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Chapter 6 Plant Biotechnology

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1 Chapter 6 Plant Biotechnology
The agricultural applications of plant biotechnology is the second objective of module 4. As you are probably aware, the impact of genetic engineering has been big in the United States, affecting the crops that farmers raise and the reliance on chemicals that are used in agriculture. We will explore how this has happened in this objective.

2 Agriculture: The Next Revolution
Biggest industry in the world ($1.3 trillion of products per year) Plant transgenesis allows innovations that are impossible to achieve with conventional hybridization methods Resistant to herbicides Pest resistant Vaccines Plant transgenesis allows innovations that are impossible to achieve with conventional hybridization methods (e.g. conventional -> strength of cotton 1.5%; insertion of a single gene > strength 60%!) 74% of all soybean crops are genetically modified 32% of all corn

3 Methods Used in Plant Transgenesis
Unique advantages of plants: The long history of plant breeding provides plant geneticists with a wealth of strains that can be exploited at the molecular level Plants produce large numbers of progeny; so rare mutations and recombinations can be found more easily Plants have been regenerative capabilities, even from one cell Species boundaries and sexual compatibility are no longer an issue

4 Methods Used in Plant Transgenesis
Selective Breeding Farmers cross plants with desirable traits to increase the likelihood of producing offspring with that trait. Genetic manipulation of plants is not new. Ever since the birth of agriculture, farmers have selected plants with desired traits. For several thousand years, farmers have been altering the genetic makeup of the crops they grow. Human selection for features such as faster growth, larger seeds or sweeter fruits has dramatically changed domesticated plant species compared to their wild relatives. Many of our modern crops were developed by people who lacked an understanding of the scientific basis of plant breeding. Click on the animation to practice conventional selective breeding. selective breeding animation

5 Methods Used in Plant Transgenesis
Protoplast Fusion to create plant hybrids Degrade cell wall with cellulase A cell lacking a cell wall is called a protoplast The protoplasts from different species of plants can be fused together to create a hybrid The fused protoplasts grow in nutrient agar for a few weeks The colonies are then transferred to media to induce root and shoot growth

6 Methods Used in Plant Transgenesis
Selective Breeding vs. Biotechnology Prior to the early 1900s, traditional plant breeding relied only on man-made artificial crosses in which pollen from one species was transferred to another sexually compatible plant. The purpose of a cross is to bring desirable traits such as pest resistance, increased yield, or enhanced taste from two or more parents into a new plant. Even though careful crossbreeding has continued to improve plants, the methods of classical plant breeding are slow and uncertain. Creating a plant with desired characteristics requires a sexual cross between two lines and repeated backcrossing between hybrid offspring and one of the parents. Isolating a desired trait in this way can take years and the repeated inbreedings can result in a loss of genetic diversity in the crops resulting from this process. Furthermore, plant breeding by conventional selective breeding depends solely on the existence of genetic variation and desirable traits within the existing population. Biotechnology promises to circumvent these historical limitations. Scientists today can transfer specific genes for any desirable traits into a plant. There are two major differences between “traditional plant breeding” and “genetic engineering.” The first is the amount of genetic material involved. When two parental plant lines are crossed using traditional breeding methods, the new plant ends up with half the genetic makeup of each parent. Thus, the desirable gene may be accompanied by many undesirable genes from that same parent. To remove the undesirable genes, continued breeding is required. In the case of genetic engineering, only the few specifically desired genes are moved into the new plant. A second difference between traditional breeding and modern biotechnology is the source of genetic material. Traditional breeding largely relies on closely related plant species. In modern biotechnology, theoretically, a gene from any living organism may be moved into any other living organism. This permits scientists to move the genes from a bacterium into a plant. Click on the animation to visualize the difference between traditional selective breeding and biotechnology. Traditional selective breeding vs. biotechnology

7 Methods Used in Plant Transgenesis
Transgene – gene for a desirable trait introduced into a novel organism Transgene construct contains a promoter, terminator, and selectable marker gene A transgenic plant contains a gene that has been artificially inserted instead of the plant acquiring the trait through pollination. The inserted gene sequence, known as the transgene, may come from another unrelated plant, or from a completely different species. Plants containing transgenes are often called genetically modified (GM) crops. The underlying reason that transgenic plants can be constructed is the universal presence of DNA. Even species that are very different have similar mechanisms for converting the information in DNA into heritable traits. Therefore, a DNA segment from bacteria can be interpreted and translated into a functional protein when inserted into a plant. Identifying and locating genes for agriculturally important traits is currently the most limiting step in the transgenic process. Once a gene has been isolated and cloned, it must undergo several modifications before it can be effectively inserted into a plant. A promoter sequence must be added for the gene to be correctly translated into a protein product. The promoter is the on/off switch that controls when and where in the plant the gene will be expressed. The most commonly used promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression throughout the life cycle of the plant in most tissues. A terminator sequence must also be added to the transgene construct to signal the cell machinery to stop translation at the end of the gene sequence. A selectable marker gene is added to the gene construct in order to identify plant cells or tissues that have successfully integrated the transgene.

