose cell wall must be penetrated and the new DNA gently moved through the cell membrane. ¨ 1. Enzymes can be used to digest the cell wall, creating an exposed membrane. Temporary holes are opened in this membrane to allow gene transfer. Plant cells with no cell wall are called protoplasts and are very susceptible to gene transfer. ¨ 2. A microorganism that naturally penetrates plant cells can be used to transfer DNA into the target cell. This technique leaves the cell wall intact.

n C. There are four methods commonly used to transfer genes and create genetically modified organisms. A technique called viral encoding does not create a genetically transformed organism but does result in an organism that produces a foreign protein. ¨ 1. Microinjection: DNA is physically injected into a cell. A small glass needle is moved through the cell membrane. After the needle has penetrated the membrane, the new DNA is simply injected into the cytoplasm. Transformed cells are grown into whole plants that exhibit the desired trait, reproduced so the offspring contains the new gene. ¨ 2. Electroporation: Placing a protoplast into an electrically charged environment can cause the cell membrane to become permeable to DNA. The technique of electroporation uses an electric charge to open holes in the cell membrane, allowing foreign DNA to enter the cell. The transformed cells are grown and propagated, with the subsequent generations exhibiting the new trait.

3. Biolistics: In this process, DNA is shot into a cell attached to microscopic metal particles. These particles are fired from a specially modified. 22 caliber gun. The particles move so fast that they can penetrate the cell membrane without doing permanent damage to it. Cells that survive this process are transformed and can be grown and propagated. ¨ 4. Vectors: A living organism, such as a virus or bacterium, or a plasmid which carries new genetic information into a target cell is a vector. The desirable gene is spliced into the DNA of the vector. The vector than penetrates the target cell as part of its natural life cycle and transforms the target cell through this infection. ¨ 5. Viral encoding: In this process, a virus is used to carry a new gene into a cell. This gene does not become part of the cell’s genetic make up and so is not transferred to future generations. While the cell is alive and infected with this virus, it will produce the protein the new gene codes for. This technique is useful in culturing single cell organisms to produce things such as insulin, antibiotics, and many vaccines. ¨

n D. Many techniques have been developed to identify genetically transformed plants. These include the use of reporter genes or marker genes, DNA probes, and immunoassays. These methods can be used to identify a genetically modified plant at any stage of development, from seed to mature plant. ¨ 1. Reporter genes: These genes are also referred to as markers or marker genes. Reporter genes code for an observable trait and are attached to the desired gene before transfer into the target organism. If the reporter gene is functioning, then the desired gene will also function. These markers are selected for traits that can be verified early in the plant’s development.

n n 2. DNA probes: This is a short piece of single-stranded DNA with the complimentary code for the desired gene. It is labeled with radioactivity. If the gene is present, the probe will stick and the radioactivity will be detected in the transformed cell. If the gene is not present, the probe will not stick so there will be no radioactivity detected. 3. Immunoassays: These are capable of detecting the presence of the actual desired gene without the use of markers or radioactivity. They accomplish this by identifying the gene product, or protein, that the desired gene produces. Immunoassays utilize techniques working with animal immune systems involving antigens and antibodies. An animal is injected with the target protein. This is registered as an antigen by the animal’s immune system. The animal produces an antibody in response to that specific antigen. These antibodies are used to detect the presence of the desired gene. These antibodies can be linked to chemicals that change color, so a simple color change can proclaim the presence of the desired gene product.

