Griffitts:Seed scarification and germination: Difference between revisions
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{{Griffitts}} | <div class="noprint"> | ||
{{Template:Griffitts}} | |||
</div> | |||
==Alfalfa== | ==Alfalfa== | ||
Note: Alfalfa seeds don't need to be scarified<br> | |||
* Calculate the number of plants you need | * Calculate the number of plants you need | ||
* Weigh out ten times more than the number you need | * Weigh out ten times more than the number you need | ||
** Use GT13R | ** Use GT13R+ | ||
** 1.7 g is approximately 1000 seeds | ** 1.7 g is approximately 1000 seeds | ||
* Place in a smooth sterile flask | * Place in a smooth sterile flask | ||
* Add 70% [[Wikipedia:ethanol|ethanol]] to substantially cover the seeds | * Add 70% [[Wikipedia:ethanol|ethanol]] to substantially cover the seeds | ||
** 70% [[Wikipedia:ethanol|ethanol]] is next to the scales | |||
* Place on a shaker at room temperature at 150 rpm for 20 minutes | * Place on a shaker at room temperature at 150 rpm for 20 minutes | ||
* Decant the ethanol and wash the seeds once with sterile dH<sub>2</sub> | * Decant the [[Wikipedia:ethanol|ethanol]] and wash the seeds once with sterile dH<sub>2</sub>O | ||
* Add | * Add Chlorox [[Wikipedia:bleach|bleach]] to substantially cover the seeds | ||
* Place on a shaker at room temperature at 150 rpm for 20 minutes | * 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<sub>2</sub> | * Decant the [[Wikipedia:bleach|bleach]] and wash the seeds 4 to 5 times with sterile dH<sub>2</sub>O | ||
* Add sterile dH<sub>2</sub> | * Add sterile dH<sub>2</sub>O to substantially cover the seeds | ||
* Place on a shaker at room temperature at 150 rpm for at least 4 hours (preferably 8) | * 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<sub>2</sub> | ** It's a good idea to wash the seeds with sterile dH<sub>2</sub>O once during this time | ||
* Decant the sterile dH<sub>2</sub> | * Decant the sterile dH<sub>2</sub>O and add fresh sterile dH<sub>2</sub>O | ||
* Dump into 100×25 mm Petri dishes | * Dump into 100×25 mm Petri dishes | ||
** Seeds should be in a monolayer with a small amount of space between them | ** Seeds should be in a monolayer with a small amount of space between them | ||
Line 24: | Line 25: | ||
** Tip the plate and give it a few seconds to be sure to get all the excess water | ** Tip the plate and give it a few seconds to be sure to get all the excess water | ||
* Wrap plate(s) in [[Wikipedia:Parafilm|Parafilm]] | * Wrap plate(s) in [[Wikipedia:Parafilm|Parafilm]] | ||
* Store in a drawer for 24 hours | * Store upside-down in a drawer for 24 hours | ||
==Other Medics== | ==Other Medics== | ||
===Scarification=== | <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. | ||
* To each of the flasks add 5 mL of sulfuric acid (H<sub>2</sub>SO<sub>4</sub>, 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<sub>2</sub>O | |||
* Add 5 mL of bleach | |||
* Place them back onto the shaker for 3 minutes | |||
* Rinse 6–8 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 24–48 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<br> | |||
NOTE: '''Remember sterility!''' | |||
==Scarification times for different ''Medicago'' seedlings== | |||
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable" | |||
! width="90" style="background:#efefef;" | species | |||
! width="60" style="background:#efefef;" | abbr. | |||
! width="90" style="background:#efefef;" | time | |||
! width="300" style="background:#efefef;" | notes | |||
|- | |||
| ''arabica'' | |||
| <center>AR</center> | |||
| 15 minutes | |||
| | |||
|- | |||
| ''blancheana'' | |||
| <center>BL</center> | |||
| <10 minutes | |||
| 10 minutes gave 100% imbibition but 0% germination | |||
|- | |||
| ''ciliaris'' | |||
| <center>CI</center> | |||
| 10 minutes | |||
| | |||
|- | |||
| ''constricta'' | |||
| <center>CO</center> | |||
| 10 minutes | |||
| | |||
|- | |||
| ''disciformis'' | |||
| <center>DI</center> | |||
| 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'' | |||
| <center>HE</center> | |||
| 20 minutes | |||
| | |||
|- | |||
| ''intertexta'' | |||
| <center>IN</center> | |||
| 10 minutes | |||
| | |||
|- | |||
| ''italica'' | |||
| <center>IT</center> | |||
| 10 minutes | |||
| | |||
|- | |||
| ''laciniata'' | |||
| <center>LA</center> | |||
| 8 minutes | |||
| | |||
|- | |||
| ''lesinsii'' | |||
| <center>LE</center> | |||
| 10 minutes | |||
| | |||
|- | |||
| ''lupulina'' | |||
| <center>LU</center> | |||
| 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'' | |||
| <center>PO</center> | |||
| 20 minutes | |||
| | |||
|- | |||
| ''praecox'' | |||
| <center>PR</center> | |||
| 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'' | |||
| <center>RU</center> | |||
| 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'' | |||
| <center>TE</center> | |||
| 8 minutes | |||
| | |||
|- | |||
| ''truncatula'' | |||
| <center>TR</center> | |||
| 10 minutes | |||
| 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> | |||
<br> | |||
==''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<sub>2</sub>SO<sub>4</sub>, 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<sub>2</sub>O | |||
* Add 5 mL of bleach | |||
* Place them back onto the shaker for 1 minute | |||
* 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 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
- 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 | 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 (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