8 Methods Used in Plant Transgenesis
Transformation by Agrobacterium Method Ti plasmid integrates into the DNA of the host cell, making it an ideal vehicle for transferring recombinant DNA to plant cells Agrobacterium tumifasciens is a species of soil-dwelling bacteria that has the ability to infect plant cells with its chromosomal DNA. This bacterium harbors a large plasmid which it can transfer into plant cells when the plant is wounded. The plasmid carries genes that trigger an uncontrolled growth of cells in the infected plant, commonly referred to as “crown gall disease”. For this reason, the plasmid is known as a tumor inducing (or Ti) plasmid. When the bacterial DNA is integrated into a plant chromosome, it effectively hijacks the plant's cellular machinery and uses it to ensure the proliferation of the bacterial population. Agrobacterium can only infect a plant through wounds. When a plant root or stem is wounded it gives off certain chemical signals. In response to those signals, the vir genes of Agrobacterium become activated and direct a series of events necessary for the transfer of the DNA from the Ti plasmid to the plant's chromosome. The DNA then enters the plant cell through the wound. It is not clear how the bacterial DNA moves from the cytoplasm to the nucleus of the plant cell, nor how the DNA becomes integrated into the plant chromosome. Because of its ability to insert foreign DNA into plant cells, Agrobacterium is used in plant genetic engineering. Ti plasmids can be isolated from Agrobacterium. Then, by using restriction enzymes, scientists can insert genes for desirable traits into the Ti plasmid. To harness the Ti plasmid as a transgene vector, scientists have removed the tumor-inducing section of the plasmid DNA, while retaining the vir genes. The recombinant Ti plasmid is transformed into Agrobacterium, where it is transferred to the fragmented plant cells and becomes integrated into the plant's chromosomes. Click on the animation to see gene transfer in plants using the Ti plasmid. Animation: Gene Transfer in Plants Using Ti Plasmid

9 Methods Used in Plant Transgenesis
Leaf Fragment Technique Small discs of leaf incubated with genetically modified Agrobacterium Ti plasmid Treat with hormones to stimulate shoot and root development The Ti plasmid gives biotechnologists an ideal vehicle for transferring DNA. To put that vehicle to use, researchers often employ the leaf fragment technique. In this method, small leaf cuttings are cultured with genetically modified Agrobacter that have been transformed with the recombinant Ti plasmid containing the gene for the desired trait. During this culture period DNA from the Ti plasmid integrates with the DNA of the host cell and the gene of interest is delivered to the host plant cell. Plant tissues are transferred to a selective medium to ensure that only plants expressing the transgene will survive. The leaf fragments are then treated with plant hormones to stimulate shoot and root development before the genetically modified plants are transferred to soil. Click on the animation to practice engineering a tomato plant that is resistant to certain insects. Animation: Engineer a Crop

10 Methods Used in Plant Transgenesis
Transformation by Gene Gun Method Blast tiny metal beads coated with DNA into an embryonic plant cell Researchers at Cornell University developed gene gun technology in Gene guns literally blast tiny metal beads coated with thousands of DNA particles into an embryonic plant cell. The coated metal particles are accelerated with air pressure and shot at plant cells in a culture dish. If the metal particle lands in the nucleus, genes can dissolve away from the metal and incorporate into the DNA of the chromosomes. Click on the animation to see a gene gun in action. Gene Gun animation

11 Methods Used in Plant Transgenesis
Chloroplast Engineering More genes can be inserted at one time Genes are more likely to be expressed DNA is separate from the nucleus Why is it more advantageous to genetically alter chloroplasts vs the nucleus?

12 Methods Used in Plant Transgenesis
Plant Breeding and Testing Intrinsic to the production of transgenic plants is an extensive evaluation process to verify whether the inserted gene has been incorporated into the plant genome without detrimental effects to other plant functions. Initial evaluation includes attention to the activity of the introduced gene, stable inheritance of the gene, and any possible unintended effects on plant growth, yield, and quality. If a plant passes these tests, most likely it will not be used directly for crop production, but will be crossed with improved varieties of the crop. This is because only a few varieties of a given crop can be efficiently transformed, and these generally do not possess all the producer and consumer qualities required of modern cultivars. The initial cross to the improved variety must be followed by several cycles of repeated crosses to the improved parent, a process known as backcrossing. The goal is to recover as much of the improved parent's genome as possible, with the addition of the transgene from the transformed parent. The next step in the process is multi-location and multi-year evaluation trials in greenhouse and field environments to test the effects of the transgene and overall performance. This phase also includes evaluation of environmental effects and food safety. Click on the animation to see an overview of crop genetic engineering. Overview of crop genetic engineering

