2Motivation for genetically engineered crops Agriculture is the biggest industrial sector in the world$1.3 trillion of products/yearOver past 40 years, world population has doubled while agricultural land area has increased by only 10%
3Plant transgenicsTransfer of genes to plants directly accelerates selective breeding practices used in the past.Cotton fiber strengthincreased 1.5% per year through conventional breedingIncreased 60% by inserting a single gene into the plantCorn and soybean have been targets of much genetic engineering
4Genetic engineering techniques applied to plants CloningMany 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 fusionIntroducing a gene into a “denuded” plant cell and generating a new plant
5Protoplast fusion Leaf fragment technique Plant A Plant B Hybrid plant Callus cellsProtoplast fusion
6Agrobacterium tumefaciens as a vector for transferring foreign genes into plant chromosome This bacterium naturally infects plant cells causing cancerous growths - crown gall diseaseInfection (vir) genes carried on Ti plasmidType of bacteria that infects plant cells, integrating into its DNA to ensure its survival causing crown gall disease in many ornamentalsTwo forms of DNA:1)bacterial chromosome2)Ti plasmid--transferred stretch of DNA “T-DNA” (20 kb long)--vir (virulence) genes that direct infection
7Infection Process Vir genes copy T-DNA Open channel in bacterial cell membrane for T-DNA to pass throughT-DNA enters plant through wound, integrates itself into plant chromosomeWhen infecting, vir genesCopy T-DNAAttach product to copied T-DNAAdd proteins along length of T-DNAOpen channel in bacterial cell membrane for T-DNA to pass throughThen 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)
8Leaf fragment technique used to introduce foreign genes into plant Small discs are cut from plant leafDiscs are cultured to start a new plantEarly in the regeneration process, the bacterium Agrobacterium tumefaciens carrying a Ti plasmid is introduced into the cultureThe plasmid DNA combines with the plant chromosomeDiscs are treated with hormones to encourage shoot and root development and then the new plant is planted in the soil
9Leaf fragment technique Make leaf discsTransfer to shoot stimulating mediumAgrobacteriumwith Ti plasmidwith foreign geneBriefly culturediscs withgeneticallymodifiedAgrobacteriumTransfer to filterpaper over nursecellsCulture 2-3 days
11Antisense 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 plantMonsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme.
12Antisense 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.
13Natural 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 insectFarmers used to spray their fields with spores of the bacteria to inoculate the plant leaf surface before it is attacked by the insect
14Insecticide 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 bacteriumThis strategy kills the pest before it kills the plant
15Plant vaccinesPlants are susceptible to diseases caused by viruses (tobacco mosaic virus)Virus surface protein induces an immune response in the plant against the virus proteinResearchers inserted the virus protein into the plant genome using the Ti plasmid/ Agrobacter vectorNow the plant produces a small quantity of the protein which elicits an immune response by the plant the way a vaccine does
16Concerns about genetically modified foods Human healthUnsuspected allergensWhat other issues are there?EnvironmentMessing up the gene pool of non-target species in the environmentLateral gene transfer in natureStill poorly understood in nature
17National biofuels initiative In 2007, the federal government increased funding for research in biofuels (ethanol and biodiesel)Idea was toproduce ethanol from cornconvert agricultural waste products (lignocellulose) to ethanolThis requires re-engineering natural biochemical pathways in plants or microbes to produce more fuel as an end-product
18How is ethanol produced? Most ethanol is produced using a four-step process:The ethanol feedstock (crops or plants) are ground up for easier processingSugar is dissolved from the ground material, or the starch or cellulose is converted into sugarMicrobes feed on the sugar, producing ethanol and carbon dioxide as byproductsThe ethanol is purified to achieve the correct concentration.
19Metabolic engineering to improve efficiency of biological production of biofuels Natural metabolic pathway1 ton of Feedstock X product A product B product C gal Ethanolproduct G gal ButanolNew enzymeGenetically engineered metabolic pathway1 ton of Feedstock X product A product D product E gal Ethanol
20Benefits of ethanolOverall, 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 useEthanol is good for the economy
21Drawbacks 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.
22SummaryVariety of techniques are available to introduce genes into plants and have the plants express the geneSuch genetic engineering is used toImprove disease resistanceFlavor of productNutrition of productShelf life of productAny other property of plant that improves its value
23Advances 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.