for about 1 h in the sterile bench with the ventilation switched
on to allow water drops from the seeds to evaporate.
4. Seal the plates with tape and place in closed cardboard boxes.
5. Stratify over night at 4
C.
6. Light-germinate for 10 days at long-day conditions.
7. Transfer nets to Arabidopsis growth agar supplemented with
5 μM norflurazon. Close lids and seal with tape.
8. Illuminate for 3 h with 100 μmol photons m
À2 s
À2 (growth
chamber conditions).
9. Collect seedlings from the metal grids with forceps. Put harvested seedlings in small aluminum foil squares folded like
envelopes, freeze in liquid nitrogen immediately after harvest
and store for carotenoid extraction (see Subheading 3.4) and
HPLC analysis (Subheading 3.5).
3.3 Carotenoid
Pathway Flux in SeedDerived Callus
This method is adopted from Ref. [10].
1. Weight 7.5 mg Arabidopsis seeds per big petri dish into a
reaction tube, add 1 ml of sterilization solution and incubate
10 min with gentle shaking at room temperature (see Note 6).
2. Centrifuge seeds for 2 min at 3000 Â g in a table top centrifuge, remove supernatant under the sterile hood, wash three
times by adding 1 ml of sterile water followed by centrifugation. After the last washing, resuspend seeds in 2 ml 0.4% (w/v)
agar-agar (see Note 7).
3. Place one sterile filter paper on a big petri dish containing SDC
medium and incubate briefly to soak some liquid. Place sterilized seeds on the filter paper by dribbling agar-agar solution
containing the seeds. Seal the plates with tape and place in
closed cardboard boxes.
4. Stratify over night at 4
C.
5. Place in growth chamber on white paper to reduce appearance
of condense water drops on lids. Germinate under long day
conditions at 100 μmol photons m
À2 s
À2 for 5 days (16 h
light/8 h dark, 26
C).
6. For norflurazon treatment transfer the filter paper including
germinated seeds on a fresh petri dish containing SDC medium
supplemented with 1 μM norflurazon. For the control, transfer
filter paper including germinated seeds on a fresh petri
dish containing fresh SDC medium without norflurazon (see
Note 8).
7. Reseal the plates with tape and place them into dark, for
example, in closed cardboard boxes. Continue incubation for
14 days at 26
C.
284
Julian Koschmieder and Ralf Welsch
on to allow water drops from the seeds to evaporate.
4. Seal the plates with tape and place in closed cardboard boxes.
5. Stratify over night at 4
C.
6. Light-germinate for 10 days at long-day conditions.
7. Transfer nets to Arabidopsis growth agar supplemented with
5 μM norflurazon. Close lids and seal with tape.
8. Illuminate for 3 h with 100 μmol photons m
À2 s
À2 (growth
chamber conditions).
9. Collect seedlings from the metal grids with forceps. Put harvested seedlings in small aluminum foil squares folded like
envelopes, freeze in liquid nitrogen immediately after harvest
and store for carotenoid extraction (see Subheading 3.4) and
HPLC analysis (Subheading 3.5).
3.3 Carotenoid
Pathway Flux in SeedDerived Callus
This method is adopted from Ref. [10].
1. Weight 7.5 mg Arabidopsis seeds per big petri dish into a
reaction tube, add 1 ml of sterilization solution and incubate
10 min with gentle shaking at room temperature (see Note 6).
2. Centrifuge seeds for 2 min at 3000 Â g in a table top centrifuge, remove supernatant under the sterile hood, wash three
times by adding 1 ml of sterile water followed by centrifugation. After the last washing, resuspend seeds in 2 ml 0.4% (w/v)
agar-agar (see Note 7).
3. Place one sterile filter paper on a big petri dish containing SDC
medium and incubate briefly to soak some liquid. Place sterilized seeds on the filter paper by dribbling agar-agar solution
containing the seeds. Seal the plates with tape and place in
closed cardboard boxes.
4. Stratify over night at 4
C.
5. Place in growth chamber on white paper to reduce appearance
of condense water drops on lids. Germinate under long day
conditions at 100 μmol photons m
À2 s
À2 for 5 days (16 h
light/8 h dark, 26
C).
6. For norflurazon treatment transfer the filter paper including
germinated seeds on a fresh petri dish containing SDC medium
supplemented with 1 μM norflurazon. For the control, transfer
filter paper including germinated seeds on a fresh petri
dish containing fresh SDC medium without norflurazon (see
Note 8).
7. Reseal the plates with tape and place them into dark, for
example, in closed cardboard boxes. Continue incubation for
14 days at 26
C.
284
Julian Koschmieder and Ralf Welsch