Plant Biotechnology Chapter 6

Motivation for genetically engineered crops Agriculture is the biggest industrial sector in the world $1.3 trillion of products/year Over past 40 years, world population has doubled while agricultural land area has increased by only 10%

Plant transgenics Transfer of genes to plants directly accelerates selective breeding practices used in the past. Cotton fiber strength increased 1.5% per year through conventional breeding Increased 60% by inserting a single gene into the plant Corn and soybean have been targets of much genetic engineering

Genetic engineering techniques applied to plants Cloning Many types of plants can regenerate from a single cell, similar to a bacterium. The resulting plant is a clone or replica of plant from which original cell was taken. Protoplast fusion Introducing a gene into a “denuded” plant cell and generating a new plant

Protoplast fusion Leaf fragment technique Plant A Plant B Hybrid plant Callus cells Protoplast fusion

Agrobacterium tumefaciens as a vector for transferring foreign genes into plant chromosome This bacterium naturally infects plant cells causing cancerous growths - crown gall disease Infection (vir) genes carried on Ti plasmid Type of bacteria that infects plant cells, integrating into its DNA to ensure its survival causing crown gall disease in many ornamentals Two forms of DNA: 1)bacterial chromosome 2)Ti plasmid --transferred stretch of DNA “T-DNA” (20 kb long) --vir (virulence) genes that direct infection

Infection Process Vir genes copy T-DNA Open channel in bacterial cell membrane for T-DNA to pass through T-DNA enters plant through wound, integrates itself into plant chromosome When infecting, vir genes Copy T-DNA Attach product to copied T-DNA Add proteins along length of T-DNA Open channel in bacterial cell membrane for T-DNA to pass through Then enters plant through wound, integrates itself into plant chromosome **Transformation: inserting foreign DNA into bacteria -- Removed tumor-inducing section of T-DNA, replace with transgene (between T-DNA border regions) http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.htm

Leaf fragment technique used to introduce foreign genes into plant Small discs are cut from plant leaf Discs are cultured to start a new plant Early in the regeneration process, the bacterium Agrobacterium tumefaciens carrying a Ti plasmid is introduced into the culture The plasmid DNA combines with the plant chromosome Discs are treated with hormones to encourage shoot and root development and then the new plant is planted in the soil

Leaf fragment technique Make leaf discs Transfer to shoot stimulating medium Agrobacterium with Ti plasmid with foreign gene Briefly culture discs with genetically modified Agrobacterium Transfer to filter paper over nurse cells Culture 2-3 days

stopped Gene gun Gene guns

Antisense technology Used to produce the Flavr-Savr tomato in 1994. Enzyme polygalacturonase breaks down structural polysaccharide pectin in wall of a plant. This is part of the natural decay process in a plant Monsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme.

Antisense molecules -- Enzyme polyglacturonase (PG) digests pectin in the wall of the plant -- Identified gene that encodes PG, removed gene from plant cells, produced complementary copy -- Agrobacter as vector, transfer new gene into tomato cells -- Gene encodes an mRNA molecule (antisense) that unites with/inactivates normal mRNA (sense molecule) for PG production.

Natural insecticide produced by soil bacterium The bacterium Bacillus thuringiensis produces a crystalline protein that if ingested by insects acts as a toxin and kills the insect Farmers used to spray their fields with spores of the bacteria to inoculate the plant leaf surface before it is attacked by the insect

Insecticide biotechnology The gene coding for the crystalline protein (Cry) was transferred from the bacterium to the plant. How can they do this? Now the plant produces the toxin protein, so don’t need to inoculate with bacterium This strategy kills the pest before it kills the plant

Plant vaccines Plants are susceptible to diseases caused by viruses (tobacco mosaic virus) Virus surface protein induces an immune response in the plant against the virus protein Researchers inserted the virus protein into the plant genome using the Ti plasmid/ Agrobacter vector Now the plant produces a small quantity of the protein which elicits an immune response by the plant the way a vaccine does

Concerns about genetically modified foods Human health Unsuspected allergens What other issues are there? Environment Messing up the gene pool of non-target species in the environment Lateral gene transfer in nature Still poorly understood in nature

National biofuels initiative In 2007, the federal government increased funding for research in biofuels (ethanol and biodiesel) Idea was to produce ethanol from corn convert agricultural waste products (lignocellulose) to ethanol This requires re-engineering natural biochemical pathways in plants or microbes to produce more fuel as an end-product

How is ethanol produced? Most ethanol is produced using a four-step process: The ethanol feedstock (crops or plants) are ground up for easier processing Sugar is dissolved from the ground material, or the starch or cellulose is converted into sugar Microbes feed on the sugar, producing ethanol and carbon dioxide as byproducts The ethanol is purified to achieve the correct concentration.

Metabolic engineering to improve efficiency of biological production of biofuels Natural metabolic pathway 1 ton of Feedstock X product A product B product C 50 gal Ethanol product G 50 gal Butanol New enzyme Genetically engineered metabolic pathway 1 ton of Feedstock X product A product D product E 150 gal Ethanol

Benefits of ethanol Overall, ethanol is considered to be better for the environment than gasoline. Ethanol-fueled vehicles produce lower carbon monoxide and carbon dioxide emissions, and the same or lower levels of hydrocarbon and oxides of nitrogen emissions. Ethanol is widely available and easy to use Ethanol is good for the economy

Drawbacks of ethanol as a biofuel Creating plant-based biofuels requires too much farmland to be practical or sustainable—land that would be better used to grow food. Producing ethanol and other biofuels takes more energy than the fuel can generate.

Summary Variety of techniques are available to introduce genes into plants and have the plants express the gene Such genetic engineering is used to Improve disease resistance Flavor of product Nutrition of product Shelf life of product Any other property of plant that improves its value

Advances in biotechnology of biofuel production will be the next greatest application of our knowledge of biology to address challenges to our society and high-maintenance lifestyles.