10% (v/v) Household Bleach
Callus Induction Medium
Gamborg's B5 Basal Medium
0.5 g/liter MES
pH 5.7
0.05 mg/liter Kinetin
0.5 mg/liter 2,4-dichlorophenoxyacetic Acid (2,4-D)
0.8% (w/v) Agar
2% (w/v) Glucose
Liquid MSA Medium
Murashige and Skoog Basal Medium
3% (w/v) Sucrose
pH 5.7
0.05 mg/liter Kinetin
0.5 mg/liter 2,4-Dichlorophenoxyacetic Acid (2,4-D)
Solid MS0 Medium
Murashige and Skoog Basal Medium
3% (w/v) Sucrose
pH 5.8
0.8% (w/v) Agar
实验步骤
1. Immerse Arabidopsis seeds in 10% Household Bleach for 20 min.
2. Rinse the seeds twice with 500 ml of sterile ddH2O and allow them to dry overnight in a laminar flow hood.
3. Push the resulting crust of seeds through a sterile tea-strainer in order to separate the individual seeds. Store the seeds in a sterile 60 mm Petri dish wrapped in Parafilm until use.
4. Lift the sterile seeds with a flamed spatula onto solid MS0 medium in screw-capped 55 x 100 mm round jars. Place the jars under fluorescent lights at about 22°C.
5. When they are large enough to handle, transfer the plants to a sterile tile and excise the roots and chop them into sections approximately 1 mm in length.
6. Proceed with either Step a OR b below:
a. Place the root pieces into Callus Induction Medium in 60 mm Petri dishes and place the dishes under fluorescent lights at about 22°C.
b. Use the largest leaves for callus initiation. Transfer each leaf to the tile and damage it with a sterile scalpel blade in order to initiate a wound response. Place the leaf into Callus Induction Medium in 60 mm Petri dishes and place the dishes under fluorescent lights at about 22°C.
7. After callus formation (2 to 3 weeks), place a pea-sized piece of callus in a 100 ml sterile conical flask with 20 ml of liquid MSA medium.
8. Cover the flask with aluminum foil and place it on a shaker set at 40 rpm in a constant temperature environment at about 22°C (either with or without light). After 7 to 10 days, the flask can be subcultured.
9. To subculture the cells, decant all of the liquid off and add 50 ml of fresh MSA. Pour the entire culture into a 250 ml flask.
10. When the volume of tissue has doubled, transfer 30% of the culture to a fresh flask. Upon subsequent subculturing, use 100 ml of medium per 250 ml flask.
