Griffitts:Seed scarification and germination: Difference between revisions
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** 70% [[Wikipedia:ethanol|ethanol]] is next to the scales | ** 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 [[Wikipedia:ethanol|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 Chlorox [[Wikipedia:bleach|bleach]] to substantially cover the seeds | * 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 [[Wikipedia:bleach|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 | ||
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* Remove the sulfuric acid | * Remove the sulfuric acid | ||
** Discard it into the sulfuric acid waste container in the fume hood | ** Discard it into the sulfuric acid waste container in the fume hood | ||
* Immediately rinse the seeds five times with dH<sub>2</sub> | * Immediately rinse the seeds five times with dH<sub>2</sub>O | ||
* Add 5 mL of bleach | * Add 5 mL of bleach | ||
* Place them back onto the shaker for 3 minutes | * Place them back onto the shaker for 3 minutes | ||
* Rinse 6–8 times with dH<sub>2</sub> | * Rinse 6–8 times with dH<sub>2</sub>O | ||
* Pour the seeds and some dH<sub>2</sub> | * Pour the seeds and some dH<sub>2</sub>O into small plates | ||
* Parafilm and store in the fridge 24 hours to allow imbibement | * Parafilm and store in the fridge 24 hours to allow imbibement | ||
** Be sure not to have excessive amounts of water in the plate | ** Be sure not to have excessive amounts of water in the plate | ||
| Line 111: | Line 111: | ||
* Remove the sulfuric acid | * Remove the sulfuric acid | ||
** Discard it into the sulfuric acid waste container in the fume hood | ** Discard it into the sulfuric acid waste container in the fume hood | ||
* Immediately rinse the seeds once with dH<sub>2</sub> | * Immediately rinse the seeds once with dH<sub>2</sub>O | ||
* Add 5 mL of bleach | * Add 5 mL of bleach | ||
* Place them back onto the shaker for 1 minute | * Place them back onto the shaker for 1 minute | ||
* Rinse five times with dH<sub>2</sub> | * Rinse five times with dH<sub>2</sub>O | ||
* Pour the seeds and some dH<sub>2</sub> | * Pour the seeds and some dH<sub>2</sub>O into small plates | ||
* Parafilm and store in the fridge 24 hours to allow imbibement | * Parafilm and store in the fridge 24 hours to allow imbibement | ||
** Be sure not to have excessive amounts of water in the plate | ** Be sure not to have excessive amounts of water in the plate | ||
* 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> | ||
Revision as of 07:50, 18 April 2008
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 Plus
- 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 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: some plants may take a little longer to germinate
| species | time | notes |
|---|---|---|
| arabica | 15 minutes | |
| constricta | 10 minutes | |
| disciformis | 10 minutes | |
| heyniana | 20 minutes | |
| italica | 10 minutes | |
| lesinsii | 10 minutes | |
| lupulina | 20 minutes | |
| polymorpha | 20 minutes | |
| praecox | 8 minutes | refrigerate in 15 mL dH2O for 36–48 hours |
| rugosa | 30 minutes | |
| tenoreana | 10 minutes | |
| truncatula A17 | 10 minutes |
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
