Biotech Home : Transgenic Plant Animation : Transcript
How to Make a Transgenic Plant
Narrator

The goal is to produce a new 'sweeter' variety of tomato. The 'sweet' gene will come from a wild, non-edible tomato plant. Plants are made up of cells, each surrounded by a cell wall. Each cell contains the genetic material, DNA.

The process begins when we cut a piece of stem from the wild tomato plant. Using a mortar and pestle, we grind up this piece of stem in liquid nitrogen to release the cell contents. The cell contents are then placed in a test tube containing reagents. After incubation, the test tube is centrifuged. During centrifugation, the cell contents separate into layers, among them, the DNA layer. To make it easier for you to visualize the process, we have colored the fluids in the test tubes. The DNA layer is harvested and transferred to a new test tube. The extraction process ends with this test tube containing a solution of wild tomato DNA. Next, a restriction enzyme, ECO R1, is added to the test tube, and mixed with the wild tomato DNA. Restriction enzymes cut at specific DNA sites along the tomato DNA sequence. ECO R1 cuts this tomato DNA sequence, creating sticky ends. Now, we've completed the preparation of the wild tomato DNA containing the 'sweet' gene.

Purified bacterial plasmids are placed in a new test tube. Plasmids are small pieces of circular DNA that are extra-chromosomal. These plasmids come from the bacteria Escherichia coli and are a tool used by scientists to transfer DNA. ECO R1 is added to the test tube and the solution is mixed.

ECO R1 recognizes and cuts plasmid DNA at the same place in the sequence as previously demonstrated in tomato DNA, creating complementary sticky ends. This completes the preparation of the plasmid DNA.

The next step is creation of recombinant DNA. Tomato and plasmid DNA preparations are mixed with ligase, to seal the sticky ends together. In a new test tube, the tomato-plasmid DNA complex is incubated with bacteria. After incubation, bacteria incorporate one of three possible outcomes. One possible outcome is that tomato DNA simply reanneals to itself. This does not yield recombinant DNA. A second possible outcome is that bacterial plasmid DNA simply reanneals to itself. This also does not yield recombinant DNA. In a third possible outcome, complementary sticky ends of the tomato DNA anneal with the bacterial plasmid DNA, creating recombinant DNA.

A bacterium will incorporate one of these three DNA outcomes. To screen for the recombinant tomato-plasmid DNA, bacteria are grown on Petri plates containing selective nutrient media. The bacterial colony containing the 'sweeter' gene, the gene we are interested in, is identified. This bacterial colony is transferred to an individual Petri plate and incubated to maximize bacterial growth. The 'sweeter' gene DNA is separated from the bacteria and purified. This completes the preparation of 'sweeter' gene DNA.

The next step is to transfer the sweet taste to edible tomato plants. Microscopic gold particles are coated with the 'sweeter' gene DNA.

A leaf from a non-sweet tomato plant is cultured sterilely on a Petri plate. The plate goes into a biolistic particle delivery system. Gold particles, coated with the recombinant DNA, are accelerated by air pressure and shot into the leaf. Microscopic gold particles enter leaf cells. The recombinant 'sweeter' gene DNA dissolves away from the gold particles and integrates into leaf-cell chromosomes of the non-sweet tomato plant. The leaf cells with the 'sweeter' gene DNA are transformed.

These leaves are placed in a new environment that encourages the transformed cells to regenerate into transgenic tomato plants. Those plants will fulfill our goal of producing sweeter tomatoes.