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AGROBACTERIUM: NATURE'S PLANT GENETIC ENGINEER

Adapted from a lesson by Dr.Steve Warshaw, Durham, NC
Submitted by Thea Sinclair


Background

The common soil bacterium Agrobacterium tumefaciens causes crown gall disease in many dicotyledonous plants by inserting a portion of its DNA into the genome of the host plant. As such, A. tumefaciens is a natural plant engineer.

Chemicals secreted from freshly wounded plant tissue attract A. tumefaciens to the wound site. The bacterium binds to the walls of the broken cells and enters these now dead cells. From there it injects a segment of its tumor-inducing plasmid DNA into adjacent living plant cells.

Before plant genetic engineers can exploit the natural genetic engineering capabilities of A. tumefaciens, they must first "disarm" the plasmid by removing its tumor-causing genes. Researchers then replace these genes with foreign gene(s) they wish to transfer to the plant. The plasmid containing the new gene(s) is returned to the bacterium, which is grown in culture. Pieces of plants to be engineered are then placed in solution with the genetically altered A. tumefaciens.

By 1994, 20 important agricultural plants had been successfully engineered using A. tumefaciens.


Summary of Activity

Part 1--In this lab students will wound the stems and leaves of a kalanchoe plant and inoculate them with A. tumefaciens. (Teachers Note: Some states require a permit to order this soil bacterium, including GA, TN, AL, and HI) Kalanchoe plants are commonly sold in plant shops as the pregnant plant, and are also available from Carolina Biological Supply. Classes can also try to have tumors form in sunflowers, bean plants and other local plants. Monocots do not seem to respond to infection. Tumors take 2-6 weeks to form.

Part 2--Students make extracts from plants that seem to avoid tumor growth, or from plants known to have antitumor properties such as the Pacific or Common yew. Inject 25 plants with the extract, then infect with A. tumefaciens. In a control set of 25 plants, just infect with A. tumefaciens. Compare the percentage of plants that grow tumors in each group. These results can be posted in Access Excellence and plants from various parts of the country can be compared.


Materials
Each lab team will need:
  • sterile toothpicks (Use autoclave or pressure cooker to sterilize toothpicks wrapped in aluminum foil)
  • a small test tube containing 5-10 ml sterile water
  • 2 small Kalanchoe plants, or other dicotyledenous potted plants (but not brassica i.e. Fast Plants)
  • an agar slant culture of A. tumefaciens (one for entire class is enough)
  • "Lysol water" (30 ml Lysol in 4 l water) or 15% chlorox solution
  • 1 inoculating needle
  • alcohol or Bunsen burner
  • alcohol swabs or rubbing alcohol and cotton balls

Procedure:
  1. Wipe down the lab table with lysol water or other disinfectant (15% chlorox works well)

  2. Use an alcohol swab to wipe the plant surface to be wounded. This can be the stem or the upper leaf surface.

  3. Pick up a sterile toothpick by touching only one end. Pierce the stem of the plant with the other end; the toothpick should go all the way through the stem.

  4. Remove the cap from the slant tube of A. tumefaciens and flame the tube's mouth. Touch a new sterile toothpick to the culture. Flame the tube again and recap. Put the toothpick into the holes made previously in the plant.

  5. In half the plants follow step 4. In the other half of the plants, use a sterile inoculating needle to apply a small amount of sterile water to the wound. Flame the needle and reflame the mouth of the test tube before replacing the cap.

  6. Keep the plants in the light and water periodically. Allow 2-6 weeks for galls to form.


Student Data Sheet

Data Sheet should include:

  • Date
  • Type of plant
  • Observation(Tumor Yes/no)

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