PROTOCOL

AEROPONIC CHAMBER for M. truncatula

(Cook lab-University of California, Davis; drcook@ucdavis.edu)

Caisson chamber:
30 gal. Rubber-Maid trash can
Plastic Type: polyproplylene
Remove handles and other hardware

Caisson top
Black lexan (plexiglass) top 3/8 thick, cut into circle of same size or slightly larger than trash can top. Modify the lid of the trash can by cutting out the top being sure to leave a small 1-2" lip. Attach the black lexan circle to the modified top with nylon screws, and seal the edges with silicone seal. Drill holes 3/16 inch diameter in the black lexan; we use a spacing of 1/2 inch, which allows growth of >1,000 plants/caisson.

Aeroponic generator:
Non-sonicating mist generators are necessary.

Model: 505 S Air Humidifier-Atomizer with float valve for direct water connection, 100-110V, 60 Hz, cat no. 10.0072.

Replacement motor sets cpl. for 505, 100-110V, 60 Hz, cat no. 22.1490.

Mr. H. S. Gamp
Export Sales Manager
Business Unit Defensor
AxAir Ltd. Talstr. 35-37
CH-8808 Pfaffikon, Switzerland
(f) 41-55-416-61-61 (o) 41-55-416-61-11

Purchasing through Switzerland will involve transfer of funds by wire to the AxAir bank account. Mr. Gamp can provide all the relevant information

The water trough supplied with the Defensor 505 has to be replaced with a plexiglass platform that will fit into the bottom of the trash can. This creates a larger reservoir for the medium. We made ours by cutting a circle from sturdy (>3/8 inch) plexiglass, and then cutting an 8 inch off-center hole through which the motor and motor stand (with legs removed) are inserted. The platform is mounted on two 6 inch legs made from 1 inch diameter plexiglass rods; the "wheel-hump" in the trash can serves as a third leg. Dichloromethane works best as a plexiglass adhesive, though chloroform works OK.

Wiring:
Remove the on-off switch and connect the wires directly. Change the wiring of the motor to move the plug point outside the tank and install an inline switch external to the caisson chamber. Use silica gel (General Electric's Silicone II brand) as sealant to seal the dome-shaped top to the motor casing, and seal all other openings through which moisture might get into the unit. Allow gel to dry overnight.

Nutrient medium (Lullien et al.):
Prepare stock solutions separately and autoclave. We make 100X stocks for the KPO4 and (the optional) NH4NO3 buffers and 1,000X stocks for the other components. For rapid induction of nodulation, start plants off in nitrate-free medium.

0.52 mM K2SO4
0.25 mM MgSO4
1 mM CaCl2
50 uM Na2EDTA
30 uM H3BO3
10 uM MnSO4
0.7 uM ZnSO4
0.2 uM CuSO4
1 uM Na2MoO4
0.04 uM CoCl2
50 uM FeSO4 (make fresh, 13.9mg/L of final medium)
5.5 mM KPO4 (1 liter of 100X pH 7.0 stock = 58.92 g K2HPO4 and 41.08 g KH2PO4).
5 mM NH4NO3 (we typically leave this out for nodulation experiments)

Depending on the tank and height of motor's platform you will require approximately 9-11 L per caisson. Add the stocks to about 8L of STERILE distilled water (although in the long run the tanks are not sterile). Add the FeSO4 and CaCl2 last, then bring up to volume with sterile water. Upon addition of CaCl, you will observe a precipitate. /p>

Preparing the caisson and loading seedlings.
Scrub throughly with soap, and rinse with water. Use 20% bleach to treat every possible component 30 min, followed by at least 3 rinses with water or until the bleach smell rinses out.

To the dry caisson top, attach saran wrap using Photomount as adhesive. (This provides a seal for the holes and support for the seedlings. If the saran wrap is not used, salt from the tank will be deposited on the hypocotyl-root junction and kill the seedlings). Using a thumbtack poke holes in the saran wrap. Load seedlings individually into these holes. For best results, seedlings should be germinated as described in the germination protocol, and loaded 24-36 hrs post germination. We inoculate seedlings 5 days after loading into the caisson with 5 ml of a 48 hr liquid culture of Rhizobium. Nodules on genotype A-17 (Jemalong) are first visible at 72 hours post inoculation using RmABS7M or near 84 hrs with Rm2011 or Rm1021.

The seedlings are extremely sensitive to low moisture. Load only those seedlings that are sure to extend through the caisson top into the mist chamber (to allow the root to act as a wick). After loading the top, spray a mist of water onto the seedlings and cover with the a transparent humidity retainer. (For this purpose we cut a second trash can to remove the top, and cover the remaining circular frame with saran wrap). The plants can be weaned away from high humidity once the roots are well established (4-5 days old). However, we usually leave the retainer in place. Remove condensation water on the inside surface of the retainer periodically. We usually mist the seedling tops each time the humidity retainer is removed; for example to examine the plants.