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Plant Detectives Manual: a research-led approach for teaching plant science

Appendix F: Seed sterilisation and plating

This procedure is a guide for seed sterilisation and plating on MS agar.

F.1) Materials

  1. 70% (v/v) ethanol (freshly prepared)
  2. 70% (v/v) ethanol in spray bottle
  3. bleach (commercial)
  4. laminar flow or chemical hood
  5. microcentrifuge
  6. MS agar
  7. P1000 micropipette
  8. P1000 micropipette tips
  9. parafilm strips (transversal cut, 2-cm wide)
  10. Petri dishes
  11. scale
  12. scissors
  13. sterile Pasteur pipette
  14. sterile Petri dishes; square Petri dishes allow for sowing more seeds
  15. sterile water
  16. vortex
  17. Tween 20
  18. water bath or oven to keep melted MS agar

F.2) Solutions

Bleach/Tween solution

 

 

 

 

25% (v/v) bleach

 

 

 

 

0.005% (v/v) Tween 20 (5 microlitres (µl) in 1 millilitre (ml))8

 

 

 

 

70% (v/v) ethanol

 

 

 

 

0.1% (w/v) agar (in water, autoclaved)

 

 

 

 

Sterile water (autoclaved)

 

 

 

 

Reagent for growing medium (MS agar)

Cat#

[initial]

[final]

Amount

Murashige – Skoog salts mixture

Sigma, M5524

 

4.3 grams (g) L–1

4.3 g

MS vitamin solution

Sigma, M3900

1,000X

1X

1 ml

agar

Sigma, A1296

 

0.7%

7 g

Bring it up to 900 ml, bring it up to pH 5.65–5.80 with 10M KOH

F.3) Procedure

 

Pouring of plates

Materials

Method

Water bath or oven

Place autoclaved MS agar in 65 ºC water or 45 ºC drying oven after removing from autoclave. Use it before it solidifies.

Laminar flow

Turn on laminar flow or chemical hood. Pouring of plates and seed sterilisation must be done in a sterile environment.

70% (v/v) ethanol in spray bottle

Spray laminar flow bench and walls with 70% (v/v) ethanol and wipe with paper towel.

Petri dishes, scissors

Spray a new bag of Petri dishes with 70% (v/v) ethanol before placing into the chamber. Open the bag once inside the laminar flow.

MS agar

Place approximately 15–20 ml of the MS agar per plate, place the lid half way, and let it cool for one hour at room temperature. It is important that cooling is done with open lids to remove excess moisture. Remember that plates will be stored at 4 ºC later on and this will cause water vapor to condense, resulting in water droplets forming on the inside of the plates, preventing light diffusion and increasing chances of contamination.

 

Seed sterilisation using bleach solution and plating

Materials

Method

Scale, seeds, spin

Weigh approximately 20–30 milligrams (mg) of seeds and place them in ‘non-sterile’ 2 ml Eppendorf tubes.

70% ethanol, vortex

Add 1 ml 70% ethanol9, vortex, incubate five minutes, spin briefly to pellet the seeds and discard supernatant.

Laminar flow

The next steps have to be performed in the laminar flow.

Sterile water, bleach

Add 750 µl of sterile water + 1 µl Tween 20 + 250 µl bleach (25% (v/v) final concentration) and incubate for three minutes10.

Sterile water

Spin briefly, wash three times in 1 ml sterile water, and resuspend in 2 ml sterile 0.1% agar. You can let it decant by gravity but a quick spin (which could be done outside the laminar flow) will hasten the procedure and create a more compact seed pellet.

Sterile Pasteur pipette

Seeds are ready to be placed on the MS agar using a long-neck glass Pasteur pipette or regular pipette. NOTE: the FINAL seed density is important to dispense individual seeds and it needs to be optimised. As a guide, using 25 mg of seeds, resuspend in 2 ml sterile 0.1% agar and use a sterile glass Pasteur pipette to evenly distribute the seeds on plates.

 

 


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