Where are transgenic plants tested? n n n A. Transgenic plants are tested in growth chambers and field trials. 1. The growth chamber is a closed environment designed to control and optimize factors that affect plant growth. This controlled environment allows researchers to test the new plants for traits that may harm the environment, speed the growth rate of the plants, and evaluate the expression of desired traits. 2. Field trials are conducted outside in a controlled, natural environment using normal production techniques. Evaluation of these trials involves much data because of the natural variability of a field. Analysis of collected data must account for the effects of weather, soil, pests, and any other naturally occurring variable.

n B. Transgenic plants are evaluated in early stage testing to determine the answers to a variety of important questions, including: ¨ 1. What traits do they express? ¨ 2. Can they pollinate other plants, producing fertile offspring which might spread the new trait into wild populations? ¨ 3. Can the transgenic plants escape to become weeds? ¨ 4. Are they effective for their intended use? ¨ 5. Will they produce unintended consequences to the environment?

n n n C. Field trials are used to test varietal differences, farming practices, and the safety of transgenic crops. 1. Varietal trials compare transformed varieties to their normal counterpart to determine the characteristics of the new varieties. These characteristics may include yield, pesticide tolerance, and pest resistance. 2. Agronomic trials identify the farming practices that will give the new varieties their best growing conditions. These can include population, row spacing, tillage practices, or fertility programs.

n 3. Safety trials are used to assess any possible risk the transgenic plant may pose. These are the same risks assessed in growth chamber trials (pollinating wild relatives, becoming a weed). But safety trials also include looking at the potential health effects on animals, including humans, that will consume these crops, and the potential for the development of pest resistance in the case of insecticidal transgenic plants.

What are theoretical benefits and risks of biotechnology? n A. Environmental benefits include: ¨ 1. The reduction of pesticide use ¨ 2. Greater survival of beneficial insects ¨ 3. Reduced exposure of farm workers to pesticides ¨ 4. Increased use of environmentally friendly herbicides such as glyphosate ¨ 5. Reduction of soil erosion ¨ 6. Reduced use of nitrogen fertilizer and the subsequent pollution from nitrates ¨ 7. Early detection of disease

n B. Global economic benefits include: ¨ 1. More predictable yields ¨ 2. Greater yields ¨ 3. Reduced cost of production due to the use of fewer inputs ¨ 4. New markets for crops with unique traits such as pharmaceutical properties ¨ 5. Improvements to the world food supply (increased protein content, new tolerance to environmental extremes, improved nitrogen fixation) ¨ 6. Increased efficiency in plant breeding

n C. Genetically modified foods may offer the benefits of: ¨ 1. Improved protein content ¨ 2. Improved flavor ¨ 3. Improved shelf life ¨ 4. More vitamins ¨ 5. Reduction of allergens or natural toxins ¨ 6. Improved fat levels ¨ 7. Reduced pesticide residue

n D. Genetically altered crops raise a number of environmental concerns including: ¨ ¨ ¨ ¨ ¨ 1. The development of insect populations resistant to this control method 2. Reduced interest in sustainable agricultural practices because of the existence of more resistant crops 3. Difficulties in controlling weeds due to transgenic herbicide resistant crops 4. The creation of new cultivars with unknown consequences as a result of modified crops breeding with wild plants 5. Increased use of certain herbicides with associated environmental risks inherent to pesticide use 6. The development of disease-resistant plants resulting in more virulent strains of the targeted pathogen 7. Poisoned wildlife 8. Reduced genetic diversity as producers become more dependant on a select group of varieties 9. Inaccurate predictions of environmental safety from field trials

n E. Economic/global concerns of biotechnology include: 1. Increased shift to more capital-intensive farming and large farms ¨ 2. Increased seed costs ¨ 3. Corporate mergers resulting in less competition among agricultural suppliers ¨ 4. Loss of ability among producers to save seed for subsequent crops ¨ n F. The concerns about biotech foods and human health include: 1. Antibiotic resistance from marker genes ¨ 2. Hidden allergens from marker genes ¨ 3. Production of new or increased levels of toxins in food crops ¨ 4. Unknown substances occurring in foods ¨

Review/Summary n n n n What are biotechnology and genetic engineering? How is genetic engineering different from traditional plant breeding? What steps are involved in engineering a plant? What methods are used to find a specific gene within an organism? How are selected genes introduced into a target organism? Where are transgenic plants tested? What are theoretical benefits and risks of biotechnology?