13 Methods Used in Plant Transgenesis
Antisense Technology Flavr SavrTM tomato introduced in 1994 Ripe tomatoes normally produce the enzyme, polyglacturonase (PG) which digests pectin Scientists isolated the PG gene, produced a complementary gene which produces a complementary mRNA that binds to the normal mRNA inactivating the normal mRNA for this enzyme When picked ripe, most tomatoes will turn to mush within days. This is because ripe tomatoes normally produce the enzyme polygalacturonase (PG), a chemical substance that digests pectin in the wall of the plant. This digestion induces the normal decay that is part of the natural plant cycle. Researchers at Calgene identified the gene that encodes PG, removed the gene from plant cells, and produced a reverse copy of the gene. Using Agrobacter as a vector organism, researchers transferred this reverse gene into tomato cells. In the cell, the gene encoded an antisense RNA molecule that would be complementary and therefore bind to the sense copy of the normal RNA. This binding makes a double stranded RNA molecule that is very unstable and inactive. With the normal mRNA inactivated, no PG is produced, no pectin is digested, and natural rotting is considerably slower. The Flavr Savr tomato was the first genetically modified food approved by the Food and Drug Administration. FlavrSavr tomatoes were available sporadically for several years, but eventually production was discontinued. The failure of the Flavr Savr has been attributed to Calgene's inexperience in the business of growing and shipping tomatoes. The variety of tomato Calgene started with was considered by farmers to be inferior, and insufficient resources were allocated to traditional plant breeding. As a result, Calgene's fields produced only 25-50% as many boxes per acre compared to most growers. Of these, only half as many as anticipated were large enough to be sold as premium-priced. Furthermore, much of the initial harvest was damaged during processing and shipping because ripe tomatoes are unavoidably more delicate than unripened ones. Equipment designed for handling peaches was purchased, and specialized shipping crates were developed, both at great expense. These costs prevented the Flavr Savr from becoming profitable, and Calgene was eventually bought by Monsanto, which was primarily interested in Calgene's ventures into cotton and oilseed. However, we can expect to see more antisense development in the near future. DNA technologists are currently working on using this technology to create a transgenic potato that resists bruising. Click on the link to see an animation of antisense technology in the Flavr Savr tomato. Antisense technology

14 Methods Used in Plant Transgenesis
RNA Interference (RNAi) Inhibits gene expression by interfering with transcription or translation of RNA molecules Currently, perhaps the most attractive strategy for preventing entry of pathogens is RNA interference (RNAi), which involves the use of short RNA molecules to interfere with the activity of a gene. This technology weakens the activity of a given target gene. Additionally, RNAi molecules designed specifically to target a pathogen’s gene can interfere either with that pathogen’s ability to infect or with some other aspect of the disease process. RNAi is sequence specific and works by silencing gene expression after the introduction of homologous double-stranded RNAs. The RNAi pathway is found in many plants and animals and is initiated by the enzyme Dicer, which cleaves long double stranded RNA molecules into short fragments of about 20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced Silencing Complex (RISC). Once the siRNAs associate with the RISC complex it is considered active and the antisense strand then guides this complex to bind to and degrade mRNA of the target pathogen. RNA viruses are possibly best suited to this approach, as theoretically both the genomic and the transcribed strands can be targeted, so it should be possible to interfere simultaneously with replication and expression of the viral pathogen. Because RNAi is sequence specific, expressing siRNAs against the viral genome should not affect any host gene functions. An alternative strategy for disease prevention would be to target host genes that play a role in pathogenesis. Click on the animation for a thorough explanation of RNAi. Protein expression silenced by RNAi Active protein expression RNAi video and animations

15 Practical Applications in the Field
Disease Resistance Vaccines for plants contain dead or weakened strains of plant viruses to turn on the plant’s immune system Transgenic plants express viral proteins to confer immunity Historically, the agricultural industry has relied on the use of fungicides worth $5.8 billion for control of fungal diseases worldwide. Infections can lead to reduced growth rate, poor crop yield, and low crop quality. Fortunately, farmers can protect their crops by stimulating a plant’s natural defenses against disease by injecting plants with a vaccine which turns on the plants version of an immune system, making it resistant to the real virus. Vaccinating an entire field of crops is no easy task and because of advancements in biotechnology is no longer always necessary. Many plants are inherently resistant to invading pathogens. Breeding for resistance to fungal, viral and other pathogenic microorganisms through traditional methods requires crossing closely related species, one of which has a gene or genes for resistance, but such resistant species often do not exist. Another approach using biotechnology involves taking a gene from the disease causing organism and inserting it into the plant so that the plant becomes protected. For example, researchers have recently inserted a gene from the tobacco mosaic virus (TMV) into tobacco plants. The gene produces a protein found on the surface of the tobacco mosaic virus and, like a vaccine, turns on the plant’s immune system. The development of transgenic disease resistant plants has also revitalized the once ravaged papaya industry in Hawaii. Researchers at Cornell University characterized the papaya ringspot virus (PRSV). The researchers were able to use recombinant DNA techniques to isolate and clone a gene from the papaya virus in the laboratory that encodes the coat proteins of the virus. This isolated gene was then transferred into cells of the papaya plant. These genetically modified papaya plants were resistant to PRSV and subsequently were responsible for rejuvenating the $45 million dollar papaya industry that had experienced a 50% decline in production due to the destruction of the papaya ringspot virus. This picture is an aerial view of a field trial in Hawaii showing healthy transgenic papaya trees surrounded by papaya trees severely infected by PRSV.

