C HAPTER 11: B IOTECHNOLOGY IN A GRICULTURE Introduction to Biotechnology, BIOL1414 Austin Community College, Biotechnology Dept

L EARNING O UTCOMES Give specific examples of agricultural and horticultural biotechnology applications, including genetically modified organism (GMO) crops, hydroponics, and plant-made pharmaceuticals Explain how genomic and plasmid DNA can be isolated from cells, including the additional steps required for plant cell DNA isolation Discuss how proteins of interest may be purified from plant samples and how DNA or protein samples may be assayed for their concentration and purity Describe the role that Agrobacterium tumefaciens plays in producing genetically modified plant crops Summarize the methods used to produce transgenic plants, and explain the selection processes for identifying transformed plant cells Describe the role of biotechnologies in food production, food processing, and food security Note about this PowerPoint – There are several links in this PPT that allow you to explore more into different topics. Some of these links are animations, movies, or exercises. Please note, you must be in the slide show to activate the links. You can press F5 any time to active the slide show and “Esc” to exit.

N EW A PPLICATIONS OF B IOTECH IN A GRICULTURE AND H ORTICULTURE Agriculture – the practice of growing and harvesting animal or plant crops for food, fuel, fibers, or other useful products Farming has changed a lot over the last few decades, and smaller farms have been displaced by large agribusinesses. Horticulture – the practice of growing plants for ornamental purposes Horticulturists use the same technologies as other agriculturists, including breeding, asexual plant propagation, and gene manipulation

N EW A PPLICATIONS OF B IOTECH IN A GRICULTURE AND H ORTICULTURE New biotechnology techniques have been applied to improve the quantity and quality of agricultural products Genetic testing Plant tissue culture DNA manipulation and gene transfer Protein manipulations Genetic engineering Plant and animal cloning With DNA fingerprinting, breeders can test parent animals and plants for several beneficial genes and recognize several undesirable genes

B ENEFITS OF S ELECTIVE B REEDING Selective breeding of animals (livestock) and plant crops has been practiced for centuries o Animals: o Improved nutritional value o Fewer feed additives o Increased growth rate o Plants: o Resistant to selected viruses o Higher nutritional content o Less fertilizer or herbicide o Less environmental impact from run-off pollution

M ETHODS U SED IN P LANT T RANSGENESIS o Selective Breeding Farmers cross plants with desirable traits to increase the likelihood of producing offspring with that trait. selective breeding animation

T HE DOWN SIDE TO S ELECTIVE B REEDING More often than not, results in offspring that do not have the desired traits or offspring that produce undesirable traits Crossing over in meiosis! Difficult to predict negative consequences and offspring Inbreeding produces a higher frequency of undesirable traits

A GRICULTURAL B IOTECHNOLOGY According to the USDA, agricultural Biotechnology uses a range of tools, including traditional breeding, altering living organisms or parts of organism to make or modify products, improve plants or animals or develop microorganisms for specified agriculture uses Includes the molecular biology tools of genetic engineering, gene manipulation and protein chemistry Using gene transfer technologies biotechnologist can add or modify specific genes to produce different proteins and offspring with predictable desired traits with less risk of unwanted characteristic

 Selective Breeding vs. Biotechnology Traditional selective breeding vs. biotechnology M ETHODS U SED IN P LANT T RANSGENESIS

Plant Breeding and Testing Overview of crop genetic engineering M ETHODS U SED IN P LANT T RANSGENESIS

G ENE M ODIFICATION – B T C ROPS Using gene modification, agriculturists have developed plants that can be grown with fewer chemicals and pesticides. Bt crops are resistant to several pests since they produce a compound toxic to insects The compound is a protein called Bt delta endotoxin and was originally discovered in a bacterial species called Bacillus thuringiensis (or Bt) By inserting the Bt gene in these plants the plants become protected from insect damage

P RACTICAL A PPLICATIONS IN THE F IELD  Genetic Pesticides 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 Animation: How Bt Affects Insects

E UROPEAN CORN BORER DAMAGE IN B T CORN European corn borer damage and fungal infection in non-Bt (left) and Bt hybrids following manual infestation with second-generation corn borer larvae

 Enhanced Nutrition Golden rice that is genetically modified to produce large amounts of beta carotene QPM: Maize with increased nutritive value QPM program in Haiti P RACTICAL A PPLICATIONS IN THE F IELD

H YDROPONICS Plants are suspended in a hydroponic tank with their roots submerged in water containing specific concentrations of macronutrients and micronutrients necessary for plant growth Also need air! Practiced for several thousands or years, but regaining popularity due to limited space and available soil for plants

P LANT P ROTEINS AS A GRICULTURAL P RODUCTS Many plants contain proteins of agricultural or medicinal value. Plant-based pharmaceutical (PBP) – a human pharmaceutical produced in plants; also called plant-made pharmaceutical (PMP)

E XTRACTING P ROTEIN FROM P LANT C ELLS Plants are sometimes dense. Grating a sample increases extraction yields. Grinding a sample in liquid nitrogen can increase yields. Removing or Weakening Cell Walls Enzymes can be used. Cell walls can be crushed. Cells can be burst open by “freeze fracture.

