The Agrobacterium Mediated Transfer of Genes

The Agrobacterium Mediated Transfer of Genes

The Agrobacterium Mediated Transfer of Genes
A naturally occurring conjugation phenomenon in Agrobacterium tumefaciens induced crown-gall disease of plants has been ably exploited by biotechnologists to genetically engineer foreign genes into dicotyledonous plants rendering them transgenic in characters such as resistance to viral diseases, herbicides or bioinsecticides.

A. Bacterial transduction with bacteriophages. A virulent phage

E. In a phenomenon called lysogeny infection with a temperate phage results in the integration of phage DNA with the bacterial Chromosome

Bacterial Transduction with Bacteriophages. A Virulent Phage

In a phenomenon called lysogeny infection with a temperate phage results in the integration of phage DNA with the bacterial Chromosome

C. The phage DNA is injected into the bacterial cell followed by disruption of bacterial DNA and the phage takes Control of bacterial cell dunctions

F. Maintained as such for several generations. occasionally phage DNA gets detached from the bacterial chromosome

The Phage DNA is Injected into the Bacterial Cell Followed by Disruption of Bacterial DNA and the Phage Takes Control of Bacterial Cell Functions

Bacterial Chromosome

D. New bacteriophages are formed and releasd by cell lysis	G. The bacterial DNA and takes control of the bacterial cell behaving like a virulent phage causing lysis of the cell with the relese of mature bacteriophages

At is a soil bacterium which infects the crown region of dicotyledonous plants (monocots are resistant) through mechanical wounds to produce crown galls. A.t harbours large extrachromosomal megaplasmids. Most of the genes required for tumour formation are located on one such 180 kb megaplasmid designated as Ti plasmid, the letters Ti denoting the tumour inducing ability of the plasmid. The Ti plasmid contains a tumour inducing region (T-DNA) which also carries genes for the synthesis of two growth hormones, the IAA and cytokinins and the genes controlling the synthesis of a group of amino acid derivatives known as opines (nopaline and octopine).

The expression of bacterial genes controlling growth hormone production is not controlled by the plant but is necessary for the-development of crown gall symptoms. The opines synthesized serve as nitrogen and carbon sources for the bacterium. However, the genes controlling the growth hormone production and opine synthesis are not essential for transferring or integration of T-DNA into the host plant cell genome.

The Ti plasmid has also a cluster of about 8 genes known as virulence genes (vir genes). This gene cluster of 35 Kb DNA is necessary for the recognition of susceptible cells on the plant surface, to excise T-DNA from the plasmid and transfer the T-DNA region to the host cell. The Vir genes are activated only by contact with cell metabolites released by the wounded plant and they are not functional or expressed in pure A.t cultures grown on synthetic media.

There are two border regions to T-DNA (LB and RB) which are known to contain genes involved in the secretion of the enzyme endonuclease that scissors off the T-strand from the Ti plasmid. Thus the genes encoded within the T-strand have all the appropriate signals for efficient transcription and translation in their eukaryotic host.

The process of infection begins with the bacterial surface components of A. t. recognizing the plant surface which is susceptible to the attack by the pathogen, followed by a process analogous to bacterial conjugation whereby a single strand of T-DNA (the T-strand) is transferred to the plant cell probably through pores in the cell wall.

Within the host cell, several copies of T-DNA are inserted at single or multiple sites in the host chromosomes and function as typical eukaryotic chromatin.

The earlier procedure to identify transformed cells was to develop plant galls or tumours from such transformed cells. Alternatively, the transformed cells were grown in hormone independent cultures. However, in later experiments, modified T-DNA and Ti plasmids have been used to facilitate rapid experimentation and development of transformed plants.

In the modified T-DNA, the genes coding for phytohormones and opines have been excised because, as stated earlier, these genes are not concerned with the transfer and integration of T-strand into the host genome but are essential for the manifestation of crown-gall symptoms.

In essence, deleting these genes from T-DNA results in the elimination (disarming) of the oncogenic (tumourous) phenotype. Foreign DNA can be inserted within the right and left border regions of an appropriate plasmid, thereby enabling the delivery of large multigenic segments of DNA into plants. In this way T-DNA region has been engineered (disarmed) to eliminate the tumour inducing genes (Fig. 102). Selection of transformed cells has been facilitated by engineering a variety of antibiotic markers selectable in plants such as resistance to kanamycin and gentamycin.

Ti plasmids have also been engineered in other Agrobacterium spp. and E. coli to facilitate efficient plasmid replication and selection. Such manipulated plasmid vectors could be used directly in transformation steps obviating the need to repeatedly infect plants with A. tumefaciens cells.

Home | Site map | Submit Article | Directory | Search