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agrobacterium transformation
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- 2. Introduction To GeneticTransformation • It is the process of transferring foreign gene. • Natural process seen in bacteria. • Two methods 1. Direct Method 2.Indirect Method
- 3. Continue… • Direct methodinvolves Electroporation, Microprojectile etc. • Indirect method i.e. Agrobacterium mediated gene transformation
- 4. General Introduction OfAgrobacterium • Round shaped gram negative bacteria. • Mostly found in soil • It causes disease in plants such as crown gall and hairy roots mostly in dicot plants
- 6. Classification of agrobacterium •On basis of pathogenicity I. Agrobacterium tumefaciens- crown gall II. Agrobacterium rhyzogenes- hairy root III. Agrobacterium radiobacter- avirulent
- 7. History • Smith andTownsend(1907)- said bacteria cause crown gall disease • Brown and stonier(1958)- proposed not whole bacteria but some parts of it cause disease • Zenen (1974)- noted virulent strain agrobacterium tumefaciens • Chilton (1977)- reported Ti and Ri plasmids
- 8. Ti-plasmid • The abilityof bacteria to cause disease is associated with Ti-plasmid • It is a large plasmid (grater than 200kb) that carries gene involved in infective process • Part of plasmid is incorporated into plant chromosomal DNA
- 9. Continue… • this segment,called T-DNA is 25-30kb in size • Contains eight genes that are expressed in plant cell and responsible for cancerous properties of host cell • Also direct synthesis of opines • In short, Agrobacterium transform the cell for its own purposes
- 10. The Ri plasmid •Ri plasmids are present in agrobacterium rhizogenes • Ri and Ti plasmid are very similar except transfer of T-DNA from an Ri plasmid to plant result in hairy root disease typified
- 11. Vector for transformation •Agrobacterium serve as a vector to transform plant cells • The bacteria can be manipulated in such a way that specific DNA sequences, along with antibiotic resistance genes, promoter sequences, and reporter genes, can be spliced into the T-DNA region of the plasmid and transferred to the plant genome
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- 13. Manipulation of plasmid •Large size of plasmid makes manipulation of interested gene very difficult (unique restriction site is impossibility) • Novel strategies have developed like I. Binary vector strategy II. Co-integration strategy
- 16. Basic protocol foragrobacterium mediated transformation • Identify suitable plant • Transformation with agrobacterium • Kill the agrobacterium • Selection • Regeneration
- 17. Methods of agrobacteriumgene transfer • Infection through wound • Co-cultivation with tissue ex-plants • In planta transformation
- 18. Infection through wound •If agrobacterium that contains engineered ti- plasmid are introduced by infection of wound in stem then only cells with resulting crown gall will posses the cloned gene • This is of little value because introduction of new gene into every cell of plant is needed
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- 22. In planta transformation •Imbibition of seeds in fresh culture of Agrobacterium • T-DNA become incorporated into plant genome • Agrobacterium enter the seedlings during germination
- 23. Killing the agrobacterium •Mostly timentin is used to get rid of agrobacterium • After 2 days of incubation , plant tissue is rinsed in solution of timentin • Also a combination of cefotaxime and timentin can be used
- 24. Selection and regeneration •Kanamycin, cytokinin and auxins are also added in the medium • Kanamycin allows only transformed plant cells to divide • Cytokinin induce shoot formation • Auxins induces formation of roots
- 25. Advantages • Natural meanof gene transfer more acceptable to those who feel natural is best • Capable of infecting intact plant, tissue or organ • Stability of transferred gene is excellent ,found to be stable over many generations
- 26. Continue… • Natural geneticengineer, manipulate host plant cell effectively and efficiently • Genes for oncogenes and opine synthesis are easy to remove to generate disarmed t-DNA vector • Relatively large length DNA fragments can be transformed
- 27. Continue… • Growing transformedroots at high density in liquid culture has been explored as potential mean of obtaining large amount of proteins from genes cloned in plants
- 28. Limitations • Time consuming •Dicots are easy to transformed such as tomato, potato ,peas but more difficult to obtain same result with monocots • Monocots lack efficient regeneration system • Have low wound response (release of acetosyringone)
- 29. Continue… • This hasbeen frustrating because monocots contains more important plants as barley rice and maize • Despite limitations efficient method has devised to transform monocots ,use of immature embryos as ex-plant
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