16 Practical Applications in the Field
Insect Control Bacillus thuringiensis (Bt) produces a protein that is toxic to plant pests Transgenic plants contain the gene for the Bt toxin and have a built-in defense against these plant pests An estimated $8 billion is used annually worldwide on chemical pesticides for control of insects that damage plants and affect human health and ecological systems. These pesticides are nonspecific, affecting all insects including beneficial ones. Pesticide poisoning is often reported in farm workers and due to the recalcitrance of pesticides, they are found in our foods and in ecosystems worldwide, far from the site of the pesticide applications. In agriculture, an estimated 30% of this market can be served by insect-protected plants, which produce a protein from a soil bacteria, Bacillus thuringiensis (Bt) whose spores contain a crystalline (Cry) protein. In the insect gut, the Cry protein breaks down to release a toxin that binds to and creates pores in the intestinal lining of the insect resulting in ion imbalance, paralysis of the digestive system, and after a few days, insect death. Crop breeders have not been able to breed high enough levels of caterpillar resistance into corn or cotton by traditional methods. With biotechnology methods, breeders have been able to insert a gene from Bt into cotton and corn to produce a protein that protects the plant against certain economically damaging insects. When caterpillars bore into the stalks and ears of corn or feed on the flowers and buds of cotton plants containing the Bt transgene, it ingests the toxin from the Cry protein and dies within in a few days. The Bacillus thuringiensis bacteria have been used as an insecticide spray by organic and conventional farmers for over 50 years and is harmless to humans and many beneficial insects. However, it can be only marginally effective when applied as a spray because it breaks down quickly and often does not get to where the insects are feeding. In the US, 30% of total agricultural insecticide use is on cotton. The National Center for Food and Agricultural Policy estimates that the use of Bt cotton will result in a reduction of 1,200 metric tons of active ingredient of insecticide. Most of the alternative insecticides have a wider spectrum of activity than Bt and are more harmful to the environment and non-target organisms like beneficial insects. Bt insect resistant crops are currently on the market for corn and cotton and have shown to not only reduce dependency on chemical pesticides, but also improve yield and profits for farmers. Click on the animation to visualize the affects of the Bt toxin on insects. Animation: How Bt Affects Insects

17 Practical Applications in the Field
Weed Management Herbicide resistance Scientists who use genetic engineering techniques for food production have the same goal as traditional breeders: making our food supply safer for consumers and the environment and less expensive to produce. An estimated 5.5 million farmers grow transgenic crops on 130 million acres in about 15 countries, led by the US and Canada. Virtually all of the biotech crops on the market today were developed to reduce crop damage by weeds, diseases, and insects. Anyone who grows a garden or maintains a lawn knows the importance of weed management. Weeds reduce the yield and quality of plants and may also contaminate the product, so most large-scale growers use herbicides. Many herbicides on the market control only certain types of weeds, and are approved for use only on certain crops at specific growth stages. Residues of some herbicides remain in the soil for a year or more, so that farmers must pay close attention to the herbicide history of a field when planning what to plant there. The rationale for herbicide-resistant crops is that the crop can be planted directly into the field, allowed to germinate with any weeds already present, and then treated with an herbicide that kills only weeds. The most common herbicide tolerant crops have been designed to be unaffected by the broad-spectrum herbicide called Roundup® (Monsanto). This herbicide works by blocking an enzyme required for photosynthesis. Roundup is a broad-spectrum herbicide that kills nearly all kinds of plants EXCEPT those that have a transgene that allows them to be tolerant of the herbicide. Through genetic engineering, scientists have created transgenic crops that express an alternate EPSPS gene from bacteria which allows the plant to use an alternative chemical pathway for photosynthesis. Therefore, a farmer can apply a single herbicide treatment to a field of these transgenic crops. The use of herbicide resistant crops reduces soil erosion and compaction because growers drive over the field fewer times. Recent data indicate that their use also results in a substantial reduction of herbicides. Farmers who plant herbicide-resistant crops are generally able to control weeds with chemicals that are milder and more environmentally friendly than typical herbicides. This approach has been especially successful in cotton and soybean crops. Approximately 70% of all soybean and cotton crops are grown with transgenes for weed management. Weed-infested soybean plot Transgenic soybean plot after Roundup treatment

