Induction of Triphysaria seedlings

Transfer seedlings from germination plates:

Prepare induction plates containing 0.25x Hoaglands, 1.0% (w/v) sucrose and 1.0% (w/v) Phytagar (GibcoBRL Life Technologies, Rockville, MD) in square petri dishes.

Clean laminar flow hood with 70% ethanol.

Obtain a germination plate with two to three week old seedlings from the 16C growth chamber room. Remove the lid of a germination plate in the flow hood only, avoid touching the edges of the plate. Sterilize the tips of the forceps by emerging in 95% ethanol, flaming, and cool the tips in the agar media. Gently remove a Triphysaria seedling from the germination plate by grasping the stem of the plant and pulling upward. *Do not mash the stem!

Place the seedling on the surface of the agar in the induction plate, about 0.5 cm from the top of the plate. Five to ten plants should be evenly spaced, in a parallel arrangement, across the surface of the media. If necessary, use the forceps to gently increase contact of the seedling root system with the surface of the agar. It is important that the roots grow down the surface of the induction plate.

Re-flame the forceps whenever a non-sterile surface is touched and/or after each induction plate is completed. Once all seedlings are transferred, label the plates and seal the edges with micropore tape (3M Health Care, St. Paul, MN).

Place sealed induction plates between the prongs of plastic test tube racks, orienting seedlings for growth (top up, roots down) and check that plates are facing the same direction. Move racks to the 22C growth room under a 16 hour light regimen. Racks should be stored at an approximate 35 angle, with the surface of the agar tilted up toward the light, facilitating root growth along the surface of the agar.

Root tip induction:

Triphysaria seedlings can be induced for haustoria formation after 5-7 days of growth on the induction plates. Unwrap plates, either in the laminar flow hood or on a clean bench top and use a sterile Pasteur pipette to add 2 ml of filter-sterilized inducer to each plate. Gently swirl or shake each plate to ensure that all root tips are contacted by inducer.

Allow induced plates to sit horizontally in the hood or on the bench for two hours before re-sealing with micropore tape (allows the inducer to sink in and facilitates contact of the roots with the surface of the agar). Re-place plates in the racks and return the racks to the 22C growth room.

After 24 hours, look at the root tips under a dissecting microscope at 0.6-4.0 x magnification. Score the plants as induced/responders (with haustoria) or un-induced/non-responders (without haustoria). Haustoria develop at the root tip and are characterized by localized swelling and root hair proliferation.