how agrobactrium transform genes into host for useful products

1. AGROBACTERIUM
TRANSFORMATION

2. Introduction To Genetic Transformation
• It is the process of transferring foreign gene.
• Natural process seen in bacteria.
• Two methods
1. Direct Method
2.Indirect Method

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• Direct method involves Electroporation,
Microprojectile etc.
• Indirect method i.e. Agrobacterium mediated gene
transformation

4. General Introduction Of Agrobacterium
• 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 and Townsend(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 ability of 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

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• 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

12. .

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 for agrobacterium
mediated transformation
• Identify suitable plant
• Transformation with agrobacterium
• Kill the agrobacterium
• Selection
• Regeneration

17. Methods of agrobacterium gene 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

20. .

21. Leaf disk method

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 mean of 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

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• Natural genetic engineer, 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

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• Growing transformed roots 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)

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• This has been 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

30. thank you