18 Practical Applications in the Field
Safe Storage avidin-blocks the availability of biotin for insects Stronger fibers Increase strength of cotton fiber by 60%

19 Practical Applications in the Field
Enhanced Nutrition Golden rice that is genetically modified to produce large amounts of beta carotene QPM: Maize with increased nutritive value Of all the potential benefits of biotechnology, nothing is more important that the opportunity to save millions of people from the crippling effects of malnutrition. One potential weapon against malnutrition is Golden rice. Golden rice is a transgenic rice that contains beta-carotene and other carotenoids needed for production of Vitamin A, essential in the prevention of blindess. According to recent estimates, 500,000 children in the world will eventually become blind because of vitamin A deficiency. Currently, health workers in developing countries carry doses of vitamin A from village to village in an effort to prevent blindness. Simply adding the nutrient to the food supply is much more efficient and effective. Another example is the production of Quality Protein Maize (QPM) which is a corn product that has 90% the nutritive value of milk. Several hundred million people in underdeveloped countries rely on maize as their principal daily food. Unfortunately maize has one significant flaw; it lacks the full range of amino acids, namely lysine and tryptophan, needed to produce proteins. Therefore diets high in maize produce a condition known as wet malnutrition where a person is receiving sufficient calories, but his or her body malfunctions due to a lack of protein. This makes conventional maize is a poor quality food staple unless consumed as part of a varied diet which is beyond the means of most people in the developing world. Researchers have introduced and adapted QPM maize with increased levels of the two amino acids, lysine and tryptophan, that makes more of maize’s protein useful to humans. According to a recent study, the heights and weights of children whose diets included QPM as their main starchy staple increased more than 20% faster than those of children who ate conventional maize. To date most QPM is produced by traditional crossbreeding methods. However, researchers have recently identified three gene loci implicated in controlling the levels of a protein synthesis factor correlated with lysine levels. Transgenic QPM could be produced at a significantly faster rate and reduced cost. Click on the link to watch a video and discover how QPM is being used to alleviate malnutrition in Haiti. QPM program in Haiti

20 Practical Applications in the Field
Future Transgenic Products RNAi to generate caffeine free coffee beans. Biotech Crop Database Whether you are for or against genetically modified food, one thing is clear: Biotech companies and university laboratories are cooking up new ideas for GM foods all the time. Click on the Biotech Crop Database to see a list of genetically modified plants by species. As previously mentioned, RNA interference (RNAi) is a new technology that is described in detail in the first. RNAi is being explored as a way to create the next wave of genetically modified foods. For example, scientists have identified the genes that lead to the production of caffeine in coffee beans and tea leaves. The demand for decaffeinated coffee is increasing because the stimulatory effects of caffeine can adversely affect sensitive individuals by triggering palpitations, increased blood pressure, and insomnia. Transgenic plants have been constructed that repress expression of genes leading to caffeine production by RNA interference. The caffeine content of these plants is reduced by up to 70% indicating that it could be feasible to produce coffee beans that are intrinsically deficient in caffeine. The U.S. Department of Agriculture lists 7,516 field tests on new GM foods currently underway. It remains unclear which if any of these foods will pass the strict series of tests that stand between the laboratories and our supermarket shelves. Nevertheless, it's fun to sneak a peak into the future. Click on the animation to find out about the next wave of genetically modified foods. Animation: Future GMOs

21 Practical Applications in the Field
The Future: From Pharmaceuticals to Fuel Plant-based petroleum for fuels Biofuel – fuel derived from biomass The broad definition of a biofuel is any fuel derived from a living or recently living organism. Biofuels in the solid form have been in use ever since man discovered fire. Wood was the first form of biofuel that was used even by the ancient people for cooking and heating. The development of the steam engine by George Stephenson in the late 1700's was the technological breakthrough that led to the industrial revolution. For the first time in human history transportation could be provided without the use of domesticated animals. Steam engines were used in steam locomotives, steam tractors and steam ships. Stationary steam engines were rapidly established in all major industries. The major fuel for steam engines was firewood. By the end of the 1800's, the demand for energy was ever increasing and firewood around industrial centers was becoming scarce. This led to a switch to coal as the major source for fuel and energy. The simultaneous development of the internal combustion engine, well drilling technology and the capacity to refine crude oil into gasoline and other liquid fuels in the late 1880’s began the fossil fuel revolution.

