Griffitts:Seed scarification and germination

Alfalfa
Note: Alfalfa seeds don't need to be scarified
 * Calculate the number of plants you need
 * Weigh out ten times more than the number you need
 * Use GT13R+
 * 1.7 g is approximately 1000 seeds
 * Place in a smooth sterile flask
 * Add 70% ethanol to substantially cover the seeds
 * 70% ethanol is next to the scales
 * Place on a shaker at room temperature at 150 rpm for 20 minutes
 * Decant the ethanol and wash the seeds once with sterile dH2O
 * Add Chlorox bleach to substantially cover the seeds
 * Place on a shaker at room temperature at 150 rpm for 20 minutes
 * Decant the bleach and wash the seeds 4 to 5 times with sterile dH2O
 * Add sterile dH2O to substantially cover the seeds
 * Place on a shaker at room temperature at 150 rpm for at least 4 hours (preferably 8)
 * It's a good idea to wash the seeds with sterile dH2O once during this time
 * Decant the sterile dH2O and add fresh sterile dH2O
 * Dump into 100×25 mm Petri dishes
 * Seeds should be in a monolayer with a small amount of space between them
 * Pull off excess water with a pipet
 * Tip the plate and give it a few seconds to be sure to get all the excess water
 * Wrap plate(s) in Parafilm
 * Store upside-down in a drawer for 24 hours

Other Medics
NOTE: Remember sterility!
 * Place twice as many seeds as you wish to plant into a 50 mL Erlenmeyer flask that has been covered with foil and autoclaved.
 * To each of the flasks add 5 mL of sulfuric acid (H2SO4, in the fume hood&mdash;be careful!)
 * Place on the shaker for the appropriate time (see table below)
 * Remove the sulfuric acid
 * Discard it into the sulfuric acid waste container in the fume hood
 * Immediately rinse the seeds five times with dH2O
 * Add 5 mL of bleach
 * Place them back onto the shaker for 3 minutes
 * Rinse 6–8 times with dH2O
 * Pour the seeds and some dH2O into small plates
 * Parafilm and store in the fridge 24–48 hours to allow imbibement
 * Be sure not to have excessive amounts of water in the plate
 * Remove the seeds from the fridge and remove the water
 * Parafilm the mini-plates, invert and place at room temperature in the dark for 24–48 hours

Lotus japonicus

 * Place twice as many seeds as you wish to plant into a 50 mL Erlenmeyer flask that has been covered with foil and autoclaved.
 * To each of the flasks add 5 mL of sulfuric acid (H2SO4, in the fume hood&mdash;be careful!)
 * Place on the shaker for 20 minutes
 * Remove the sulfuric acid
 * Discard it into the sulfuric acid waste container in the fume hood
 * Immediately rinse the seeds once with dH2O
 * Add 5 mL of bleach
 * Place them back onto the shaker for 1 minute
 * Rinse five times with dH2O
 * Pour the seeds and some dH2O into small plates
 * Parafilm and store in the fridge 24 hours to allow imbibement
 * Be sure not to have excessive amounts of water in the plate
 * Remove the seeds from the fridge and remove the water
 * Parafilm the mini-plates, invert and place at room temperature in the dark for one day

Melilotus spp.
NOTE: This protocol has not been tested. Other online protocols call for 45 minutes in bleach.
 * Place twice as many seeds as you wish to plant into a 50 mL Erlenmeyer flask that has been covered with foil and autoclaved.
 * To each of the flasks add 5 mL of ethanol
 * Place on the shaker for 5 minutes
 * Dump off the ethanol
 * Immediately rinse the seeds once with dH2O
 * Add 5 mL of bleach
 * Place them back onto the shaker for 30 minutes
 * Rinse five times with dH2O
 * Pour the seeds and some dH2O into small plates
 * Parafilm and store in the fridge 24 hours to allow imbibement
 * Be sure not to have excessive amounts of water in the plate
 * Remove the seeds from the fridge and remove the water
 * Parafilm the mini-plates, invert and place at room temperature in the dark for one day

Trifolium spp.
NOTE: This protocol has not been tested. Other online protocols call for 10 minutes in sulfuric acid and no ethanol or bleach steps.
 * Place twice as many seeds as you wish to plant into a 50 mL Erlenmeyer flask that has been covered with foil and autoclaved.
 * To each of the flasks add 5 mL of ethanol
 * Place on the shaker for 1 minute
 * Dump off the ethanol
 * Immediately rinse the seeds once with dH2O
 * Add 5 mL of bleach
 * Place them back onto the shaker for 10 minutes
 * Rinse five times with dH2O
 * Pour the seeds and some dH2O into small plates
 * Parafilm and store in the fridge 24 hours to allow imbibement
 * Be sure not to have excessive amounts of water in the plate
 * Remove the seeds from the fridge and remove the water
 * Parafilm the mini-plates, invert and place at room temperature in the dark for one day