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Plant Genetic Engineering Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another Why do scientists want to change gene instructions? to produce needed chemicals to carry out useful processes to give an organism desired characteristics THE SCIENCE OF GENETIC ENGINEERING Isolate desired gene for a new trait from any organism Gene inserted into plasmid. Isolate plasmid DNA Introduce modified plasmid into bacterium for replication. Grow in culture to replicate. DNA of interest is EVERYWHERE Plant Genetic Engineering Product Concepts and Technical Feasibility Building the Transformant Plant Transformation Selection Plant Breeding Seed Production and Marketing Detection of GMO Crops in the Commodity Chain Plant transformation getting DNA into a cell getting it stably integrated getting a plant back from the cell Requirement 1. a suitable transformation method 2. a means of screening for transformants 3. an efficient regeneration system 4. genes/constructs Vectors reporter genes ‘genes of interest’ Promoter/terminator selectable marker genes Plant Transformation DNA Delivery to Target Cells Selection and Regeneration Event Selection Transformation methods DNA must be introduced into plant cells Technique Methods Indirect 1. Agrobacterium mediated gene transfer 2. Viral vector Direct 1. Lipid-mediated method, 2. Calcium-phosphate mediated, 3. Dextran-mediated, 4. Electroporation, 5. Biolistics, 6. Polybrene, 7. Laser transfection, 8. Gene transfection enhanced by elevated temperature. Method depends on plant type, cost, application Agrobacterium tumefaciens plasmid • A large (~250kbp) plasmid called Tumor-inducing (Ti) plasmid) • Plasmid contains genes responsible for the crown gall disease • Portion of the Ti plasmid is transferred between bacterial cells and plant cells T-DNA (Transfer DNA) • There are 2 x 23bp direct repeat border Agrobacterium tumefaciens Ti plasmid T-DNA 23 kb tra vir genes for transfer to the plant bacterial conjugation pTi ~200 kb opine catabolism Genetic engineering of the Ti plasmid Some considerations: Ti plasmid is 200 kb - too big for easy cloning need to remove tumor-inducing genes from T-DNA T-DNA Disarm Ti plasmid YFG Disarmed Ti plasmid Cloning vector VIR genes Agrobacterium Remember: – Only VIR genes are required for T-DNA transfer – Only LB and RB are required for T-DNA insertion into genome The binary Ti plasmid system Binary vector system Binary vector system Agrobacterium-mediated transformation Transformation by the help of agrobacterium Agrobacterium is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants Agrobacterium tumefaciens Agrobacterium Plant cell Genomic DNA Genomic DNA Ti plasmid (carries the gene of interest) Restriction enzyme A Restriction enzyme A + Empty plasmid Gene of interest Ti plasmid with the gene of interest Agrobacterium tumefaciens Ti plasmid with the new gene cell’s DNA + Agrobacterium Transformation Plant cell The new gene Transgenic plant Cell division Biolistics ‘gene gun’ DNA is coated onto gold (or tungsten) particles (inert) Gold is propelled by helium into plant cells if DNA goes into the nucleus it can be integrated into the plant chromosomes Cells can be regenerated to whole plants “Gene Gun” Technique DNA coated golden particles Gene gun Cell’s DNA Plant cell A plant cell with the new gene Transgenic plant Cell division Tearless Onion Colorful Cauliflowers Purple tomatoes Blue Roses Herbicide Resistance A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds. Herbicides such as RoundupTM and Liberty LinkTM are able to kill a wide range of weeds and have the advantage of breaking down easily. Development of herbicide resistant crops allows the elimination of surrounding weeds without harm to the crops. ROUNDUP (Glyphosate) TOLERANCE ( HERBICIDE TOLERANCE IN CROPS) Glucose ROUNDUP (Glyphosate ) 3 phosphoglycerate Tryptophan Glycolysis EPSP SYNTHASE Phosphoenol pyruvate EPSP SYNTHASE Tyrosine Phenylalaline ROUNDUP (Glyphosate ) In transgenic plant, herbicide can not bind the mutant of EPSP synthase (Example: RR-Cotton, RR-Soybean) Insect Resistance Various insect resistant crops have been produced. Most of these make use of the Cry gene in the bacteria Bacillus thuringiensis (Bt); this gene directs the production of a protein that causes paralysis and death to many insects. Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis GENE FOR Bt TOXIN WAS TRANSFERRED TO OBTAIN BT TRANSGENIC PLANTS PLANT SYNTHESIZES INACTIVE PROTOXIN INSECT FEEDS ON TRANSGENIC PLANT PROTEINASE DIGESTION IN INSECT GUT MAKES THE ACTIVE TOXIN Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death Virus Resistant Crops Papaya infected with the papaya ringspot virus Virus resistance gene introduced The Freedom II squash has a modified coat protein that confer resistance to zucchini yellows mosaic virus and watermelon mosaic virus II. Scientists are now trying to develop crops with as many as five virus resistance genes Delayed Fruit Ripening Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. It is produced by blocking the polygalacturonase (PG) gene, which is involved in spoilage. PG is an enzyme that breaks down pectin, which is found in plant cell walls. Plants were transformed with the anti-sense PG gene, which is mRNA that base pair with mRNA that the plant produces, essentially blocking the gene from translation. Flav’r Sav’r tomato “Rot-Resistant Tomato” Anti-sense gene complementary to polygalacturonase (PG) PG = pectinase accelerates plant decay/rotting Golden Rice Normal rice Transgenic technology produced a type of rice that accumulates beta-carotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. “Normal” rice “Golden” rice Gernayl Gernayl diphosphate (GGPP) Phytoene synthase Phytoene Phytoene desaturase Lycopene Lycopene cyclase Beta carotene Complete biochemical pathway in the rice for production of betacarotene, a precursor for vitamin A. Edible Vaccines Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects. Approved Transgenic plants Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco Carnations Potato Flax Papaya Chicory Rice Melon