A DVANCES IN A GRICULTURE THROUGH DNA T ECHNOLOGY As in medicine and industry, agricultural scientists began developing the processes necessary to genetically engineer agricultural products on the molecular level. Plant crop scientists, in particular, have produced several new and unique crops using DNA technology.

I SOLATING P LANT G ENOMIC DNA Isolating Plant genomic DNA: 1. Lysis buffer is used to break open the cells 2. Protein is precipitated and centrifuged out with the cellular debris 3. RNase is added to destroy any RNA 4. Genomic DNA is precipitated out with ethanol or isopropanol (with centrifugation) Extracting DNA is more challenging in plants than with bacteria or animal cells Some protocols add steps for removing pectin and cellulose barrier Some plant molecules can interfere with down stream applications such as PCR (polyphenolics and polysaccharides)

o Transgene – gene for a desirable trait introduced into a novel organism Transgene construct contains a promoter, terminator, and selectable marker gene M ETHODS U SED IN P LANT T RANSGENESIS

U SING A. TUMEFACIENS TO G ENETICALLY E NGINEER P LANTS Transgenic plants – the plants that contain genes from another species; also called genetically engineered or genetically modified plants Transforming Agrobacterium. Before A. tumefaciens can be used to transform a plant, its Ti plasmid must be transformed with the gene(s) of interest.

Ti Plasmid. The Ti plasmid has two selection genes on it, NPT II and beta-D-glucuronidase (GUS), so that when it gets into plant cells, the plasmid transfer can be recognized. Cells receiving this plasmid will be able to survive on kanamycin-containing agar (from NPT II expression). They will also be able to convert a white carbohydrate in the medium to a blue color (due to GUS expression), which makes the entire colony blue, allowing the researcher to ascertain successful DNA transfer.

During A. tumerfaciens-mediated plant transformation, the bacterium attaches to the outside of the cell and inserts part of or the entire, Ti plasmid into the cell

o Transformation by Gene Gun Method o Blast tiny metal beads coated with DNA into an embryonic plant cell Gene Gun animation M ETHODS U SED IN P LANT T RANSGENESIS

A RABIDOPSIS THALIANA, A M ODEL O RGANISM FOR P LANT G ENETIC E NGINEERING Arabidopsis thaliana has been the target of plant genetic engineering studies. Biology & chemistry of this plant is well understood – DNA sequenced These plants are easy to transform Explore! Click here! Explore! Click here! Learn more about Arabidopsis! Learn more about Arabidopsis!

B IOTECHNOLOGY IN F OOD P RODUCTION AND P ROCESSING Advances in agricultural biotechnology have impacted food production and processing. The results are improved food supplies, increased nutritional content of some foods, and increased food safety and security.

D ETECTION OF P ESTICIDES IN F OOD Testing for microbial and pesticide contamination is standard in all plant and animal food products Testing includes the use of cell culture, DNA testing (PCR, sequencing, microarrays), and ELISA Also testing for naturally occurring and environmental toxins in food – such as aflatoxins (mold that grows on grain) Allergen testing (gluten) Species contamination in food processing

 Human Health Allergens Antibiotic resistance H EALTH AND E NVIRONMENTAL C ONCERNS

Environmental Impact Effect on non-target species Super weeds H EALTH AND E NVIRONMENTAL C ONCERNS

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 H EALTH AND E NVIRONMENTAL C ONCERNS

P LANT B IOTECHNOLOGY

 The Future: From Pharmaceuticals to Fuel Plant-based petroleum for fuels Biofuel – fuel derived from biomass T HE F UTURE OF A GRICULTURE

T YPES OF B IOFUELS  Bioethanol – alcohol made by fermenting plant based sugar compounds Video tour of an Ethanol Plant

T YPES OF B IOFUELS  Biodiesel - vegetable oil or animal fat derived diesel fuel Comparison of biodiesel output for different feed stocks

Q UESTIONS AND C OMMENTS ?

R EVIEW Q UESTIONS Your Turn! Put your name at the top of a sheet of paper, answer these questions and hand in: 1. Name two advantages of growing plants hydroponically. 2. What are the steps to isolate plant genomic DNA from plant tissue? What are some issues that may come up? 3. Why is the bacterium, A. tumefaciens, of interest to biotechnologists? 4. Describe how the Ti plasmid is used by biotechnologists to create transgenic plants. 5. What does GMO stand for? Explain how Monsanto Roundup Ready ® soybeans are an example of a GMO. 6. Why are so many plant genetic-engineering experiments conducted with Arabidopsis, even though it has little, if any, economic value? 7. List some food-borne pathogens that may be detected by biotechnologies. What are some other food-borne molecules that biotechnologists test for in food? 8. Summarize the roles of the USDA, FDA, and EPA in biotechnology food products and processing. 9. Why all the fuss about genetically modified food? What are some ethical concerns?