Long-distance Auxin Transport Assay (Giel van Noorden)

 

This assay tests the transport of auxin from the shoot apex down to the root, using radioactively labeled auxin, H³-IAA. It measures how much gets loaded in the shoot and gets transported to the root.

 

1. Pour 15 cm round petri plates with Fahräeus medium.
2. Place pregerminated seedlings on plate, grow vertically in growth chamber.
3. Inoculate at 3 days. Put 10 ml drop per root. Run the drop along bottom of root. Want plants dryish, so no parafilm!
4. Start assay 24 hours after inoculation with rhizobium.
5. Add 2 ml drop of H³-IAA to plant apex – right between cotyledons. I plates are dryish, IAA stays on as a droplet. No parafilm! If drop touches agar, assay won’t work (H³-IAA will diffuse through agar!). Pull roots off if they are touching other roots, and lay straight on the agar.

 

Adding the drop of H³-IAA to the shoot apex.

 

The plants on the right have a 2 ml drop between the cotyledons, on the shoot apex.

 

6. After 3 hours, cut plant on a mat using special knife, with razor blades sandwiched between plexi glass tubes so that they are a known distance apart. Line top of knife up just below the cotyledons. There will be 8 segments, plus the root tip and shoot.
7. To keep from contaminating between samples, rinse knife between roots in ethanol and wipe mat with tissue in between roots.
8. Move cut roots segments each to a scintillation vial in scintillation fluid. The shoot and root tip will be placed in the last vial and counted, to confirm loading. Rinse forceps in ethanol in between each segment. It also helps to find root bits stuck to forceps.