8. Collect seed-developed calli from filter paper either with forceps or by pushing the plates upside down on a table for several
times to collect seed-derived calli on the lid. Put harvested
seed-derived calli 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).
3.4 Carotenoid
Extraction
1. Put harvested leaves, seedlings or seed-derived calli in small
aluminum foil envelopes. Freeze envelopes with samples in
liquid nitrogen immediately after harvest. When all samples
are collected, grind plant material with a mortar and pistil in
liquid nitrogen, return powder into aluminum envelope and
lyophilize overnight (see Note 9).
2. Weigh lyophilized powder in a 15 ml plastic tube: 5 mg for
leaves, 20 mg for seed-derived calli and 10 mg for seedlings
(dry weight). Note the exact weight.
3. Add 100 μl of 10 mM Tris (pH not adjusted), vortex (see
Note 10).
4. Add 2 ml of acetone and exactly 100 μl of internal standard
(α-tocopheryl acetate, 2 mg/ml in acetone) to each sample.
Prepare three aliquots with 100 μl each from same standard
dilution in reaction tubes, dry in a SpeedVac and dissolve in
100 μl chloroform/methanol (2:1, v/v), like samples (see
below). Store at À20
C until start of HPLC analysis. These
samples serve as external controls to determine standard recovery during extraction and HPLC analysis (see Note 11).
5. Apply short pulses of sonication to mix and extract sample.
6. Centrifuge for 5 min at 3000 Â g.
7. Transfer supernatant to a new 15 ml tube.
8. Add another 2 ml of acetone to the pellet and reextract sample
by sonication.
9. Centrifuge for 5 min at 3000 Â g.
10. Combine supernatant with supernatant from step 7.
11. Repeat steps 8 to 10.
12. Add 2 ml PE:DE (2:1; v:v) to combined supernatants (~ 6 ml)
and mix by vortexing.
13. Add water up to 14 ml.
14. Close tube tightly and mix samples by inverting tube several
times.
15. Centrifuge for 5 minutes at 3000 Â g.
16. Transfer upper organic phase to a new 15 ml tube.
Quantification of Carotenoid Pathway Flux
285
times to collect seed-derived calli on the lid. Put harvested
seed-derived calli 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).
3.4 Carotenoid
Extraction
1. Put harvested leaves, seedlings or seed-derived calli in small
aluminum foil envelopes. Freeze envelopes with samples in
liquid nitrogen immediately after harvest. When all samples
are collected, grind plant material with a mortar and pistil in
liquid nitrogen, return powder into aluminum envelope and
lyophilize overnight (see Note 9).
2. Weigh lyophilized powder in a 15 ml plastic tube: 5 mg for
leaves, 20 mg for seed-derived calli and 10 mg for seedlings
(dry weight). Note the exact weight.
3. Add 100 μl of 10 mM Tris (pH not adjusted), vortex (see
Note 10).
4. Add 2 ml of acetone and exactly 100 μl of internal standard
(α-tocopheryl acetate, 2 mg/ml in acetone) to each sample.
Prepare three aliquots with 100 μl each from same standard
dilution in reaction tubes, dry in a SpeedVac and dissolve in
100 μl chloroform/methanol (2:1, v/v), like samples (see
below). Store at À20
C until start of HPLC analysis. These
samples serve as external controls to determine standard recovery during extraction and HPLC analysis (see Note 11).
5. Apply short pulses of sonication to mix and extract sample.
6. Centrifuge for 5 min at 3000 Â g.
7. Transfer supernatant to a new 15 ml tube.
8. Add another 2 ml of acetone to the pellet and reextract sample
by sonication.
9. Centrifuge for 5 min at 3000 Â g.
10. Combine supernatant with supernatant from step 7.
11. Repeat steps 8 to 10.
12. Add 2 ml PE:DE (2:1; v:v) to combined supernatants (~ 6 ml)
and mix by vortexing.
13. Add water up to 14 ml.
14. Close tube tightly and mix samples by inverting tube several
times.
15. Centrifuge for 5 minutes at 3000 Â g.
16. Transfer upper organic phase to a new 15 ml tube.
Quantification of Carotenoid Pathway Flux
285