4. Sterile bench.
5. 0.4% (w/v) agar-agar.
6. Arabidopsis growth agar plates. Add per liter: 4.33 g Murashige and Skoog medium, 0.5 g 2-(N-morpholino)ethanesulfonic acid/KOH, pH 5.7, 4 g Phytagel. After autoclaving, poor
into large petri dishes (145 mm diameter). Prepare as many
plates without and with 5 μM norflurazon. Norflurazon is
added from stock solution (see above) to the autoclaved
medium before pouring the plates.
7. Rectangular stainless steel nets (e.g., V2A-315/200) of
3 Â 3 cm in size with a mesh size of 0.315 mm and 0.2 mm
wire thickness. Wrap in small batches of, for example, six pieces
in aluminum foil and sterilize by autoclaving. Place in drying
oven to evaporate water before use.
8. Cold chamber or fridge at 4
C.
9. Phytochamber or light source with 100 μmol photons m
À2 s
À2 .
2.3 Carotenoid
Pathway Flux in SeedDerived Callus
1. Arabidopsis seeds.
2. Sterilization solution (see above), sterile water.
3. Table top centrifuge.
4. Seed-derived callus medium (SDC medium). For 1 L, dissolve
the following components: 4.33 g Murashige and Skoog
medium, 30 g sucrose, 0.5 mg 2,4-dichlorophenoxyacetic
acid (50 μl of 10 mg/mL stock in 1 M KOH), 2 mg indole3-acetic acid (20 μl of 100 mg/ml stock in 1 M KOH), 0.5 mg
2-isopentenyladenine (50 μl of 10 mg/ml stock in 1 M KOH).
Adjust pH to 5.8 with KOH. Add 4 g Phytagel per L medium
and autoclave, let cool to about 40
C, add 1000 μl 1000Â
Gamborg B5 vitamins per liter medium and poor into big petri
dishes (145 mm diameter). Prepare as many plates without and
with 1 μM norflurazon. Norflurazon is added from stock solution (see above) to the autoclaved medium before pouring the
plates.
5. 0.4% (w/v) agar-agar.
6. Sterile filter paper, forceps.
2.4 Carotenoid
Extraction
and Analysis
1. Liquid nitrogen, mortar, pistil (alternatively: microdismembrator), aluminum foil.
2. Freeze dryer, speed vacuum evaporator, sonicator, table top
centrifuge, centrifuge for 15 ml plastic tubes, vortex evaporator
or nitrogen steam.
3. 15 ml plastic reaction tubes.
4. 100 mM Tris (pH not adjusted).
282
Julian Koschmieder and Ralf Welsch
5. 0.4% (w/v) agar-agar.
6. Arabidopsis growth agar plates. Add per liter: 4.33 g Murashige and Skoog medium, 0.5 g 2-(N-morpholino)ethanesulfonic acid/KOH, pH 5.7, 4 g Phytagel. After autoclaving, poor
into large petri dishes (145 mm diameter). Prepare as many
plates without and with 5 μM norflurazon. Norflurazon is
added from stock solution (see above) to the autoclaved
medium before pouring the plates.
7. Rectangular stainless steel nets (e.g., V2A-315/200) of
3 Â 3 cm in size with a mesh size of 0.315 mm and 0.2 mm
wire thickness. Wrap in small batches of, for example, six pieces
in aluminum foil and sterilize by autoclaving. Place in drying
oven to evaporate water before use.
8. Cold chamber or fridge at 4
C.
9. Phytochamber or light source with 100 μmol photons m
À2 s
À2 .
2.3 Carotenoid
Pathway Flux in SeedDerived Callus
1. Arabidopsis seeds.
2. Sterilization solution (see above), sterile water.
3. Table top centrifuge.
4. Seed-derived callus medium (SDC medium). For 1 L, dissolve
the following components: 4.33 g Murashige and Skoog
medium, 30 g sucrose, 0.5 mg 2,4-dichlorophenoxyacetic
acid (50 μl of 10 mg/mL stock in 1 M KOH), 2 mg indole3-acetic acid (20 μl of 100 mg/ml stock in 1 M KOH), 0.5 mg
2-isopentenyladenine (50 μl of 10 mg/ml stock in 1 M KOH).
Adjust pH to 5.8 with KOH. Add 4 g Phytagel per L medium
and autoclave, let cool to about 40
C, add 1000 μl 1000Â
Gamborg B5 vitamins per liter medium and poor into big petri
dishes (145 mm diameter). Prepare as many plates without and
with 1 μM norflurazon. Norflurazon is added from stock solution (see above) to the autoclaved medium before pouring the
plates.
5. 0.4% (w/v) agar-agar.
6. Sterile filter paper, forceps.
2.4 Carotenoid
Extraction
and Analysis
1. Liquid nitrogen, mortar, pistil (alternatively: microdismembrator), aluminum foil.
2. Freeze dryer, speed vacuum evaporator, sonicator, table top
centrifuge, centrifuge for 15 ml plastic tubes, vortex evaporator
or nitrogen steam.
3. 15 ml plastic reaction tubes.
4. 100 mM Tris (pH not adjusted).
282
Julian Koschmieder and Ralf Welsch