Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 1
1. Agrobacterium-mediated. 2. Biolistics or gene gun.
Systems used for genetic transformation
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 2
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.
crown gall
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 3
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.
Plasmid
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 4
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).
Plasmid
New gene construct
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 5
The next step is to use special enzymes (blue arrows) to insert the gene construct into the Ti plasmid of the bacterium.
Plasmid
New gene construct
New gene integrated into the Ti plasmid
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 6
Plasmid
Plant 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.
Nucleus
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 7
Plasmid
Nucleus
The Agrobacterium plasmid moves into the cell and locates the nucleus.
Plant cell
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 8
The new gene from the plasmid is integrated on to a section of the chromosome and even though it is a foreign gene it can be expressed as if it were part of the plant’s genome.
New gene inserted into chromosome
Nucleus
Plant cell
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 9
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.
Genetic transformation - Agrobacterium
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 10
Genetic transformation – Agrobacterium 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.
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 11
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.
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 12
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 13
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 14
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 15
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 16
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 17
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.
Genetic transformation - Arabidopsis
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 18
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.
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 19
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.
Genetic transformation – Gene gun
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 20
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.
Microprojectiles
Macroprojectile
Genetic transformation – Gene gun
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 21
Microprojectiles
Membrane
Stop plate
Target tissue
Pressure builds up behind the macroprojectile that forces it against the membrane until the membrane ruptures.
Genetic transformation – Gene gun
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 22
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.
Microprojectiles
Membrane
Stop plate
Target tissue
Genetic transformation – Gene gun
Tissue Culture
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 23
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.
Genetic transformation – Gene gun
