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.

Thursday, 6 August 2015

Agrobacterium tumefaciens and crown gall disease

Agrobacterium tumefaciens and crown gall disease

Introduction :

—                      1000’s of plant species are susceptible; they include especially rose, nut trees, grape, many shrubs and vines and perennial garden plants

Symptoms:
                       roundish, rough-surfaced galls, several inches or more in diameter, usually at or near the soil line, or on roots and lower stems
The galls,  at first cream coloured or greenish, later turn brown or black.
Crown gall, disease of plants caused by Agrobacterium tumefaciens.

           Avoiding replanting for that period; removing severely infected plants (including as many roots as possible); protecting against injury; keeping down weeds; controlling root-chewing insects and nematodes; cutting away large galls on trees, and disinfecting the wounds

Agrobacterium tumefaciens




Ti plasmid

  —The ability to cause crown gall disease is associated with the presence of the Ti 
     (tumorinducing) plasmid within the bacterial cell. This is a large (greater than 200 kb) plasmid
  —that carries numerous genes involved in the infective process
  —The ability to cause crown gall disease is associated with the presence of the Ti plasmid within the bacterial cell.
  —This is a large (greater than 200 kb) plasmid
That carries numerous genes involved in the infective process
  —This segment, called the T-DNA, is between 15 and 30 kb in size, depending on the strain.
  It is maintained in a stable form in the plant cell and is passed on to daughter cells as an integral part of the chromosomes
  —A remarkable feature of the Ti plasmid is that, after infection, part of the molecule is integrated into the plant chromosomal DNA 
 —But the most remarkable feature of the Ti plasmid is that the T-DNA contains eight or so genes that are expressed in the plant cell and are responsible for the cancerous properties of the transformed cells.
  —These genes also direct synthesis of unusual compounds, called opines, that the bacteria use as nutrients.
  —In short, A. tumefaciens genetically engineers the plant cell for its own purposes