22 Types of Biofuels Bioethanol – alcohol made by fermenting plant based sugar compounds Bioethanol is a biofuel made by fermenting the sugar components of plant materials into alcohol. This sort of fermentation is the same metabolic process that is used to convert carbohydrates into alcoholic beverages such as beer and wine. Distillation of the fermentation broth yields pure ethanol (bioethanol) that can be used as a transport biofuel. Bioethanol is most commonly derived from corn crops. Cellulosic ethanol, on the other hand is a biofuel produced from wood, grasses, or the non-edible parts of plants. Switchgrass is the major cellulosic biomass material being studied today, due to its high productivity per acre. In 2008, there was only a small amount of switchgrass dedicated for ethanol production. In order for it to be grown as a large-scale producer of biofuel, it must compete with existing uses of agricultural land, mainly for the production of crop commodities. Currently, corn is easier and less expensive to process into ethanol in comparison to cellulosic ethanol. The ethanol produced can be used as a fuel for vehicles in its pure form, but it is usually used as a gasoline additive to increase octane and improve vehicle emissions. Click on the link to take a video tour of an ethanol plant. Video tour of an Ethanol Plant

23 Types of Biofuels Biodiesel-vegetable oil or animal fat derived diesel fuel Biodiesel is made from vegetable oils, animal fats or recycled greases. Biodiesel is typically made by chemically reacting these lipids with an alcohol in a process known as transesterification. Biodiesel can be used in pure form or may be blended with petroleum diesel at any concentration in most injection pump diesel engines to reduce levels of particulates, carbon monoxide, and hydrocarbons from diesel-powered vehicles. Since the passage of the Energy Policy Act of 2005 biodiesel use has been increasing in the United States. Biodiesel has better lubricating properties and much higher cetane ratings than today's lower sulfur diesel fuels. Approximately 85% of biodiesel production comes from the European Union. A variety of oils can be used to produce biodiesel. Current worldwide production of vegetable oil and animal fat is not sufficient to replace liquid fossil fuel use. Feedstock yield efficiency per unit area affects the feasibility of ramping up production to the huge industrial levels required to power a significant percentage of vehicles. Click on the link to compare the biodiesel output of several different feed stocks. Comparison of biodiesel output for different feed stocks

24 Biofuel Production Generation 1 Biofuels: Food Sources
A 2008 survey titled Ethanol 2020: A Global Market Survey provides an analysis and review of major ethanol markets, leading producers, feed stock price trends, import-export trends, government targets as well as challenges and opportunities worldwide. This survey identifies three transitional generations of biofuels emerging in the next ten years. Biofuels can be classified as first, second, or third, generation based on the feedstock and the method of fuel production . First-generation, or 1G, biofuels are created largely from feedstocks that have traditionally been used as food, such as corn, wheat and vegetable oils. The feedstock yields starch that can be fermented into bioethanol and can then be pressed to yield vegetable oil that can be used in biodiesel. Most of the current infrastructure is built to process biofuels from first generation sources.   This is primarily because the technology already exists to make this fuel in a more efficient manner than the other generations of biofuels. 

25 Biofuel Production Corn Used for Ethanol production 1985-2008
The United States produces and consumes more ethanol fuel than any other country in the world. Despite the increase in production and plants in the US of corn ethanol, many people have concerns that the production of corn-based fuels will lead to food shortages and argue that the process is too inefficient to make a significant dent in our energy needs. According to a study published by Cornell University, 21 pounds of corn are needed to produce just one gallon of ethanol and farm production of that amount of corn requires half a gallon of fossil fuels. Corn would be in competition with other crops for land that could be used for food production and these crops are the staple grains in the diets of many people, especially in less developed countries. Other concerns are the large amount fertilizers and pesticides that would be necessary to irrigate these crops. There is also speculation that dependence on first generation biofuels will lead to an increase in food prices due to the demand for biofuel grain crops.  Much of the controversy about the use of corn as a biofuel has been disputed by biofuel producers. They argue that most corn goes to feeding livestock, not people. Furthermore, corn is not a good source of complete nutrition, being deficient in a couple of amino acids necessary to convert the starch to protein. Also, the spent mash from ethanol fermentations is more nutritious than is the corn feedstock used in fermentation, and is in fact being sold back to the farmers to feed their livestock.  Livestock fed this spent mash often have fewer diseases than livestock fed conventional feed due to the microorganisms in the spent mash which take out excess starch and replace it with a higher protein content and more balanced amino acid ratios.  The microorganisms also put vitamins into the spent mash which has been sold in health food stores as "yeast extract“ for decades.  

26 Biofuel Production Biotechnology Solutions for Generation 1
The soybean The US Department of Energy recently funded a collaboration of researchers from 18 US institutions to sequence the genome of the most important source of protein and oil: the soybean. By analyzing the sequence, scientists have identified more than 46,000 genes in the soybean genome, of which over 1000 are involved in lipid metabolism. These genes and their associated pathways are the building blocks for soybean oil content and represent targets that can be modified to bolster output and lead to the increase of the use of soybean oil for biodiesel production. While biodiesel from soybean oil represents a cleaner, renewable alternative to fossil fuels with desirable properties as a liquid transportation fuel, there simply is not enough oil produced by the plant to be a competitive gasoline on a gallons-of-fuel yield per acre. The availability of the soybean genome may provide some key solutions. With the combination of bionformatics, biochemistry and genetics new species can now be created with an improved level of protein and oil content. Now that the relevant genes have been identified, genetic modifications can be engineered into the genome using recombinant DNA techniques. Experts estimate that this approach can improve soybean oil production up to 40%.

