General protocol and scenario

Biological materials:

Lens culinaris (cv Anicia, AOC Verte du Berry, CIBELE company, France) seeds

Lentil seeds will be flown on the International Space Station (ISS) and will be installed in the EMCS facility:

Scenario:

Germination will be induced by seed hydration in microgravity.

After a germination period in microgravity, different gravity levels will be applied to the samples for different time periods.

The experiment will investigate two gravity levels (0.01g and 2g). However, because of the position of the seeds on the centrifuge, there will be a gravity-gradient within each culture chamber, the lowest g-value perceived by the seeds being 0.005g, the highest being 2g (as illustrated below).

Run #1:

  • 4 Experimental Unit (EUE) will be centrifuged at 0.01g for 9 hours, after 21 hours of growth in microgravity
  • 4 EUEs will be grown in microgravity for 30 hours. (control samples)

Run #2:

  • 4 EUEs will be centrifuged for 15 minutes at 2g
  • 4 EUEs will be centrifuged for 5 minutes at 2g

run2

 

Fixatives:

  1. Fixative 1: Glutaraldehyde 4% in potassium phosphate buffer
  2. Fixative 2: Paraformaldehyde 3% + glutaraldehyde 0.5% in potassium phosphate
    buffer
  3. Fixative 3: Paraformaldehyde 2% + glutaraldehyde 0.5% +
    potassium pyroantimonate 2% in potassium phosphate buffer
  4. Fixative 4: RNA later

The ground reference experiment shall consist of a continuous nominal 1 g cultivation of 4 EUEs. The ground reference experiment will be performed 3-4 weeks later.

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