Sterile Medicago seeds

Sulfuric Acid Scarification

1. Scarify seeds in small flasks with enough concentrated sulfuric acid to cover for 5-12 minutes
(we use the shorter time for sensitive mutants) — seeds are ready when small black spots are visible
2. Decant acid into a hazardous waste bottle
3. Rinse seeds with sterile water 4-5 times, putting first rinse in the hazardous waste bottle
4. Surface sterilize in concentrated Clorox + 1 drop Tween for 3 minutes
5. Add at least an equal volume of water
6. Decant liquid into sink with running water
7. Rinse with sterile water 4-5 times
8. Imbibe at room temp on an orbital shaker for 3-6 hours, changing sterile water several times
9. At this point seeds can plated for germination or can be stored in water at 4oC for 24-72 hours to improve germination, especially for recently harvested seed). Can also perform second sterilization with 10% bleach solution (make fresh) for 1 minute.
NOTES : Several rinses are needed to remove the Clorox, but it is possible to use a shorter time or even skip imbibing on the shaker, especially if seeds are to be put in the cold.

Modified from Sharon Long’s lab:¬†http://cmgm.stanford.edu/biology/long/files/protocols/Germination.pdf