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Plant Genetic Engineering ( Plant Transformation)

Plant Genetic Engineering ( Plant Transformation). Nono Carsono , PhD. Dr. rer . nat Suseno Amien Anas , PhD. Genetic transformation/modification of plant?. Introduction of exogenous DNA into a plant cell - transient : no incorporation of exogenous DNA ( transgenes ) into the genome

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Plant Genetic Engineering ( Plant Transformation)

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  1. Plant Genetic Engineering(Plant Transformation) Nono Carsono, PhD. Dr. rer. natSusenoAmien Anas, PhD.

  2. Genetic transformation/modification of plant? • Introduction of exogenous DNA into a plant cell - transient: no incorporation of exogenous DNA (transgenes) into the genome - stable: incorporation into genome Transgene - the genetically engineered gene added to a (plant) species Product - Transgenic plant: plant containing transgenes introduced by genetic engineering/modification/ transformation (not classical breeding) • Transformation of multicellular organisms: - can not directly transform every cell - transformation involves one cell which then regenerates an entire organism Stable expression of GFP Transient expression of GFP

  3. Why use Genetic Transformation? • Accelerate the breeding process • Introduce/enhance desired trait in an established genetic background • Extend the gene pool • Select genes from any Kingdom (with care, especially if potential for entry into the food chain)

  4. Diff. between conv. breeding and genetic engineering

  5. Bt Corn Reduces:Insecticide MycotoxinApplication Production

  6. stress-inducible promoter driving drought- and cold-responsive transcription factor wild type What are the Uses of GM Plants? • Research • Largest number of transgenic plants are currently created for research purposes • Knock-outs, over-expression, modified proteins K. Yamaguchi-Shinozaki, JIRCAS, Japan

  7. Field following application What are uses of GM Plants? • Commercial Applications • Altered agronomic traits • Disease/insect resistance • Virus resistance • Herbicide resistance • Salt/drought tolerance • Cold tolerance • Enhanced yields, other quantitative traits • Phytoremediation • Production of vaccine Application of Roundup herbicide 2007 time

  8. Other uses of GM Plants? • Bioreactors / Molecular farming • Therapeutic proteins • Human lactoferrin to treat iron deficiencies • Antibodies • Vaccine production • Antigen expression • HepC, HIV Dow AgroSciences Achieves World’s First Registration for Plant-Made Vaccines Indianapolis, IN - January 31, 2006 Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company, (NYSE: DOW), announced today that it has received the world's first regulatory approval for a plant-made vaccine from the United States Department of Agriculture (USDA) Center for Veterinary Biologics. This approval represents an innovative milestone for the company and the industry...

  9. Greater public acceptance when the technology is shown to more greatly benefit consumers? Other uses of GM Plants? • Functional foods (humans and livestock) • Today: Golden rice • Vitamin A enriched • Future directions: • Boosted antioxidants • Elevated content of specific minerals • Removal of food allergens, carcinogens

  10. Golden Rice Scientists from Swiss and German universities have engineered two genes from daffodil and one bacterial gene into rice to produce provitamin A. (daffodil) Provitamin A biosynthesis pathway GGPP Phytoene Lycopene beta-Carotene = provitamin A Phytoene synthase (psy) Phytoene desaturase (crtl) (bacteria) Lycopene ß-cyclase (lcy) Funding: Rockefeller Foundation, Swiss Federal Institute Of Technology, European Community Biotech Program (daffodil)

  11. How to make Transgenic Plants An Overview of the Genetic Engineering/modification cycle

  12. Termination sequence Bt coding sequence Promoter Prerequisites for genetic transformation • Available gene of interest (incl. selectable marker and other regulatory sequences)- isolation and construct design • Efficient method for genetic transformation- gene transfer • Efficient method for regeneration (whole plant)- plant regeneration ? How the direction of transcription? Right or left?

  13. CODING SEQUENCE PROMOTER poly A signal INTRON Plant Selectable Marker Gene Plasmid DNA Construct • bacterial genes • antibiotic marker • replication origin Building the Transgenes ON/OFF Switch Makes Protein stop sign Plant Transgene

  14. 1. Gene of interest • The coding region and its controlling elements 2. Selectable marker • Distinguishes transformed/untransformed plants 3. Insertion sequences • Aids Agrobacterium insertion Transformation Cassettes Contains

  15. Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos • Develop shoots • Root the shoots Transformation Steps Prepare tissue for transformation Introduce DNA • Agrobacterium or gene gun Culture plant tissue Field test the plants • Multiple sites, multiple years

  16. Delivering the gene to the plant genome: Gene transfer methods • Direct transfer of DNA - PEG-polyethylene glycol - Electrophoration • Transfer of DNA via carrier - Microinjection - Particle bombardment • Transfer of DNA via vector - Agrobacteriumtumefaciens - Viral vector

  17. Cloned Gene in Vector DNA Molecule Biolistic bombardment (gene gun) Transformation of Agrobacterium Protoplast transformation followed by cell wall regeneration Agrobacterium-mediated transformation of plant cell Migration and integration of gene into nucleus Plant cells grown in tissue culture Regeneration of genetically modified plant from tissue culture

  18. Biolistic / Gene Gun

  19. Plasmids Transformed cell Donor DNA Plasmid vector Selectable antibiotic resistance marker Donor DNA cut with EcoRI Donor DNA fragments Vector cut with EcoRI Add DNA ligase Tetracycline-resistant Bacterial colony from transformed cell Introduce into E. coli Recombinant DNA

  20. Non-transgenics Transgenic plant The Next Test Is The Field Herbicide Resistance

  21. Before After Example: Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn

  22. Roadmap Bioengineering Plants for the Future Plant production platforms • Vitamines • Fatty acids • Enzymes • Bio-polymers • Pigments • Pharmaceutical products • Fibers Health food and quality • Amino acids • Oil • Starch Plant protection Efficient agriculture Stress resistance • Viruses • Nematodes • Fungi • Insects • Cold • Drought • Salinization • Bt technology • Herbicide resistance etc 1997 2005 2015 2025

  23. Nature Biotechnology 25: 271 (2007)

  24. Nature Biotechnology 25: 271 (2007)

  25. Nature Biotechnology 25: 271 (2007)

  26. Transgenic Confirmation (Genotypic analysis) • PCR for rapid screening • Southern blot for precise gene detection • Northern blot for transcription analysis • Western blot for translation analysis, together with Ab-binding or enzymatic analysis • Mendelian analysis for insertion locus and linkage analysis • In situ hybridization for precise insertion locus analysis • DNA methylationanalysis for silencing potential analysis

  27. Regulatory principles of transgenic crops: • Scientifically based, based on information of organism, used technology and effects to humans and environment • Product-based approach, use existing product-based legislation • Familiarity and substantial equivalence, experience with the use of that species. The determination is based on scientific literature and practical experience with the plant and similar plant varieties. 4. Case-by case, allow the development of knowledge that could inform criteria and requirement over time.

  28. Regulatory principles: • Step-wise fashion, products should be assessed throughout the chain of development : From laboratory to greenhouse and finally large-scale field trial • Transparency • Precautionary principle/approach, derived from Rio Declaration, regulatory groups can make decisions about products based on scientific uncertainty. • Harmonization, sharing of or acceptance of another group’s review

  29. Questions Do not forget..next week student presentation!

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