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

• 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—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
  

Scarification times for different Medicago seedlings

species	time	notes
arabica	15 minutes
constricta	10 minutes
disciformis	10 minutes
heyniana	20 minutes
italica	10 minutes
lesinsii	10 minutes
lupulina	20 minutes
polymorpha	20 minutes
praecox	8 minutes
rugosa	30 minutes
tenoreana	10 minutes
truncatula	10 minutes	this works for all ecotypes

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—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