SEED GERMINATION
for Medicago truncatula

(Cook lab-U.C. Davis; drcook@ucdavis.edu)

1. Seed storage:

M. truncatula seeds maintain their viability best when stored in their pods at room temperature. Once removed from the fruits, seeds should be useds within one month. For a protocol to remove seeds from pods efficiently (and painlessly), see the protocol for construction of a seed mill.

2. Acid scarification:

Soak seeds in 5 volumes of conc. H₂S0₄for 5-10 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). 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. Surface sterilization:

Add 5 volumes of commercial grade bleach (~5% sodium hypochlorite). Leave the seeds in bleach for 3 minutes. Under sterile conditions (e.g. laminar flow hood) decant the bleach and begin rinsing the seed using sterile distilled H₂O. Repeat the rinse 6 to 8 times.

4. Cold treatment:

Place the seed at 4°C for 36 hrs in about 3 volumes of standing sterile water. During this period, replacement of water with pre-chilled sterile water (2 to 3 times) will help subsequent germination.

5. Germination:

Rinse the seed with sterile deionized water at room temperature, 6 to 8 times. During rinsing, shake seed very gently on a rotary shaker being careful not to damage the radicle.

For small batches of seed (up to 300), we germinate seed in petri plates. Transfer treated seed to a petri plate and drain most of the water, leaving just enough prevent desiccation during germination. Wrap the petri plate in parafilm and invert so that the seeds adhere to the top of the plate (this will give very uniform seedling growth, with straight roots that are ideal for loading into aeroponic chambers). Place seed in a dark cabinet at room temp. Seed will be germinated in 12 to 24 hrs.