Griffitts:Seed scarification and germination

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Contents

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

Medicago seeds after crushing the pod.
Medicago seeds after crushing the pod.
  • 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
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