David Hildebrand Plant Physiology, Biochemistry & Molecular Biology Crop Science Nutritional Sciences Agronomy Department University of Kentucky

Development of Crop Plants - History Domestication of crops We have been modifying our crops for 10,000 years through Selection. All crops we grow today have undergone extensive genetic change from their wild ancestors. Crops, strains and genes have moved around the globe. Domestication of corn creating a new species in the process – Zea mays Domestication of corn creating a new species in the process – Zea mays Thanks to C.S. Prakash for some of the pictures on this and the next couple of slides.

Improving Our Crop Plants Developing Modern Varieties of Crops –Hybridization Crosses with Wild Relatives Hybrids –Mutation Irradiation Chemicals –Cell Culture Embryo Rescue Somaclonal variation

Chihuahuas and Great Danes… Products of Modern Breeding

Modern Genetic Modification Inserting one or few genes to achieve desired traits. GM Crops –Relatively Specific –Changes are Subtle –Allows Flexibility –Expeditious

Are “genetically modified” GM crops and foods new? Not in the general sense. We have been genetically modifying plants and animals for a very long time or since the dawn of civilization! Most all our crop plants are extensively genetically modified compared to their wild relatives. We have been genetically modifying plants and animals for a very long time or since the dawn of civilization! Most all our crop plants are extensively genetically modified compared to their wild relatives..

Are “genetically modified” GM crops and foods new? Members of the same genus or species of corn (Zea mays) or soybeans (Glycine max) do not even exist in the wild. Modern bread wheat is a mixture of recombined genes from 3 different wild species and the strawberry you buy in stores is a mixture of genes from 2 species. All of these major crop species are a result of human directed genetic modification of the original wild organisms.

The domestication of animals has also been accompanied by extensive human-directed genetic modification. This is exemplified with the domestic dog, Canis familiaris. Modern genetics including genomics indicates that all domestic dogs are originally derived from the wolf, Canis lupus. Thus the Siberian Husky and Mexican Chihuahua are derived from the same original specie with the only difference being the extent of the genetic modification!

Traditional Breeding Crossing individuals with desirable characteristics (e.g. yield) and selecting among the progeny. Genes recombine in a random fashion and finding superior progeny has been as much art as science. Needs excellent management since it’s a numbers game and genotype often masked by environmental influences. Traditional Breeding Crossing individuals with desirable characteristics (e.g. yield) and selecting among the progeny. Genes recombine in a random fashion and finding superior progeny has been as much art as science. Needs excellent management since it’s a numbers game and genotype often masked by environmental influences. For specific characteristics such as fatty acid % of oil, naturally occurring mutants screened for and if not found induced by chemical mutagenesis or irradiation.

Various new breeding tools have been developed to assist breeding in the last 50 years. Mutagenesis is one such technique that has been utilized in the breeding of many of the food plants developed in the last decade. Crops produced this way are considered GMOs. Modern biotechnology provides a new tool for breeding plants and animals with much greater precision.

Various new breeding tools have been developed to assist breeding in the last 50 years. Mutagenesis is one such technique that has been utilized in the breeding of many of the food plants currently utilized. Crops produced this way are considered GMOs. Modern biotechnology provides a new tool for breeding plants and animals with much greater precision.

Various new breeding tools have been developed to assist breeding in the last 50 years. Mutagenesis is one such technique that has been utilized in the breeding of many of the food plants developed in the last decade. Modern biotechnology provides a new tool for breeding plants and animals with much greater precision.

How do GE crops differ from conventionally derived varieties or hybrids? How do GE crops differ from conventionally derived varieties or hybrids?

Conventional: Wide Hybridization introduces 20,000 to 100,000 potentially negative genes in order to obtain one desirable disease resistance gene. Induced mutagenesis has been used for decades to create genetic variants.

Genetic Engineering: Introduce one (or a few) foreign “good” genes into the best accepted cultivar background.

