Procedure Outline

Notes and References

M truncatula Seed Sterilization and Imbibition

Add counted seeds to sterile Falcon tube

Use 15 ml Falcon tube for 200 seeds or less; 50 ml tube for 200-300 seeds.

Protocol outlined here works for both sterile seedling production (used for transformation experiments) and pot growth (used for seed collection).  For root transformation, start with 24-36 seeds per construct being tested. We generally want to analyze 16 transgenic roots.

Scarify with concentrated H₂SO₄

Completely cover seeds with concentrated H₂SO₄ (~2 ml) and mix well to ensure all seeds exposed to the acid.

Incubate 6-7 min.

In laminar hood or equivalent sterile space, remove H₂SO₄ with sterile pipet and dispose of appropriately.  Wash seeds 5x using 10-12 ml sterile deionized H₂O for each wash. Mix the tube several times during each wash. During first wash, disperse seeds quickly after adding the water to prevent a rapid temperature increase.  Water can be decanted from seeds.

Wearing a lab coat while doing this is a good idea.  Use a sterile 1 ml platic pipet tip to mix acid and seeds.  Seeds will stick to plastic (sides of tube, etc.) and need to tapped down to bottom of tube.  Six minutes is minimum time necessary to break through waxy cuticle.

This and all subsequent steps need to be performed under sterile conditions for seedling use in transformation.  Remove as much H₂SO₄ before adding water since the acid/water mixture will generate large amounts of heat. 

Sterilize in commercially available bleach (sodium hypochlorite)

Add 2-3 ml commercial bleach with sterile pipet so that seeds are covered and gently mix for 2 minutes. 

Remove bleach and wash in sterile deionized H₂O 5x as described previously. 

Keep working amount of bleach in a covered bottle instead of using large jug.  Seeds will float so decant H₂O carefully or remove with sterile pipet.

Imbibe seeds

Add 10 ml of sterile deionized H₂O and cover tube in foil (dark treatment).  Place on orbital or rocking platform (~ 50 rpm or equivalent) for 2 hrs, changing H₂O every 30 min.

If growing plants in pots for seed production, go to M. truncatula Growth for Seeds following this step.  If growing plants for transformation, continue to next step.

Cold treat seeds

Store covered tube with seeds and H₂O at ~4^oC for 48-96 hours. Alternatively, plate seeds on sterile 1% agar (see below) and store inverted plates wrapped in aluminum foil in refrigerator for 24-48 hours.

Temperature of typical refrigerator is fine.  Seeds can be treated at 4^oC for 1-7 days, but germination may be poorer at the two extremes. Cold treatment will result in greater and more synchronized germination.  Proceed next to M. truncatula Seedling  Transformation.

Total time:

2.5 hours (30 minutes for scarification and sterilization; 2 hours for imbibition)