Agrobacterium mediated gene transfer

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Agrobacterium mediated gene transfer

  1. 1. AgrobacteriumMediatedPlantTransformation 30th April’2012 Presented by, Prabhu Thirusangu, Research Scholar,
  2. 2. Transformation – the process of obtaining transgenic plants Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants. Most common genes (and traits) in transgenic or biotech crops herbicide resistance Insecticide resistance Bt genes in field corn (maize) virus-resistance (coat-protein) genes Transgenic plants – An introduction
  3. 3. Plant Transformation Methods Physical Chemical Biological Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Silica/carbon fibers Lazer mediated SAT PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers A. Tumefaciens A. Rhizogenes Virus-mediated
  4. 4. 4 Introduction to Agrobacteria… A rod shaped, Gram negative soil bacterium A natural genetic engineer 2 species  A.tumefaciens (produces a gall)  A. rhizogenes (produces roots) Oncogenes (for auxin and cytokinin synthesis) + Opines • Disease producing Agrobacterium are higly pathogenic and do not benefit the plant • In the presence of exudates (e.g. acetosyringone) from wounded plants, Virulence (VIR) genes are activated and cause the t-DNA to be transferred to plants. Everything between the left and right border is transferred.
  5. 5. Scientific classification • Kingdom :Bacteria • Phylum :Proteobacteria • Class :Alproteobacthaperia • Order :Rhizobiales • Family :Rhizobiaceae • Genus :Agrobacterium • Species :A. tumefaciens
  6. 6. Ti plasmid and virulence genes • A Ti plasmid is a circular piece of DNA found in almost all Agrobacteria • Three main regions T-DNA region(Between right and left T-DNA border) Oncogene Opine Virulence region Opine catabolism region
  7. 7. Cellular process of Agrobacterium–host interaction Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
  8. 8. Molecular involvement in t-DNA trANsFEr…
  9. 9. Images of Crown Gall Tumor
  10. 10. Approved Traits..... • Glufosinater herbicide • Sethoxydimr herbicide • Bromoxynilr herbicide • Glyphosater herbicide • Sulfonylurear herbicide • Bt gene • Male-sterility • Modified fatty acid • Flower colour • Flower life • Delayed fruit ripening • Virus resistance
  11. 11. Binary vector t-DNA VIR genes Plasmid DNA Bacterial ChromosomeBacterial ORI Ampicillin resistance Construction of vector with disired genes
  12. 12. Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants
  13. 13. Procedure for plant transformation IMAGE: Mol bio of the cell by Albert (pg no:599)
  14. 14. Regeneration, Selection And Detection... Regeneration: for shoot organogenesis, cytokinin (lower amounts of auxin) are required Selection: two antibiotics are required • an antibiotic to kill the Agrobacterium, while not affecting the plant's cell growth and division • a second antibiotic allows growth of transformed shoots (w/selectable marker) but inhibits growth of untranformed plant cells. Detection of the "trait" gene PCR methods can detect the presence of the "trait" DNA protein detection methods are used where a gene product is produced that defines the trait verification of the incorporation of the trait gene into the plant's chromosome • by Southern hybridization • by demonstrating transfer of the trait to the original transformant's progeny
  15. 15. Approved Transgenic plants..... • Soybean • Corn • Cotton • Oil Seed rape • Sugarbeet • Squash • Tomato • Tobacco • Carnations • Potato • Flax • Papaya • Chicory • Rice • Melon
  16. 16. Types of genetic modification >99% of all transgenic crops are either herbicide or insect resistant <1% have other traits 0 5 10 15 20 25 Herbicide Insect resistance Others Millionsofhectares
  17. 17. Problems and potential
  18. 18. Ø Scientists can insert any gene they want into the plasmid in place of the tumor causing genes and subsequently into the plant cell genome Ø By varying experimental materials, culture conditions, bacterial strains, etc. scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn Ø This method of gene transfer enables large DNA strands to be transferred into the plant cell without risk of rearrangement whereas other methods like the Gene Gun have trouble doing this Ø The vast majority of approved genetically engineered agriculture has been transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer Ø Original problems existed in that Agrobacterium tumefaciens only affects dicotyledonous plants Ø Monocotyledon plants are not very susceptible to the bacterial infection Benefits and Problems with Agrabacteria….
  19. 19. Transformed plants will help the world to meet the human demands for food, energy, medicine, shelter, clothing, and a cleaner environment. Gene transfer technology is being used to produce plants capable of hyper accumulating toxic metals from soil and water, thus contributing to a cleaner environment Increases in plant production are needed to meet growing demands for food. Transformed Plants offers significant advantages over traditional plant breeding in developing improved crops. CoNClusioN…..
  20. 20. References Introduction to plant biotechnology by H.S Chawla Molecular biology of the cell by Albert et al Online References http://www.nepadbiosafety.net/for-regulators/resources/subjects/biotechnology/ plant-transformation-agro http://www.bio.davidson.edu/people/kabernd/seminar/2002/method/dsmeth/ds.htm http://arabidopsis.info/students/agrobacterium/uses.html http://microbewiki.kenyon.edu/index.php/Agrobacterium_tumefacien0073 http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/CrownGall.aspx http://www.ag.ndsu.edu/pubs/plantsci/crops/a1219w.htm
  21. 21. By, T.Prabhu, 30th April’2012

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