Growth and hybridization of Triphysaria in soil
Mix 50% (w/w) sand and 50% (w/w) Sunshine Mix #1 soil (Sun Gro Horticulture, Inc., Bellevue, WA).
Fill a 4” pot with the sand/soil mixture and sub-irrigate with 0.25 X Hoagland’s until saturated (this may take several hours). Sub-irrigation helps avoid compaction. Up to four seedlings may be placed in a single pot.
Plant several rye grass, Arabidopsis, Medicago, or other host seeds toward the center of each pot and cover with a ~0.5 cm layer of sand. Sprinkle lightly with water. Place pots (up to ten pots) in a perforated black plastic tray. Cover with a clear cover..
Lay three 10 ml plastic pipets in the bottom of a second, non-perforated tray. Make a single, small hole near each end of the bottom of this tray. Lay the perforated tray containing the pots on top of the pipette supports so that there is an approximately 1cm space between the top and bottom trays.
Place the tray in the controlled environment chamber, each end resting on a 1” x 1” x 12” block of wood. The elevation of the tray will facilitate drainage.
Water by sub-irrigation twice a week and monitor growth of the host plant. Remove plastic cover when seeds emerge.
After the host has emerged (1-2 weeks), transplant Triphysaria seedlings into pots. Use forceps to make a hole in the soil about 1-2 cm from the emerged host plant. Using the forceps, carefully remove a Triphysaria seedling from the germination or induction plate and place its roots into the hole. Avoid bringing clumps of agar along with the seedling. Use the forceps to fill the hole with soil and lightly compact .
Water the freshly transplanted seedling from above to increase contact between the soil and the roots. Label the seedling with a pedigree # (consult database for most current number) using a plastic tag/marker and Sharpie pen.
Transplanted seedlings should be hardened off over 2-3 weeks. Cover trays containing new transplants with clear plastic lids or create a plastic wrap dome (careful not to touch seedlings with the plastic). Every few days, lift the cover slightly to increase air-flow and remove cover when plants show visible signs of new growth.
Sub-irrigate bi-weekly with 0.25x Hoaglands solution. If fungal or algal growth becomes apparent, cut back on the nutrient irrigation and add more sand to the surface of the soil. Plants are very sensitive to drying and soggy soil. Try to maintain a “damp sponge” feeling soil water content.
Older plants may need to be staked using twist-ties and small wooden sticks.
Triphysaria has four anthers, two pairs found behind the style, hidden in the galea. Each anther will have a pollen sac at the top. Using the tip of a sharp scalpel, gently scrape the pollen sac and look for yellow-orange pollen grains. You might need to use magnifying glasses. Immature pollen sacs will not yield pollen and old, dessicated sacs will yield a white, grainy pollen (may or may not be viable).
Place the yellow-orange pollen on the stigma of the maternal plant.
Clean the scalpel with 95% ethanol after each pollination.
Use a paper tag to note the pedigree #’s of the plants used in each cross, the date, which plant was pollen donor/acceptor, and the number of flowers pollinated.
Triphysaria seedlings will develop flowers within 1-2 weeks of transplanting. Crosses may be made over the course of 6-12 weeks and seed capsules appear within 2-3 weeks of pollination.
Harvest seed capsules when they begin to brown and open at the tip. Each capsule may contain up to 25-30 seeds.
Store and dry seed capsules in paper envelopes for several weeks at room temperature (at 37°C to speed up the process). Clean seeds and count them, then store in well-labeled microfuge tubes at 4°C.
18 hour day and 6 hr night cycle at 20ºC and 16ºC respectively. Use fluorescent lights.
Relative humidity about 80%