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Through the process of genetic transformation, scientists are able to put new genes into plants.

Photo showing an example of Agrobacterium transformation.

Agrobacterium transformation

Photo showing an example of gene gun tranformation.

Gene gun transformation

Photo showing an example of Arabidopsis transformation.

Arabidopsis transformation

Genetic transformation - Agrobacterium

Agrobacterium tumefaciens (crown gall) and Agrobacterium rhizogenes (hairy root) are bacteria that form characteristic diseases in plants. Biologists take advantage of these bacteria as vectors to insert genes into plants.

This technology is a common strategy for transformation of cells for genetic engineering.

Photo of a plant with crown gall on it pointed to.

The Agrobacterium plasmid is a circular piece of DNA that the bacterium uses to insert its genes into the plant's genome.

In nature, the bacterium inserts genes that cause the plant to make a tumor and produce novel amino acids for the bacterium to use.

Illustration of a plasmid as a ring-like structure.

The biologist develops a gene construct that will eventually be expressed in the transgenic plant.

The new gene usually has a section that allows for the selection of transformed cells along with the new gene of interest (like herbicide resistance or flower color).

Illustration showing a plasmid as a ring, and a section of new gene construct as a new section of a ring.

The next step is to use special enzymes (blue arrows) to insert the gene construct into the Ti plasmid of the bacterium.

Illustration showing the new gene construct integrated into the Ti plasmid.

The Agrobacterium plasmid infects the plant cell and places the gene of interest into the plant's DNA in the nucleus.

If this is done properly, the new gene will be expressed in the transformed plant.

Illustration showing a plant cell, with it's nucleus pointed out, and the altered plasmid outside of the cell.

The Agrobacterium plasmid infects the plant cell and places the gene of interest into the plant's DNA in the nucleus.

If this is done properly, the new gene will be expressed in the transformed plant.

Illustration of a plant cell with the altered plasmid entering into its nucleus.

The Agrobacterium plasmid infects the plant cell and places the gene of interest into the plant's DNA in the nucleus.

If this is done properly, the new gene will be expressed in the transformed plant.

Illustration of a plant cell with the new gene inserted into the a chromosome of the nucleus.

Usually the transformed cells are separated from non-transformed cells in tissue culture.

This means that adventitious shoots must be regenerated from the transformed and micropropagated to get a whole plant that expresses the new genes.

Photo of shoots growing in container of tissue culture.

Steps in a transformation protocol

  • Step 1. Make gene construct that includes gene of interest; a selectable resistance gene and an appropriate gene promoter sequence.
  • Step 2. Place new gene in Agrobacterium vector.
  • Step 3. Infect plant tissue. This may be an organ like a cotyledon or leaf disc or it may be callus.
  • Step 4. Select cells that have been transformed. This can be accomplished because along with the gene of interest, a resistance gene has been included as a selectable marker. This is usually resistance to an antibiotic or a herbicide.
  • Step 5. Regenerate shoots from transformed cells through a micropropagation system.
  • Step 6. New whole plants usually become part of a standard breeding program, where the novel gene is used to create commercial introductions.

Genetic transformation - Gene gun

One technique used to engineer or transform plant tissue is with a gene gun. Technically, this is call biolistics.

The gene gun pictured here is used to "shoot" genes into plant cells.

Photo of a gene gun.

The technician places the target tissue into the gene gun. The target tissue might be a developing cotyledon, seed tissue, or even callus.

Once the new gene is inserted in the tissue it will have to be isolated from cells that were not transformed and then new shoots regenerated in a micropropagation system.

The recovered plants expressing the new gene can then be used as a parent in a breeding program to get elite breeding stock to inherit the new gene.

Photo of a technician placing target tissue into a gene gun.

Microprojectiles are small metal particles coated with DNA.

These are placed on the surface of a larger macroprojectile prior to loading into the gene gun.

Illustration showing a macroprojectile being coated with microprojectiles.

Pressure builds up behind the macroprojectile that forces it against the membrane until the membrane ruptures.

Illustration showing a macroprojectile covered with microprojectiles at the top. Beneath it is a membrane, then below that is a stop plate with a hole in it. Beneath all of them is a dish with the target tissue in it.

The larger macro-projectile crashes into the stop plate but the smaller DNA-coated micro-projectiles continue on and enter the plant tissue.

The foreign DNA in the cell can be incorporated into the plant's gene during plant cell division.

Illustration similar to the preceding one, but now the membrane has ruptured. The macroprojectile has been stopped by the stop plate, while the microprojectiles have entered into the target tissue.

You can use the indicator gene called GUS to see which cells become transformed after using the gene gun. The GUS gene codes for an enzyme that turns a chemical substrate blue in the tissue.

Each blue spot represents a plant cell that has been transformed.

Photo of tissue which has numerous blue spots indicating transformed plant cells.

Genetic transformation - Arabidopsis

Arabidopsis is the most important model system in plant science. Its entire genome has been mapped and many of the important genes in this plant have been identified.

Molecular genetic studies with Arabidopsis have already identified many gene functions in plants.

Molecular genetics relies on sexual crosses mutagenesis and genetic transformation to identify genes.

Photo of an Arabidopsis plant.

Arabidopsis transformation relies on Agrobacterium-mediated gene insertion except that there is no tissue culture step.

In this case, whole plants will be exposed to Agrobacterium with the hope that the developing seeds will be genetically transformed.

The next several pages shows how this is done.

Photo of a technician with several Arabidopsis plants.

In preparation for transformation, Agrobacterium cells are grown in culture.

These cells contain the plasmid with the new genes that will be placed into the Arabidopsis plants.

Photo of technician holding a container with Agrobacterium cells in it.

A bathing solution is made from the suspension of Agrobacterium cells.

This will serve as the medium into which the Arabidopsis plants will be treated.

Photo of technician creating the bathing solution.

Inside the developing flowers are the ovules that will become the seeds. The Agrobacterium cells can infect this part of the Arabidopsis plant and transfer the genes to the growing seed embryo. A percentage of these seeds will contain the new gene.

When these seeds are germinated on a selection medium, those with the new gene will grow and develop into whole transformed plants. In the ideal situation, these plants will transfer the gene to the next seed generation.

Photo of plant with the developing flowers pointed out.

The actual procedure is very simple.

The Arabidopsis plants at the appropriate stage of flower development are inverted and submerged into the Agrobacterium suspension to permit infection.

Photo of technician holding plant upsidedown over the Agrobacterium suspension liquid.

After treating the plants for the appropriate amount of time to permit infection, the plants are removed form the suspension and moved to the greenhouse or growth chamber to allow seed set.

Mature seeds are harvested and tested to uncover those seedlings that have been genetically transformed.

Photo of multiple plants sitting upsidedown in shallow tray of suspension liquid, and being removed by technician.