Thursday, 20 August 2015

Ti plasmid Plant transformation

Plant transformation:


                Plant transformation vector are plasmids that have been specially designed to facilitate the generation of transgenic plant.
                The most commonly used for the vector is binary vector because of their ability to replicate in both E.coli and a common lab bacterium.
And Agarobacterium tumefaciens used to insert the recombinant DNA into plant


Three key elements:

                Plasmid selection
                Plasmid replication
                Transfer DNA

Three major group of gene vectors for plants:


                1.Ti plasmids of Agarobacterium tumefaciens
                2.Caulimoviruses
                3.Geminiviruse

Organization of Ti plasmid:


                Ti plasmids are large (90-150*10 to power of 6 dalttons) circular double stranded DNA.
                Molecular weight is 120-160 megadaltons.
                When exposed to a temperature above 37*c, it undergoes denaturation and looses its 

tumorogenic property.


                It has an oncogenic region which produces some unusual amino acids.
                Ti plasmid has a region encoding for the synthesis of opine.
                It has also a sequence for opine catabolism which produces some essential enzymes that helps in opine catabolism.

A.tumefaciens


                It induces grown gall disease
                It is a Gram –ve  Rod shape bacteria
                Enter through plant wounds easily
                Also forms tumours
                It is a soil bacterium

T-DNA region:


               
            T-DNA contains two type of genes.
The oncogenic genes, encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumour formation.
 And the gene encoding for the synthesis of opines.
These compounds, produced by condensation between aminoacids and sugars, are synthesized and excreted by the grown gall cells and consumed by A.tumefacies as carbon and nitrogen sources.
Virulence gene:
          The gene responsible for the transfer of the T-DNA region into the host plant are also situated on the Ti plasmid, in an 40-kb region outside the T-DNA known as the virulence region

Procedure for prevention or protecting:


Leaf (explants)



Surface sterilization
(70% ethanol, 0.1% sodium hypo chloride )




Culture overnight in liquid medium with agarobacterium(Ti-plasmid transferred) to infect the leaf
Cells is grown in suspension medium (add shoot inducing solid medium) high in cytokinin culture for 2days




Shoot including solid medium+kanamycin+carbenicillin culture for about 20days



                                                                    
Shoot formation




                         Shoot callus is transfer to root inducing medium                                                                         
 


                                                                    
Root inducing solid medium (high in auxin+kanamycin+carbenicillin)                                                  



                                                                                                                                                                                                                                                                
Transfer plantlets to soil
 


                                                                          

Agarobacterium cells attached to the leaf. Kanamycin, carbenicillin to kill division and differentiation of the  transformed cells.