Griffitts:Seed scarification and germination
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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 dH_{2}O
- 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 dH_{2}O
- Add sterile dH_{2}O 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 dH_{2}O once during this time
- Decant the sterile dH_{2}O and add fresh sterile dH_{2}O
- 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 (H_{2}SO_{4}, 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 dH_{2}O
- Add 5 mL of bleach
- Place them back onto the shaker for 3 minutes
- Rinse 6–8 times with dH_{2}O
- Pour the seeds and some dH_{2}O 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 | 15 minutes | ||
blancheana | <10 minutes | 10 minutes gave 100% imbibition but 0% germination | |
ciliaris | 10 minutes | ||
constricta | 10 minutes | ||
disciformis | 10 minutes | ||
doliata | >10 minutes | 10 minutes gave <20% germination | |
granadensis | >10 minutes | 10 minutes gave <20% germination | |
heyniana | 20 minutes | ||
intertexta | 10 minutes | ||
italica | 10 minutes | ||
laciniata | 8 minutes | ||
lesinsii | 10 minutes | ||
lupulina | 20 minutes | imbibe for 2 days and germinate for 2 days | |
minima | <8 minutes | 8 minutes gave 100% imbibition, but <30% germination | |
noeana | 10 minutes | ||
orbicularis | >10 minutes | 10 minutes gave <10% germination | |
polymorpha | 20 minutes | ||
praecox | 8 minutes | ||
radiata | >15 minutes | 15 minutes gave <50% imbibition and <30% germination | |
rigiduloides | <10 minutes | 10 minutes gave 100% imbibition, but 0% germination | |
rotata | >10 minutes | 10 minutes gave <10% germination | |
rugosa | 30 minutes | ||
sauvagei | <10 minutes | 10 minutes gave 100% imbibition, but <20% germination | |
scutellata | <10 minutes | 10 minutes gave 100% imbibition, but <25% germination | |
secundiflora | >10 minutes | 10 minutes gave <10% germination | |
soleirolii | <10 minutes | 10 minutes gave 100% imbibition, but <30% germination | |
tenoreana | 8 minutes | ||
truncatula | 10 minutes | this works for all ecotypes imbibe for 2 days and germinate for 2 days | |
turbinata | <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 (H_{2}SO_{4}, 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 dH_{2}O
- Add 5 mL of bleach
- Place them back onto the shaker for 1 minute
- Rinse five times with dH_{2}O
- Pour the seeds and some dH_{2}O 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 dH_{2}O
- Add 5 mL of bleach
- Place them back onto the shaker for 30 minutes
- Rinse five times with dH_{2}O
- Pour the seeds and some dH_{2}O 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 dH_{2}O
- Add 5 mL of bleach
- Place them back onto the shaker for 10 minutes
- Rinse five times with dH_{2}O
- Pour the seeds and some dH_{2}O 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