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

Isolate desired gene for a new trait from any organism Isolate plasmid DNA Gene inserted into plasmid. Introduce modified plasmid into bacterium for replication. Grow in culture to replicate. THE SCIENCE OF GENETIC ENGINEERING

DNA of interest is EVERYWHERE

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 Genetic Engineering

getting DNA into a cell getting it stably integrated getting a plant back from the cell Plant transformation Requirement 1.a suitable transformation method 2.a means of screening for transformants 3.an efficient regeneration system 4.genes/constructs VectorsPromoter/terminator reporter genesselectable marker genes ‘genes of interest’

DNA Delivery to Target Cells Selection and Regeneration Event Selection Plant Transformation

Transformation methods DNA must be introduced into plant cells Method depends on plant type, cost, application TechniqueMethods Indirect1.Agrobacterium mediated gene transfer 2.Viral vector Direct1.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.

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 Ti plasmid Agrobacterium Genomic DNA Plant cell the gene of interest Genomic DNA (carries the gene of interest) + Ti plasmid with the gene of interest Gene of interest Empty plasmid A Restriction enzyme A

Agrobacterium Ti plasmid with the new gene Plant cell cell’s DNA Transgenic plant Cell division The new gene + Transformation Agrobacterium tumefaciens

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 division A plant cell with the new gene Transgenic plant Plant cell Cell’s DNA

Herbicide Resistance A problem in agriculture is the reduced growth of crops imposed by the presence of unwanted weeds. Herbicides such as Roundup TM and Liberty Link TM 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.

EPSP SYNTHASE Phosphoenol pyruvate Tryptophan Tyrosine Phenylalaline ROUNDUP (Glyphosate ) Glucose 3 phosphoglycerate Glycolysis EPSP SYNTHASE ROUNDUP (Glyphosate ) In transgenic plant, herbicide can not bind the mutant of EPSP synthase (Example: RR-Cotton, RR-Soybean) ROUNDUP (Glyphosate) TOLERANCE ( HERBICIDE TOLERANCE IN CROPS)

Insect Resistance Corn hybrid with a Bt gene Corn hybrid susceptible to European corn borer 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.

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis GENE FOR Bt TOXIN WAS TRANSFERRED TO OBTAIN BT TRANSGENIC PLANTS PLANT SYNTHESIZES INACTIVE PROTOXIN 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 INSECT FEEDS ON TRANSGENIC PLANT

Papaya infected with the papaya ringspot virus Virus resistance gene introduced Virus Resistant Crops 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

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. 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. 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. Delayed Fruit Ripening

“Rot-Resistant Tomato” Anti-sense gene  complementary to polygalacturonase (PG) PG = pectinase  accelerates plant decay/rotting Flav’r Sav’r tomato

Normal rice “Golden” rice Golden 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

Phytoene Phytoene synthase Gernayl Gernayl diphosphate (GGPP) Phytoene desaturase Lycopene Lycopene cyclase Beta carotene Complete biochemical pathway in the rice for production of beta- carotene, 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 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 Soybean Corn Corn Cotton Cotton Oil Seed rape Oil Seed rape Sugarbeet Sugarbeet Squash Squash Tomato Tomato Tobacco Tobacco Carnations Carnations Potato Potato Flax Flax Papaya Papaya Chicory Chicory Rice Rice Melon Melon