Genetic Engineering: Genetic Engineering: Introduce one (or a few) foreign “good” genes into the best accepted cultivar background. The main thing that is new with genetic engineering is that species barriers can now readily be bridged.

Genetic Engineering: Genetic Engineering: Introduce one (or a few) foreign “good” genes into the best accepted cultivar background. The main thing that is new with genetic engineering is that species barriers can now readily be bridged. This opens new opportunities and depending on how it is used requires new safeguards.

The Underlying Science of Genetic Engineering Restriction Enzymes & Recombinant DNA Restriction Enzymes & Recombinant DNA Gene Discovery, Isolation and Cloning Gene Discovery, Isolation and Cloning Move Foreign Gene or Transgene from Any Organism to Any Other Organism

KEY ELEMENT OF BIOTECHNOLOGY/ GENETIC ENGINEERING Can Use Recombinant DNA Methods To Move A Gene From Any Organism To Any Other Organism

What is Recombinant DNA?

DNA_Extraction.swf

Gene Cloning

Agrobacterium tumefaciens A. tumefaciens Chromosome T-DNA Ti plasmid T-DNA Chromosomal DNA Crown Gall Plant crown gall tissue naturally transgenic Plant chromosomes containing T-DNA

How Do You Introduce a Foreign Gene into a Recipient Organism ? Overview

Methods Used to Date for Plant Transformation Agrobacterium tumefaciens and rhizogenes Gene Gun Microprojectiles (PDS) Electroporation of protoplasts Microinjection Pollen Tube Pathway Silica Carbide fibers Microlaser Viruses [some native genes replaced]

plasmid Desired DNA Recombinant plasmid Agrobacterium tumefaciens containing Wt Ti plasmid A. tumefaciens containing engineered Ti plasmid Plant cell inoculated with A. tumefaciens Plant cell containing Desired DNA Cultured plant cells Regenerant Adult plant expressing desired trait (DNA) Inserting foreign genes into plant cells. A plasmid containing DNA is cut with a restriction enzyme & DNA of desired gene (red) inserted. Desired gene then inserted into Ti (tumor-inducing) plasmid naturally found in A. tumefaciens. Plant cell inoculated with A. tumefaciens containing engineered Ti plasmid + the desired DNA transfers desired DNA + t-DNA into plant chromosomes. Plantlets with desired trait then regenerated.

The Gene Gun PDS1000 Microparticle Delivery System Helium chamber Rupture disk Macrocarrier DNA coated gold particle Stopping screen Focusing device Target tissue Gene gun From Collins lab

Biolistic Transformation Before impact DNA coated gold particle After impact During impact ? Plant Cell Wall

How do Genes do Their Job?

GENE EXPRESSION DNA Transcription mRNA Translation Protein

Product AProduct B Structural Gene Enzyme (Protein)

Roundup Ready Crops Monsanto web page:

Roundup (Glyphosate) is a very strong inhibitor of EPSP 1 Synthase. Glyphosate Shikimate 3-phosphate EPSP PEP 1 EPSP = 5-ENOLPYRUVYLSHIKIMATE 3-PHOSPHATE

Sulfonylurea Tolerant Soybeans - STS - These cultivars are resistant to certain sulfonyl ureas (SUs), a family of herbicides which are most effective against broadleaf weeds. STS herbicides used over soybean varieties that have the STS gene offer the benefit of using broad spectrum sulfonylurea broadleaf herbicides without injuring young soybean plants. Labeled sulfonylurea herbicides include Synchrony STS¹, Reliance STS¹, Classic¹, Pinnacle¹, Canopy¹, Canopy XL¹ and Concert¹. The STS gene was incorporated into soybean germplasm using conventional breeding methods. The STS gene was incorporated into soybean germplasm using conventional breeding methods. SU tolerant gene induced by EMS mutagenesis (US patent # 5,084,082). Bx breeding

SUs inhibit the essential plant enzyme acetolactate synthase or ALS. Animals do not have ALS. Mechanism of action of SUs  -keto butyrate pyruvate acetolactate ALS isoleucine CO 2

Bt crops

Grower Advantages GM crops on the market today do not directly increase & can decrease costs to farmers. According to Kline & Company corn pesticides can be reduced as much as 70% when growers use GM corn. This can save growers > $200 million/yr in pesticide costs. Also EPA EPA: "The average net benefit of $3.31 per acre on million acres of Bt corn planted in 1999 (a year of low corn borer pressure) leads to the national estimate of $65.4 million." The net benefit to sweet corn producers was calculated at $5.38 per acre in 1999.

