Griffitts:Seed scarification and germination: Difference between revisions

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==Other Medics==
==Other Medics==
<div class="noprint">[[Image:Griffitts-Medicago seeds.JPG|right|thumb|Medicago seeds after crushing the pod.]]</div>
* Place twice as many seeds as you wish to plant into a 50 mL Erlenmeyer flask that has been covered with foil and autoclaved.  
* 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<sub>2</sub>SO<sub>4</sub>, in the fume hood&mdash;be careful!)
* To each of the flasks add 5 mL of sulfuric acid (H<sub>2</sub>SO<sub>4</sub>, in the fume hood&mdash;be careful!)
Line 42: Line 43:
* Remove the seeds from the fridge and remove the water
* 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&ndash;48 hours<br>
* Parafilm the mini-plates, invert and place at room temperature in the dark for 24&ndash;48 hours<br>
<br>
NOTE: '''Remember sterility!'''
<br>
 
<br>
==Scarification times for different ''Medicago'' seedlings==
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable"
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable"
|+'''Scarification times for different ''Medicago'' seedlings'''
|-
! width="90" style="background:#efefef;" | species
! width="90" style="background:#efefef;" | species
! width="60" style="background:#efefef;" | abbr.
! width="90" style="background:#efefef;" | time
! width="90" style="background:#efefef;" | time
! width="300" style="background:#efefef;" | notes
! width="300" style="background:#efefef;" | notes
|-
|-
| ''arabica''
| ''arabica''
| <center>AR</center>
| 15 minutes
| 15 minutes
|  
|  
|-
| ''blancheana''
| <center>BL</center>
| <10 minutes
| 10 minutes gave 100% imbibition but 0% germination
|-
| ''ciliaris''
| <center>CI</center>
| 10 minutes
|
|-
|-
| ''constricta''
| ''constricta''
| <center>CO</center>
| 10 minutes
| 10 minutes
|  
|  
|-
|-
| ''disciformis''
| ''disciformis''
| <center>DI</center>
| 10 minutes
| 10 minutes
|  
|  
|-
| ''doliata''
| <center>DO</center>
| >10 minutes
| 10 minutes gave <20% germination
|-
| ''granadensis''
| <center>GR</center>
| >10 minutes
| 10 minutes gave <20% germination
|-
|-
| ''heyniana''
| ''heyniana''
| <center>HE</center>
| 20 minutes
| 20 minutes
|
|-
| ''intertexta''
| <center>IN</center>
| 10 minutes
|  
|  
|-
|-
| ''italica''
| ''italica''
| <center>IT</center>
| 10 minutes
| 10 minutes
|
|-
| ''laciniata''
| <center>LA</center>
| 8 minutes
|  
|  
|-
|-
| ''lesinsii''
| ''lesinsii''
| <center>LE</center>
| 10 minutes
| 10 minutes
|  
|  
|-
|-
| ''lupulina''
| ''lupulina''
| <center>LU</center>
| 20 minutes
| 20 minutes
| imbibe for 2 days and germinate for 2 days
|-
| ''minima''
| <center>MI</center>
| <8 minutes
| 8 minutes gave 100% imbibition, but <30% germination
|-
| ''noeana''
| <center>NO</center>
| 10 minutes
|  
|  
|-
| ''orbicularis''
| <center>OR</center>
| >10 minutes
| 10 minutes gave <10% germination
|-
|-
| ''polymorpha''
| ''polymorpha''
| <center>PO</center>
| 20 minutes
| 20 minutes
|  
|  
|-
|-
| ''praecox''
| ''praecox''
| <center>PR</center>
| 8 minutes
| 8 minutes
|  
|  
|-
| ''radiata''
| <center>RA</center>
| >15 minutes
| 15 minutes gave <50% imbibition and <30% germination
|-
| ''rigiduloides''
| <center>RI</center>
| <10 minutes
| 10 minutes gave 100% imbibition, but 0% germination
|-
| ''rotata''
| <center>RO</center>
| >10 minutes
| 10 minutes gave <10% germination
|-
|-
| ''rugosa''
| ''rugosa''
| <center>RU</center>
| 30 minutes
| 30 minutes
|  
|  
|-
| ''sauvagei''
| <center>SV</center>
| <10 minutes
| 10 minutes gave 100% imbibition, but <20% germination
|-
| ''scutellata''
| <center>SC</center>
| <10 minutes
| 10 minutes gave 100% imbibition, but <25% germination
|-
| ''secundiflora''
| <center>SE</center>
| >10 minutes
| 10 minutes gave <10% germination
|-
| ''soleirolii''
| <center>SO</center>
| <10 minutes
| 10 minutes gave 100% imbibition, but <30% germination
|-
|-
| ''tenoreana''
| ''tenoreana''
| 10 minutes
| <center>TE</center>
| 8 minutes
|  
|  
|-
|-
| ''truncatula''
| ''truncatula''
| <center>TR</center>
| 10 minutes
| 10 minutes
| this works for all ecotypes
| this works for all ecotypes<br>imbibe for 2 days and germinate for 2 days
|-
| ''turbinata''
| <center>TU</center>
| <10 minutes
| 10 minutes gave 100% imbibition, but <5% germination
|}
|}
<br>
<br>
Line 119: Line 216:
* Remove the seeds from the fridge and remove the water
* 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<br>
* Parafilm the mini-plates, invert and place at room temperature in the dark for one day<br>
==''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<sub>2</sub>O
* Add 5 mL of bleach
* Place them back onto the shaker for 30 minutes
* Rinse five times with dH<sub>2</sub>O
* Pour the seeds and some dH<sub>2</sub>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<br>
==''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<sub>2</sub>O
* Add 5 mL of bleach
* Place them back onto the shaker for 10 minutes
* Rinse five times with dH<sub>2</sub>O
* Pour the seeds and some dH<sub>2</sub>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<br>

Latest revision as of 10:46, 22 July 2011


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
  • 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.
  • 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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