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
  

NOTE: Remember sterility!

Scarification times for different Medicago seedlings

species	abbr.	time	notes
arabica	AR	15 minutes
blancheana	BL	<10 minutes	10 minutes gave 100% imbibition but 0% germination
ciliaris	CI	10 minutes
constricta	CO	10 minutes
disciformis	DI	10 minutes
doliata	DO	>10 minutes	10 minutes gave <20% germination
granadensis	GR	>10 minutes	10 minutes gave <20% germination
heyniana	HE	20 minutes
intertexta	IN	10 minutes
italica	IT	10 minutes
laciniata	LA	8 minutes
lesinsii	LE	10 minutes
lupulina	LU	20 minutes	imbibe for 2 days and germinate for 2 days
minima	MI	<8 minutes	8 minutes gave 100% imbibition, but <30% germination
noeana	NO	10 minutes
orbicularis	OR	>10 minutes	10 minutes gave <10% germination
polymorpha	PO	20 minutes
praecox	PR	8 minutes
radiata	RA	>15 minutes	15 minutes gave <50% imbibition and <30% germination
rigiduloides	RI	<10 minutes	10 minutes gave 100% imbibition, but 0% germination
rotata	RO	>10 minutes	10 minutes gave <10% germination
rugosa	RU	30 minutes
sauvagei	SV	<10 minutes	10 minutes gave 100% imbibition, but <20% germination
scutellata	SC	<10 minutes	10 minutes gave 100% imbibition, but <25% germination
secundiflora	SE	>10 minutes	10 minutes gave <10% germination
soleirolii	SO	<10 minutes	10 minutes gave 100% imbibition, but <30% germination
tenoreana	TE	8 minutes
truncatula	TR	10 minutes	this works for all ecotypes imbibe for 2 days and germinate for 2 days
turbinata	TU	<10 minutes	10 minutes gave 100% imbibition, but <5% germination

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
  

Melilotus spp.

NOTE: When tested (once) this protocol had nearly 100% imbibition but <10% germination. 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 24–48 hours
  

Trifolium spp.

NOTE: When tested (once) this protocol had nearly 100% imbibition (for T. hybridum, T. pratense, and T. repens) but <10% germination. 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 24–48 hours