Plant Incorporated Pesticides (PIP’s) Scientists in industry and academia have been developing corn and soybean lines that produce their own natural insecticides to resist insect losses. Since 1996, six types of Bt corn have been commercialized to protect against attack by European and southwestern corn borer. Other types of Bt corn are being evaluated that prevent losses to corn rootworm, black cutworm, and fall armyworm.

Transgenic virus resistant papaya: Main hope for controlling papaya ringspot virus in Hawaii

From a DuPont Webpage

Zygotic embryo Somatic Embryo (SE) Blue SEs expressing introduced GUS gene

Soybean Engineering H2OH2OH2OH2O N2N2N2N2 CO 2 h CH 3 (CH 2 ) n COOR

A much higher linolenic acid (18:3) content would enhance the drying characteristics of soybean oil such as in printing inks. New molecular techniques have provided the most efficient method to accomplish this.

Soybean Seed 40% Protein Animal Feed, etc. 20% Oil Edible Oil Products, Limited Industrial Use

Stokesia laevis Vernonia galamensis Engineering Soybeans for Epoxy Fatty Acid Accumulation Take genes from wild plant(s) with high epoxy fatty acid accumulation in seed oil and put in commercial oilseed such as soybeans.

MaizeCantaloupeWheatCarnationPlum TreesChrysanthemum PotatoFlaxMellonEggplantGrapeCranberry TomatoChicoryPetuniaStrawberrySpruceOilseed Rape SoybeanRiceCucumberBarleyCabbageSweet Potato CottonPoplarApplePeanutTrefoilFodder Rape MustardSquashCauliflowerEucalyptusKiwi TobaccoSunflowerSugarcaneCarrotRose Sugar BeetRapeseedSweetgumServiceberryPapaya AlfalfaBirchWalnutPepperAsparagus CanolaLettuceRaspberryFescueWatermelon Some GE Plants in Field Trials From Collins lab

What’s Being Tested (US field releases, cumulative since 1987 SOURCE: US DEPARTMENT OF AGRICULTURE From Collins lab

Our new century is predicted to be the “Century of Biology” as the last century was the “Century of Chemistry & Physics”. Until ~ 100 years ago most building materials, everyday materials in the home, fuels and clothing came from our farms and forests. In the last century we have seen a major shift to such materials coming from petrochemicals rather than plants and animals. Petrochemicals come from what were once plants and animals but are not a readily renewable resource. Now with modern technology, particularly biotechnology, we can derive more and more of the materials humankind needs in the future from plants and animals produced by our farmers. Thus in the future, farmers should play an increasingly vital role in our economic prosperity in addition to their essential role in food production.

From our state government leaders to our state universities, Kentucky is working to be a significant player in this “new economy”. Biotechnology has already spawned a number of new companies in KY (see URL below for a list):

What has happened to the farmer’s share of the food dollar? 1999: 20.6%, 2000: 19.9%, 2001: 21%, 2002e: 20% Adding value to crops can reduce or reverse this trend

What is the Future of Biotechnology? Value Added Extended Uses Genomics Bioinformatics Diagnostics Sustainability/Environment Acceptance

Biotechnology Industrial Areas Agriculture Pharmaceuticals Health Care Food Energy Environment

Harvesting Tobacco Biomass for Pharmaceutical Production See: The Kentucky Tobacco Research and Development Center See: The Kentucky Tobacco Research and Development Center

