Transgenic Plants

Objectives Students will be able to: Identify transgenes Describe the processes and techniques used to produce transgenic plants

Genetically Altered? Scenario #1 An insect feeds on corn stalks. To prevent losses from the insect plant breeders select plants with the strongest stalks to be used as parents for the next generation. A plant is created that is resistant to the insect. Scenario #2 An insect feeds on corn stalks. To prevent losses from the insect plant breeders isolate a gene that kills insects once they consume the plant. This gene is placed in the DNA of plants. A plant is created that is resistant to the insect.

Traditional Breeding vs. Biotechnology http://passel.unl.edu/pages/animation.php?a=traditional_breeding _biotechnology.swf&b=1010427834

What is a transgene? A gene that has been genetically altered http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=958077244&topicorder=2&maxto=7&minto=1

Genetic Engineering In order to create a transgene, the following steps must be completed: 1. Extract DNA- Scientists must find a desired trait. Once the trait is found it can be isolated through a series of steps 2. Gene Cloning: After the DNA is extracted many copies of the gene must be made 3. Gene design: Gene is cut apart with enzymes and certain regions are replaced.

3. Gene Design The newly designed gene must have: Promoter- controls location of insertion and level of expression Coding Region- desired gene *Selectable Marker- method to determine if desired gene was successful Termination Sequence- end of gene http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=959031259&topicorder=4&maxto=9&minto=1

4. Transformation Once the gene has been designed, transformation can begin The process of genetically changing a living thing A genetically engineered plant has been transformed and is referred to as a transgenic plant. http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=958077244&topicorder=2&maxto=7&minto=1

Successful Transformations Copies of the transgene must be placed into the nucleus without killing the cell. At least one copy of the transgene must insert into the chromosome The transformed cell must replicate it chromosomes (including the new gene) and then divide. This must repeat until the transformed cell develops into an entire transgenic plant This transformed plant must have some characteristic that makes it easy to distinguish as a transgenic plant. This could be a selectable marker gene http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=958077244&topicorder=2&maxto=7&minto=1

How do plant breeders insert a transgene? Gene Gun Agrobacterium Microfibers ‘Whiskers’ Electroporation

Gene Gun Used to create ‘Round-up Ready Soybean’ Steps: Plant cells are grown in tissue culture Microscopic pieces of gold are coated with hundreds of copies of the desired gene DNA is shot into the tissue culture cells Disadvantage: Either zero or multiple copies will insert into the chromosome.

Gene Gun Animation http://passel.unl.edu/pages/animation.php?a=GeneGun.swf

Determining Success Selection Media & Selectable Markers When tissue culture cells are place on this media, cells that have not been transformed and do not contain the gene conferring resistance to the substance (selectable marker) will die leaving only those cells that are transgenic This helps to determine if the gene gun attempt was successful

Agrobacterium tumefaciens Method Most commonly used Soil bacterium that works as a natural bio-engineer Invades plants through wounds Bacteria as extra piece of DNA (Ti Plasmid)

Agrobacterium tumefaciens Biology Once the bacteria enters the plant a portion of the Ti plasmid enters the plant cell and inserts itself into the plant’s chromosomes. This turns on the genes of the Ti plasmid. The genes cause the plant to make nutrients for the growth of the bacterium. The bacterium takes over the plant, which eventually dies.

How could Agrobacterium tumefaciens be used to create a transgenic plant? Remove the genes from the Ti plasmid that create nutrients and replace them with a desired gene Can be used to infect and transform plant cells growing in tissue culture

Transformation with Agrobacterium Video Clip https://ge.unl.edu/oomycete/step-2-transformation-transgene- design/transformation-with-agrobacterium/

Microfibers ‘Whiskers’ Simple Used with tissue culture cells Microfibers or ‘whiskers’ that look like tiny needles are coated with hundreds of copies of the gene. Tissue culture cells and microfibers are placed in a solution and shaken vigorously The tiny whiskers stab the plant cells potentially delivering the new DNA into the nucleus without killing the cell

Electroporation Quick pulses of electricity open tiny pores in the walls of the plant cell The DNA is small enough to fit through the tiny pores. DNA is mixed in a solution with plant cells to allow the DNA to pass through the pores.

5. Backcross Breeding Once a successful transformation event has occurred transgenic plants are crossed with a high performing line to create a line with both high performance and the transgenic trait. The offspring are crossed back to the high performing line repeatedly to increase their performance while maintaining the transgenic trait.