27 Biofuel Production Generation 2 Biofuels: Non-Food Sources
In response to first generation limitations, a second-generation of biofuels was developed from nonfood feedstocks. The goal of second generation biofuel processes is to extend the amount of biofuel that can be produced sustainably by using biomass consisting of the residual non-food parts of current crops, such as stems, leaves and husks that are left behind once the food crop has been harvested. Other feedstocks include switch grass, jatropha and grasses that bear little grain and industry waste such as woodchips, skins and pulp from fruit pressing. Second generation biofuel crops have many advantages over first generation biofuels.  The most obvious is the lack of competition between feeding and fueling ourselves.  Another advantage is that they can be grown on marginal lands that could not support food crops.  This would lessen competition with food crops for land.  They also require less fertilizer and pesticide inputs. Cellulosic ethanol is the most developed second-generation biofuel and is produced from the cellulose or cell wall of plant cells. One of the major challenges for biologists is to find chemical enzymes that can efficiently break down cell walls which contain cellulose and lignin. Despite higher oil yield, feedstock costs remain high. This is partially because of the high cost of storage due to the large amount of space required and the limitation that they must be kept dry. Despite these challenges, second-generation biofuels can widen the feedstock options and produce an increase in available fuel for the market, with the potential for greater greenhouse gas emission savings compared to first-generation biofuels.

28 Biofuel Production Biotechnology Solutions for Generation 2
GM Jatropha doubles oil yield Jatropha, a waxy-leafed plant that produces round fruits and oily seeds, has been touted as a promising future source of biofuel due to its high oil yield and easy conversion to biodiesel. Unfortunately, yields of jatropha are stubbornly limited, making the cultivation of the plant for biofuel less than cost-effective. Many jatropha farms are struggling, as the plant has failed to grow on the meager supply of water that can be made available for a non-food crop. We all know what we can do with a promising crop that turns out to be something of a disappointment…that’s right, genetically modify it! SG Biofuels of San Diego, CA has developed a genetically modified version of the plant that doubles its seed oil yield and is easily grown in places like Guatemala and India whose governments have made substantial investments in growing the plant.

29 Practical Applications in the Field
Metabolic Engineering Manipulation of plant biochemistry to produce nonprotein products or to alter cellular properties

30 Health and Environmental Concerns
Human Health Allergens Antibiotic resistance The introduction of transgenic crops and foods into the existing food production system has generated a number of questions about possible negative consequences. People with concerns about this technology have reacted in many ways, from participating in letter-writing campaigns to demonstrating in the streets to vandalizing institutions where transgenic research is being conducted. These are complex issues and have powerful emotional appeal, particularly as they relate to human health. Opponents of genetically modified crops envision scenarios where novel proteins may trigger dangerous reactions in unsuspecting customers. However, at this time there is no evidence that genetically engineered foods are any more likely to cause allergic reactions than conventional foods. According to a recent report from the American Medical Association, very few proteins have the potential to trigger allergic reactions. In fact, biotechnology may someday help prevent allergy related deaths. Researchers are now working to produce peanuts that lack the proteins that can trigger violent allergic reactions. Allergies are not the only concern. The use of antibiotic resistant markers in the development of transgenic crops has raised concerns about whether transgenic foods will play a part in our loss of ability to treat illnesses with antibiotic drugs. One aspect of this topic is the risk of horizontal gene transfer, that is, transfer of DNA from one organism to another. Transfer of a resistance gene from transgenic food to microorganisms that normally inhabit our stomach and intestines, or to bacteria that we ingest along with food, could help those microorganisms to survive an oral dose of antibiotic medicine. Although horizontal transfer of DNA does occur under natural circumstances and under laboratory conditions, it is probably quite rare in the acidic environment of the human stomach. According to a recent report in the journal Science, there is only a miniscule chance that an antibiotic resistant gene could ever pass from a plant to bacteria.

