Biology Reference
In-Depth Information
2.2 Common
Solutions
Prepare all cultivation media using deionized water.
1. Stock solutions of plant hormones, 2,4-dichlorophenoxyacetic
acid (2,4-D) and naphthalene-1-acetic acid (NAA), both in
concentration 0.1 g/l: dissolve 10 mg of 2,4-D or NAA in
1-1.5 ml of 96 % ethanol and refi ll with hot deionized water
to 100 ml. Stir at constant temperature (80 °C) for at least 4 h
until the chemicals are fully dissolved.
2. Stock solution of thiamin: dissolve 100 mg thiamin in 100 ml
deionized water. Store 2 ml aliquots in small tubes (epi-tubes)
at −20 °C.
3. Modifi ed Murashige-Skoog (MS) medium for suspension-
cultured BY-2 and LE cells: 30 g/l sucrose, 4.3 g/l Murashige
and Skoog salts (Sigma M5524), 100 mg/l inositol, thiamin
(1 ml of stock solution per l), 2,4-D (2 ml of stock solution
per l), 200 mg/l KH
2
PO
4
, pH 5.8 (adjust with 3 M KOH).
Autoclave at 121 °C for 20 min under 0.1 MPa (
see
Note 1
).
4. V4 medium for suspension-cultured VBI-0 cells: prepare com-
ponent solutions: (a) fi nal volume 500 ml: 7.5 g/l KCl, 6 g/l
NaNO
3
, 2.5 g/l MgSO
4
× 7H
2
O, 1.25 g/l NaH
2
PO
4
× 2H
2
O;
(b) fi nal volume 500 ml: 7.5 g/l CaCl
2
× 2H
2
O; (c) fi nal vol-
ume 100 ml: 1 g/l ZnSO
4
× 7H
2
O, 0.1 g/l MnSO
4
× 4H
2
O,
0.03 g/l CuSO
4
× 5H
2
O, 1 g/l H
3
BO
3
, 0.01 g/l KI, 0.03 g/l
AlCl
3
× 6H
2
O, 0.03 g/l NiCl
2
× 6H
2
O; (d) fi nal volume
500 ml: 3.72 g/l Na
2
EDTA, 2.78 g/l FeSO
4
× 7H
2
O; (e) fi nal
volume 100 ml: 0.01 g/l B1 (thiamin), 0.01 g/l B6 (pyridox-
ine), 0.05 g/l B3 (nicotinic acid), 0.3 g/l glycine. Mix the
following components for fi nal volume 1,000 ml: component
A (100 ml), component B (10 ml), component C (1 ml),
component D (10 ml), inositol (100 mg/l), casein hydroly-
sate (1 g/l), sucrose (30 g/l), NAA (10 ml of stock solution;
1 mg/l), 2,4-D (10 ml of stock solution; 1 mg/l), component
E (10 ml). Adjust pH to 5.7. Refi ll with deionized water to
fi nal volume. Autoclave at 121 °C for 20 min under 0.1 MPa.
5. Solid (agar) medium for suspension-cultured cells: before
autoclaving, add agar (6 g/l) to the particular liquid medium
described above and autoclave at 121 °C for 20 min under
0.1 MPa. Pour the medium under sterile conditions into
sterile Petri dishes (about 20 or 40 ml to 60 or 90 mm diameter
plates, respectively) and leave to solidify.
1. Sterilized by autoclaving at 120 °C for 20 min: Erlenmeyer
fl asks (100 and 250 ml) and cylinder, covered with double alu-
minium foil, regular pipette tips, and pipette tips cut off by ca.
1-1.5 cm, suitable for 1, 5, or 10 ml pipettes (
see
Note 2
).
2. Metal spatula, parafi lm strips, ethanol in a beaker, and gas burner.
2.3 Cultivation
of Suspension (Liquid)
and Callus (Agar)
Cultures
3. Sterile plastic Petri dishes (60 or 90 mm diameter).
Search WWH ::
Custom Search