PROTOCOL

SEED GERMINATION for M. truncatula

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

1. Soak seeds in 5 volumes of conc. H2S04 for 5-12 minutes with intermittent gentle vortexing. (The actual treatment time depends on the condition of the seeds, with 8 min. being a reasonable place to start).
2. Carefully decant as much of the acid as possible and add water to rinse seeds. (Addition of water to sulfuric acid generates heat, so it is important to decant as much of the acid as possible, and douse seeds with a large volume of water). Rinse seeds further 4-X with water.
3. Add 5 volumes of commercial grade bleach (~5% sodium hypo chlorite). Leave the seeds in bleach for 2-3 minutes. Under sterile conditions (e.g. laminar flow hood) decant the bleach and begin rinsing the seed using sterile diH20. Repeat the rinse 6 to 8 times. (Many people have eliminated the bleach step from the germination protocol).
4. Soak seed in sterile water 4-6 hours. Change water several times during this rinsing period. During this time, seed should begin to swell up from absorption of water. Spread seed on to moist filter paper (2-3 sheets thick) placed in a petri-plate. Seal petri-plate with parafilm and invert petriplates so that seed is hanging from the roof of the plate (seed is held in place by surface tension).
5. Cold treatment: place the seed at 4°C for 36-48 hrs to synchronize germination.
6. Room temperature incubation- remove cold treated to room temperature, in the dark for 12-24hrs. Seedlings germinate with roots hanging down from the roof of petri-dishes. Seedlings 1-2 cm in length are well suited for planting.