Agrobacterium tumefaciens is a soil bacterium known for its natural ability to transfer a segment of its DNA into plant cells. Transgenic plants are those that have had specific genes from other organisms introduced into their own genetic material through genetic engineering techniques. This process results in new traits that do not occur naturally in the plant species. Agrobacterium tumefaciens plays a central role in creating these genetically modified plants.
How Agrobacterium Naturally Transfers DNA
Agrobacterium tumefaciens naturally infects wounded plants, causing a plant disease known as Crown Gall. This disease manifests as tumor-like growths, or galls, typically found at the bases of stems. The bacterium achieves this by transferring a specific segment of its genetic material, called transfer DNA (T-DNA), into the plant cell’s genome.
This T-DNA is located on a large circular DNA molecule within the bacterium, known as the tumor-inducing (Ti) plasmid. Upon sensing chemical signals released from wounded plant tissue, the bacterium activates a set of virulence (vir) genes on the Ti plasmid. These vir genes encode proteins that facilitate the excision of the T-DNA and its transfer into the plant cell through a specialized transport system.
Once inside the plant cell, the T-DNA integrates into the plant’s chromosomes. The genes carried within this transferred T-DNA instruct the plant cells to produce plant hormones, leading to uncontrolled cell division and tumor formation. Additionally, these genes direct the plant to synthesize unique compounds called opines, which serve as a specific food source for the Agrobacterium.
Adapting Agrobacterium for Biotechnology
Scientists have repurposed Agrobacterium’s natural DNA transfer system for genetic engineering. This adaptation involves a process called “disarming” the bacterium. The disease-causing genes responsible for tumor formation and opine synthesis are removed from the T-DNA region of the Ti plasmid.
The modified Ti plasmid is then engineered to carry foreign genes, such as those for pest resistance or herbicide tolerance, along with a selectable marker gene. These foreign genes are inserted into the T-DNA region. The bacterium still retains its ability to transfer DNA into plant cells, but now it delivers beneficial genes instead of those that cause disease.
Often, this system utilizes a “binary vector” approach, where the modified T-DNA carrying the foreign genes is placed on a smaller plasmid. The vir genes, which are necessary for the DNA transfer process, remain on a separate, disarmed Ti plasmid within the same Agrobacterium cell. This separation simplifies the genetic manipulation while maintaining the bacterium’s transfer capability.
Using Agrobacterium to Create Transgenic Plants
Creating transgenic plants using Agrobacterium involves a series of laboratory steps, beginning with the preparation of plant material. Small pieces of plant tissue, known as explants, are typically used because their cells are often more receptive to genetic transformation and can be regenerated into whole plants. These explants might be pre-cultured on a nutrient medium to prepare them for the transformation process.
The prepared plant explants are then subjected to co-cultivation, where they are incubated with the engineered Agrobacterium suspension. This incubation period allows the bacteria to attach to the plant cells and transfer the modified T-DNA into the plant cell nuclei. After co-cultivation, the explants are typically washed to remove excess bacteria and transferred to fresh media.
Following co-cultivation, a selection step is performed using selective media. This media contains agents that only allow the growth of plant cells that have successfully integrated the new gene. Untransformed cells, lacking the resistance gene, will not survive on this selective medium.
The surviving transformed cells are then encouraged to regenerate into whole plants through tissue culture techniques. This involves culturing the cells on specialized media containing plant hormones to induce the formation of callus (a mass of undifferentiated cells), followed by the development of shoots and roots.
Why Agrobacterium Remains Indispensable
Agrobacterium tumefaciens remains a widely used method for plant genetic transformation due to several advantages. The bacterium exhibits a broad host range, capable of transforming many dicotyledonous plants, some monocotyledonous plants, and even gymnosperms, making it adaptable for various crop species. This versatility allows researchers to apply similar transformation strategies across diverse plant types.
The efficiency of Agrobacterium-mediated transformation is another factor. It can effectively transfer large DNA fragments into plant cells, and the integration of these genes into the plant genome is precise. This process results in stable gene transfer, where the introduced gene is reliably passed on to subsequent plant generations.
The method is also cost-effective and relatively straightforward to implement compared to some other transformation techniques. The minimal DNA rearrangements observed during Agrobacterium-mediated transfer contribute to the integrity and predictable expression of the introduced genes. These combined features underscore Agrobacterium’s continued importance in plant biotechnology.