31 Health and Environmental Concerns
Environmental Impact Effect on non-target species Super weeds In addition to concerns relating to human health a number of opponents of genetically modified food express concerns about damage to the environment. The Monarch butterfly became a symbol for the anti-biotech movement in the 1990’s because of a laboratory experiment with Bt corn pollen that suggested that the pollen produced by the transgenic corn could be deadly to monarch butterflies. The results were expected. Researchers had known for years that, in large doses, the toxin naturally produced by B. thuringiensis could be harmful to butterflies. Still, the report set off a firestorm of controversy. When researchers took their experiments out of the laboratory and into the field, many of their concerns quickly faded. Several studies found that few butterflies in the real world would be exposed to enough pollen to cause any harm. After two years of study, the USDA announced that Bt toxin posed little risk to monarch butterflies in real world situations. Likewise, the EPA made its assessment of the potential risks of Bt corn to non-target species like the Monarch butterfly under field conditions. The EPA also did not find that non-target insects like the Monarch would be exposed to sufficient amounts of Bt protein to cause an unreasonable deleterious effect, nor that Bt crops would threaten the long term survival of a substantial number of individuals in the populations of these species. This is in contrast to chemical pesticides which are known to not only severely affect non-target insects but also severely affect animal populations and human health. Unlike the Bt toxin, these chemicals remain in foods and ecosystems for long periods of time. However, worries about the environment impact of transgenic crops have not disappeared. Just as genes for antibiotic resistance could theoretically spread from plants to bacteria, genes for pest or herbicide resistance could potentially spread to non-targeted plant species creating so called superweeds. Because many crops, including squash, canola, and sunflowers, are close relatives to weeds, crossbreeding occasionally occurs, allowing genes from one plant to mix with the genes of the other. At this time, however, few experts predict any sort of explosion of genetically enhanced weeds. In fact, after intensive use over 25 years, only five weed species have developed resistance. However, in response to these concerns, the EPA has mandated measures to reduce the risk of resistance development. As technology advances, it is important that scientists and regulatory agencies assess the impacts of both new and existing technologies for farmworker and consumer safety and for any environmental effects on plants, animals, and water systems. All available evidence to date shows that foods from biotech crops are as safe as foods from non-biotech crops.

32 Health and Environmental Concerns
Regulations US Department of Agriculture (USDA) Safe to grow Food and Drug Administration (FDA) Safe to consume Environmental Protection Agency (EPA) Poses little or no environmental risk The system for regulating transgenic crops in the US is complex and evolving. Transgenic crops are regulated at every stage in their development, from research planning through field testing, food and safety evaluations, and international marketing. Over the years, federal agencies have developed policies relevant to their particular responsibilities, including agriculture. In 1986, the US Office of Science and Technology Policy published its “Coordinated Framework Notice” which declared the cooperative effort of the USDA, FDA, and EPA in regulating biotech crops. The Animal and Plant Health Inspection Service (APHIS) branch of the USDA must determine whether a transgenic plant is likely to become a pest or have negative agricultural or environmental effects. The agency regulates the import, transportation, and field testing of transgenic seeds and plants through notification and permitting procedures. Researchers must notify APHIS of their intention to transport or field test a transgenic plant. Field testing must be conducted in many locations over several years and requires procedures to minimize the spread of the transgene and keep it out of the food supply. To commercialize a transgenic plant, the researcher petitions APHIS for non-regulated status. This requires extensive data on the introduced gene construct, effects on plant biology, and effects on the ecosystem, including spread of the gene to other crops. After the crop is on the market, APHIS has the authority to halt its sale if there is evidence that the plant is becoming a pest. The FDA has authority under the Federal Food, Drug, and Cosmetics Act to determine the safety of foods or food ingredients. FDA staff consult with the plant developer, review safety and nutritional data, and request additional data considered appropriate for each product. At the end of the consultation and review process, the FDA sends a letter to the developer stating that the agency is satisfied with the data regarding food safety of the product and the food is granted “Generally Recognized as Safe” (GRAS) status. After a product is on the market, the FDA has the authority to remove a product from the market if it is deemed unsafe. The EPA regulates transgenic plants that are engineered for pest resistance. To implement its oversight of transgenic crops, the EPA examines data characterizing the transgene and reviews the environmental effects of the proposed transgene, including effects on non-target species. The EPA then grants an experimental use permit to begin field testing. After years of field trials, the EPA reviews all of the data and determines whether the introduced gene or its product are toxic. The next phase of the process is commercialization, during which the EPA grants deregulated status and the product is approved for market.

33 Plant Biotechnology If you eat the same foods as most Americans, you probably are consuming some foods from biotech crops. Because genetically engineered corn, soybean, and cotton have been so widely planted by farmers, about 60-70% of all processed foods now contain at least one ingredient from a genetically engineered plant. Most of the transgenic crop varieties currently grown by farmers are either herbicide tolerant or insect pest resistant. As part of the ongoing dialogue about biotechnology and other emerging technologies, it is important for the public to stay informed and engaged. The food and agriculture system is complex and dynamic. Because of the complexity of the ethical, scientific, technological and economic questions, no one group of experts can resolve the issues. To find ways of integrating the benefits of agricultural biotechnology into our food systems and our society, without being overcome by any risks associated with these new technologies, requires broad-based discussion among many groups. These discussions should include broad education in the science of biotechnology, the system of food production and distribution, and the political context in which regulatory decisions are made.

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