Tobacco Plants Produce Cancer Vaccine Non-Hodgkin’s B-cell lymphoma is the most prevalent form of lymphoma, a malignancy which affects the lymph system and is the sixth most common cause of cancer- related deaths in the US. Researchers have now established that a therapeutic vaccine protein produced by modified tobacco plants is effective in preventing the growth of non-Hodgkin’s lymphoma cells - at least in laboratory mice. Eighty percent of the mice receiving the vaccine survived the lymphoma, while untreated mice died within three weeks after contracting the disease. As reported in the Proceedings of the National Academy of Sciences, the work was a collaborative effort between scientists at Stanford and Biosource Technologies, based in Vacaville, Calif. The researchers removed malignant B cells from laboratory mice, and then isolated the gene coding for the surface markers specific to those cells. They inserted the gene into a tobacco mosaic virus, and exposed tobacco plants to the virus. As the virus spread through the tobacco leaves, the plants produced the desired B-cell protein, which was extracted and injected into mice which had received lethal dosages of tumor cells. Biosource hopes to begin clinical trials of the vaccine in humans within a year, if the vaccine passes necessary safety tests. To speed development, the company launched a new division called Biosource Antigenics to focus initially on the vaccine. Robert Erwin, CEO of Biosource, says that the plant-based expression system has proven itself to be faster than other technologies for producing effective vaccines. He says the Antigenics division will use high- throughput robotics to provide human and animal health vaccines companies with a means to rapidly prototype lead vaccine designs. Source: AgBiotech Reporter - February 1999

Fields of... brown. Improving the drought tolerance of corn could make dried-out crops like this one a thing of the past. CREDIT: RICHARD HAMILTON SMITH/CORBISS New Ways to Protect Drought-Stricken Plants Anne Simon Moffat. Science 296: , May With drought an ever-present threat, researchers are identifying genes that can help plants tolerate arid conditions in hopes of using them to produce hardier crops. Tomato plants carrying a foreign gene that protects their cells from salt-induced dehydration thrive in a 200-mM salt solution, whereas unaltered plants wither. CREDITS: E. BLUMWALD/UNIVERSITY OF CALIFORNIA, DAVIS

Studies have shown that ethanol: Reduces tailpipe carbon monoxide emissions by as much as 30% Reduces exhaust VOC (volatile organic compounds) emissions by 12% Reduces toxic emissions by 30% Reduces particulate matter (PM) emissions by more than 25% (Particulate matter has been found to penetrate deeply into human lungs.) Corn can be genetically engineered to improve the efficiency of ethanol production.

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From Collins lab

fbi.gov/programs/lab/labhome.htm From Collins lab

Researchers test a pilot-scale system for using vegetation in soil remediation. Welcome Page The Hazardous Substance Research Center/South and Southwest is a competitively awarded, peer-reviewed research consortium led by Louisiana State University with the cooperation of the Louisiana State University Georgia Institute of TechnologyGeorgia Institute of Technology and Rice University toRice University address critical hazardous substance problems, especially as they relate to contaminated sediments. The South & Southwest Center was established in October 1991 under Section 311(d) of CERCLA to conduct research and technology transfer designed to promote risk-based management and control of hazardous substances for the nation and regions 4 and 6 the Environmental Protection Agency (EPA). The contaminated sediments and dredged materials of concern contain organics, metals, and conventional pollutants. These environmental contaminants are either suspended in the water column or stored on the bottom of rivers, bayous, lakes, harbors, estuaries, freshwater wetlands, and in ocean waters of the continental shelf. Research theme areas include: From Collins lab

Issue: Impact on non-target organisms There has been much media attention given to the potential impact of GM crops on non- target organisms. An ideal pest control tactic would be one that controls the pest, but does not harm other non-target organisms in any way. Non-target organisms include all organisms except for the pest to be controlled. Examples of non-target organisms would be mammals, fish, birds, reptiles and other insects. Lady Beetle Lacewing Monarch From Ric Bessin

