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

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!

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

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

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

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 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

NOTE: This protocol has not been tested. Other online protocols call for 10 minutes in sulfuric acid and no ethanol or bleach steps.