Genetic Modification in Nature

A Natural way of Genetically Modifying Organisms, Agrobacterium tumefaciens

. By Jared Keefer
 Crown gall caused by transfected DNA from Agrobacterium tumefaciens
Genetically modified organisms may not be as "unnatural" as some may think. The process of altering the DNA of one organism for the benefit of another organism is not a man-made idea. Agrobacterium has been accomplishing this feat for millions of years.
Agrobacterium tumefaciens is a remarkable species of soil-dwelling bacteria that has the ability to infect plant cells with a piece of its own DNA. When the agrobacterial DNA is integrated into a plant chromosome, it effectively uses the plant's own cellular machinery to ensure the proliferation of the agrobacterial population.
In 1977, University of Washington microbiologists; Mary-Dell Chilton, Eugene W. Nester, and Milton P. Gordon were the first to prove that it was DNA from a plasmid in Agrobacterium that was transferred and integrated into plant DNA. This was the cause of crown gall tumors. They also identified the particular plasmid DNA segments and deduced the first genetic map of one of the tumor-inducing plasmids. Many gardeners and orchard owners are quite familiar with Agrobacterium tumefaciens, because it causes crown gall diseases in many ornamental and fruit plants.
The Crown Gall Group, here at the University of Washington, in collaboration with other researchers, has been extensively studying Agrobacterium since 1999. They have recently finished sequencing the genome of Agrobacterium strain C58 and have published the sequence in the December 14th, 2001 issue of Science. The bacterial genome sequence will enable scientists to gain a greater insight into this organism's properties, especially those that enable it to genetically engineer its host's cells.
The Agrobacterium tumefaciens cell contains two DNA containing structures; a bacterial chromosome and another structure known as a Ti (tumor-inducing) plasmid. The process of this natural genetic engineer contains many steps but is rather direct. First, Agrobacterium only infects a plant through wounds in the plants roots or stems. Diagram of Agrobacterium tumefaciens cell, Source: Ohio State UniversityWhen wounded, the plant gives off certain chemical signals. In response to these signals, a cascade of Agrobacterium vir (virulence) genes activate and direct a series of events necessary for the transfer of the T-DNA (transfer DNA) from the Ti plasmid to the plant's chromosome. Different vir genes copy the T-DNA, prepare the T-DNA for passage into the host, and then open a channel in the bacterial cell membrane, through which the T-DNA passes. The T-DNA then enters the plant cell through the wound. Finally, with the exact process is still unknown, the T-DNA then moves from the cytoplasm to the nucleus of the plant cell and the T-DNA becomes integrated into the plant chromosome. One theory for this final step is that the T-DNA waits until the plant DNA is being replicated or transcribed, and then inserts itself into the exposed plant DNA1.



Genetic Modification in the Laboratory

Laboratory Methods for Genetically Modifying Organisms

By Jared Keefer
Everyone, in one way or another, knows about Genetically Modified Organisms. Yet, not everyone is familiar with the actual process of genetic engineering. GMO's are produced by transforming the organisms. Transformation is the heritable change in a cell or organism brought about by the uptake and establishment of introduced DNA. There are two predominant procedures of transforming genes in organisms: the "Gene Gun" method and the Agrobacterium (Link Agrobacterium to the Agrobacterium page) method.

"Gene Gun" method

The "Gene Gun" method is also referred to as micro-projectile bombardment or biolistics (ballistics using biological components). This technique is used for in vivo, within a living organism, transformation and has been especially useful in transforming monocot species like corn and rice.
This approach literally shoots genes into plant cells and plant cell chloroplasts. DNA is coated onto small particles of gold or tungsten approximately two microns in diameter. This technique can also be used for RNA, if desired. The particles are placed in a vacuum chamber and the plant tissue to be The Helios Gene Gun by BioRad engineered is placed below the chamber. The particles are propelled at high velocity using a short pulse of high pressure Helium gas into any target cell or tissue.

Click here for a descriptive animation of the "Gene Gun" method.

Agrobacterium method

Transformation via Agrobacterium (Link to the Agrobacterium page) has been successfully practiced in dicots, broadleaf plants, soybeans and tomatoes, for many years. Recently it has been adapted to and effective in monocots like grasses, corn and rice. In general, the Agrobacterium method is considered preferable to the gene gun, because of a greater frequency of single-site insertions of the foreign DNA, which allows for easier monitoring. In this method, the tumor inducing (Ti) region is removed from the T-DNA (transfer DNA) and replaced with the desired gene and a marker, which is then inserted into the organism. The marker is used to find the organism which has successfully taken up the desired gene. Tissues of the organism are then transferred to a medium containing an antibiotic or herbicide, depending on which marker was used. The Agrobacterium present is also killed by the antibiotic. Only tissues expressing the marker will survive and possess the gene of interest. Thus, subsequent steps in the process will only use these surviving plants. In order to obtain whole plants from these tissues, they are grown under controlled environmental conditions in tissue culture. This is a process of a series of media, each containing nutrients and hormones. Once the plants are grown and produce seed, the evaluation of the progeny process begins. This process entails selection of the seeds with the desired traits and then retesting and growing to make sure that the entire process has been completed successfully with the desired results. Click here for a descriptive animation of Agrobacterium Method