One group of non-target organisms that need to be encouraged is the natural enemies of our crop pests. Natural enemies are composed of a wide array of parasitic and predatory insects and other arthropods. Control of crop pests by natural enemies is referred to as biological control. Plant pesticides produced by genetically modified crops are more selective than pesticide sprays, thus not harming natural pest predators. In this way, genetically modified crops that produce their own plant pesticides are more compatible with biological control. Issue: GM Crops Compliment Biological Control From Ric Bessin

Bt crops -- EPA Assessments "Bt Biopesticides Registration Action Document," the EPA's review contains 283 pages and can be viewed in its entirety at: “the Bt protein behaves as would be expected of a dietary protein, is not structurally related to any known food allergen or protein toxin, and does not display any oral toxicity when administered at high doses." "(Data) provide a weight of evidence assessment indicating no unreasonable adverse effects of Bt Cry proteins expressed in plants to non-target wildlife or beneficial invertebrates,whether they are earthworms, springtails, parasites, predators, pollinators or soil microbial and invertebrate flora." “...widespread cultivation of Bt crops may have huge benefits for monarch butterfly survival." From Ric Bessin

Concerns Concerns Social and Economic ConsiderationsSocial and Economic Considerations Ethical ConcernsEthical Concerns Environmental ImpactEnvironmental Impact Also since biotechnology impacts our food supply a fourth concern has been voiced Social and Economic ConsiderationsSocial and Economic Considerations Ethical ConcernsEthical Concerns Environmental ImpactEnvironmental Impact Also since biotechnology impacts our food supply a fourth concern has been voiced Like all new technologies there are concerns with the commercial application of biotechnology. These concerns can be divided into three areas [although in many cases the concern crosses more than one area]:

Food SafetyFood Safety Food SafetyFood Safety Extensive studies indicate that biotech-derived foods are as safe or safer than conventional foods. Also although regulatory oversight is evolving, it is more extensive for biotech foods than any others. In balance biotech crops are considered to be beneficial to the environment. We should work to ensure that biotech has maximum benefit on sustainable agricultural development.

U.S. Labeling Policy for Food Biotechnology FDA safety standards are consistent for all foods. –Must label the presence of common allergens not normally found in certain foods –Must demonstrate scientifically that allergens are not present in modified food. –If nutritional content or composition has been changed, product must be labeled accordingly.

Social and Economic Considerations Among the major social and economic concerns that have been raised are: Social and Economic Considerations Among the major social and economic concerns that have been raised are: Is biotechnology scale-neutral or does it mainly benefit the larger and wealthier farmers? Can it provide a market advantage for large multinational corporations? Do just a few corporations control much of the intellectual property associated with agricultural biotechnology? Might biotechnology increase farmer’s reliance on seed companies and cause them to be less likely to use seeds of varieties they have traditionally used? Can it exacerbate over-production making farming even less profitable than it already is? Is it a “tool of capitalism”?

Ethical Concerns Some of the major ethical concerns that have been voiced are: Ethical Concerns Some of the major ethical concerns that have been voiced are: In many ways the social and economic concerns are also ethical considerations. Also in some ways adoption of biotechnology represents a break from tradition. ]Should we mess with Mother Nature [or are we]? Are genetically engineered plants and animals “natural”? Should anyone be allowed to own or patent a living organism [or does this actually occur]?

About Us Bioethics Health and Food Safety Farm Impact Research and Science News Environmental Topics FAQs Glossary Just for Kids / Teachers BREI is a multi-disciplinary team of research, extension and teaching professionals from the College of Agriculture. Comments and suggestions are welcome, send to BREI. Copyright © 2000, University of Kentucky, College of AgricultureBREI Sharing information about agricultural biotechnology through: Publications Visual Media Education Curriculum Web-based Information In-service Training

Charles Darwin... “ I t is not the strongest species that survive, nor the most intelligent, but the ones most responsive to change” “I’m all for progress; it’s change I don’t like” - Mark Twain Slide from C.S. Prakash

Thanks