Elementary plant Biochemistry and Biotechnology (2+1)

Indirect gene transfer method

Agrobacterium mediated transformation

• Agrobacterium tumefaciens is a soil borne, Gram-negative bacterium, causative agent of ‘crown gall’ disease, an economically important disease of grapes, walnuts, apples and roses. The ability to cause grown gall (tumorous tissue growth) depends on the ability of Agrobacterium spp. to transfer bacterial genes into the plant genome. This is an unique example of inter-kingdom gene transfer and biotechnologists made use of this unique capability in development of plant transformation methods.


• Crown gall formation depends on the presence of a plasmid in Agrobacterium tumefaciens known as the “Ti” (tumour inducing) plasmid. Part of this plasmid (the T (transfer) DNA region) is actually transferred from the bacterium into the plant cell, where it becomes integrated into the plant genome.
  


• The T-DNA carries genes involved in both hormone (auxin and cytokinin) biosynthesis and the biosynthesis of novel plant metabolites called opines and agropines. The production of auxin and cytokinin causes the plant cells to proliferate and so form the gall. The opines and agropines are used by Agrobacterium. Opines and agropines are not normally part of plant metabolism and are very stable chemicals, which provide carbon and energy source that only Agrobacterium can use.

Molecular mechanism of T-DNA transfer

• Wounded plant cells release phenolic substances and sugars that are sensed by VirA protein, which activates VirG protein by phospharylation. VirG induces the expression of all the genes in the vir region of the Ti plasmid. Gene products of the vir genes are involved in variety of process. VirD1 and VirD2 proteins are involved in single stranded T-DNA production, protection and export and VirB products forms the transfer apparatus (molecular bridge).
  


• The single stranded T-DNA (associated with VirD2) and VirE2 are exported through the transfer apparatus. In the plant cells, the T-DNA coated with VirE2 and VirD2 interacts with different plant proteins which are then attached to the T-DNA and influence transport and integration. The T-DNA/VirD2/VirE2/plant protein complex enters the nucleus through the nuclear port complex. Integration into the plant chromosome occurs through illegitimate recombination.


Agrobacterium mediated plant transformation protocol:

1. Explant: The explant used in transformation experiments must be capable of producing whole plants by regeneration and should contain high proportion of cells that are competent for transformation.
2. Vector construction: Vectors used in this system are derivatives of Ti plasmid. They are, however, extensively modified so that most of the features of a natural Ti plasmid are removed, only the left and right border sequences being used to ensure transfer of the T-DNA region between them. The vector also contains a selectable marker on the T-DNA so that transformed plants can be identified, as well as a separate selectable marker (outside the T-DNA) to enable identification of the transformed bacteria. Virulence genes required for transfer of the T-DNA either located on in this same plasmid or separate plasmid.


The basic protocol used for Agrobacterium mediated transformation

1. Suitable plant tissue, to be used as a source of explants, is removed from the donor plant and sterilized. For tobacco transformation, leaves are ideal.
2. The leaf tissue is cut into small pieces (using a scalper or cork-borer) and placed into a culture of Agrobacterium (Which contains the recombinant vector) for about 30 minutes, a process known as co-cultivation. During this process, the bacteria attaches to the plant cells, since the medium contains Acetosyrengone – a phenolic compound (released when the plants wounded). The explants are subsequently removed from the bacterial culture, excess bacterial culture blotted off and then placed on to the solid MS medium that contains no selective agent.
3. The incubation of the explants with Agrobacterium. is allowed to continue for 2 days to allow transfer of the T-DNA to the plant cells. This step is called as co-cultivation.
4. The explants are removed from the medium and washed in an antibiotic solution (such as cefotaxime) that kills Agrobacterium.
5. The explants are transferred to fresh solid medium. This medium is supplemented with a selective agent (depend on which selectable marker gene is present in the T-DNA of the vector) to prevent the growth of non-transformed plant cells.
6. The selected transformed explants are then transferred to shoot and root regeneration medium.
7. Finally transformed plants were confirmed by PCR, southern blotting for the presence of the foreign gene and by Northern and Western Blotting for the expression of the foreign gene.