Plant Genetic Engineering

1. Plant Genetic Engineering

2. Why do scientists want to change gene instructions? • to produce needed chemicals • to carry out useful processes • to give an organism desired characteristics Genetic Engineering The process of manipulating and transferring instructions carried by genes from one cell to another

3. THE SCIENCE OF GENETIC ENGINEERING 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.

4. DNA of interest is EVERYWHERE

5. 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

6. Plant transformation • getting DNA into a cell • getting it stably integrated • getting a plant back from the cell Requirement • a suitable transformation method • a means of screening for transformants • an efficient regeneration system • genes/constructs • Vectors Promoter/terminator • reporter genes selectable marker genes • ‘genes of interest’

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

8. Transformation methods DNA must be introduced into plant cells Method depends on plant type, cost, application

9. Transformation by the help of agrobacterium Agrobacterium-mediated transformation Agrobacterium is a ‘natural genetic engineer’ i.e. it transfers some of its DNA to plants

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

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

12. 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

13. Cell’s DNA DNA coated golden particles Gene gun Plant cell A plant cell with the new gene Cell division Transgenic plant “Gene Gun” Technique

14. 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.

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

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

17. 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 INSECT FEEDS ON TRANSGENIC PLANT Toxin binds a receptor on the gut epithelial cells, forms a channel on the membrane. This causes electrolyte leakage and insect death

18. 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

19. 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.

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

21. 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

22. Gernayl Gernayl diphosphate (GGPP) Phytoene synthase Phytoene Phytoene desaturase Lycopene Lycopene cyclase Beta carotene Complete biochemical pathway in the rice for production of beta-carotene, a precursor for vitamin A.

23. 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.

24. Soybean Corn Cotton Oil Seed rape Sugarbeet Squash Tomato Tobacco Carnations Potato Flax Papaya Chicory Rice Melon Approved Transgenic plants