Materials and Reagents

Arabidopsis thaliana seedlings (~10 days after germination)
Note: Amount of anthocyanin per seedling weight is higher in young seedlings.
Bleach solution
Sterile dH₂O
Methanol
Acetic acid
Murashige and Skoog medium salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
Sucrose
2-Morpholinoethanesulfonic acid (MES)
Agar (for plant culture)
Modified Murashige and Skoog medium (see Recipes)
Extraction buffer (see Recipes)

Equipment

Procedure

Add bleach solution to Arabidopsis thaliana seeds and shake gently for 7 min.
Rinse three to five times with sterile dH₂O.
Sow surface-sterilized seeds on modified MS medium.
Grow the plants under long day (16 h light/8 h dark) or continuous light condition (50~70 µmol/m²/s) at 22 °C for 7~10 days.
Measure fresh weight of 10~15 seedlings.
Note: Remove surface moisture with paper towel before measurement.
Freeze seedlings with liquid nitrogen and grind in a mortar and pestle.
Add 5 volumes (based on fresh weight) of extraction buffer and mix thoroughly.
Centrifuge at 12,000 x g for 5 min at room temperature.
Transfer supernatant to new tube.
Centrifuge at 12,000 x g for 5 min at room temperature.
Transfer supernatant to new tube.
Measure absorbances at 530 and 637 nm.
Calculate anthocyanin content (Abs₅₃₀/g F.W.) by [Abs₅₃₀ - (0.25 x Abs₆₅₇)] x 5.
Note: To correct contribution of chlorophyll to the absorbances at 530 nm, the formula Abs530 - (0.25 x Abs657) was used. Multiplication of “5” can be changed depending on volumes of added extraction buffer in step 5. For example, if 10 volumes of extraction buffer was added, multiply “10”.

Recipes

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References