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 (see Hint #1).
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.
11. Subculture at weekly intervals, even if the tissue appears to be growing more slowly. Adjust the amount of tissue used to inoculate a new flask in accordance with the growth rate.
Solutions
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
BioReagents and Chemicals
Agar Sucrose Kinetin 2,4-Dichlorophenoxyacetic Acid Gamborg's B5 Basal Medium Glucose MES Murashige and Skoog Basal Medium Household Bleach
Protocol Hints
1. It does not seem to matter which face of the leaf is placed downwards on the solid Callus Induction Medium.
Pollen Development&
