7. Cell counting chamber (Neubauer chamber or Bu ¨rker
chamber).
2.3 Transformation
of Fusarium
Sterilize all media and material in an autoclave at 120
C for
20 min.
1. Darken medium: 15 g corn steep liquor, 30 g sucrose, 2 g
(NH 4 ) 2 SO 4 , and 7 g of CaCO 3 in 1 l of distilled water.
Autoclave.
2. ICI medium: 10 g glucose, 1 g (NH 4 ) 2 SO 4 , 5 g KH 2 PO 4 , 1 g
MgSO 4 ·7H 2 O, and 2 ml of Geissman microelement solution
in 1 l distilled water [21]. Autoclave.
3. Geissman microelement solution: 0.1 g FeSO 4 ·7H 2 O; 0.015 g
CuSO 4 ·5H 2 O; 0.161 g ZnSO 4 ·7H 2 O; 0.01 g MnSO 4 ·7H 2 O;
0.01 g (NH 4 ) 6 Mo 7 O 20 ·4H 2 O in 100 ml of distilled
water [21].
4. Osmostabilization buffer: 1.2 M KCl, 0.66 M CaCl, pH 5.5.
Dissolve 35.97 g of KCl and 2.94 g of CaCl 2 in 400 ml of
distilled water. Adjust pH to 5.5 and autoclave.
5. Enzyme solution: Mix 200 mg of Lysing enzymes from Trichoderma harzianum (Sigma), 150 mg of Driselase from basidiomycetes (Fluca), 15 mg of lyticase (Sigma), and 10 mg of BSA
in 50 ml of osmostabilization buffer. Mix with a magnetic
stirrer bar during at least 30 min. Sterilize through a 0.42 μm
filter.
6. STC 2Â: sorbitol 2.4 M, 20 mM Tris–HCl, pH 8, 100 mM
CaCl 2 .
7. 30% PEG Tris–HCl: 30% PEG 8000, 10 mM Tris–HCl, pH 8,
50 mM CaCl.
8. Selective medium: DG medium with 50 mg/l hygromycin
(Roche).
9. Regeneration medium: Prepare a 2Â regeneration medium
containing 100 mg of yeast extract, 100 mg of NZ amine A,
and 1.2 g of agar in 50 ml of water. Autoclave. Let it cool until
55
C and keep it warm in a heat cabinet until preparation of
the dishes. Then mix with 50 ml of 1.6 M sucrose solution
prewarmed at 42
C.
10. Regeneration top agar: Prepare regeneration 2Â medium
replacing agar by the same amount of agarose. Add the same
amount of 1.6 M sucrose (2Â), mix and maintain the regeneration top agar in a bath at 55
C. Add 40 μl of 50 mg/ml
hygromycin (Roche) per 5 ml of regeneration top agar.
11. Sterile borosilicate filter, porosity 1 and 0, 50-ml volume,
ROBU
® (Glasfilter-Ger€ ate GmbH).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
347
chamber).
2.3 Transformation
of Fusarium
Sterilize all media and material in an autoclave at 120
C for
20 min.
1. Darken medium: 15 g corn steep liquor, 30 g sucrose, 2 g
(NH 4 ) 2 SO 4 , and 7 g of CaCO 3 in 1 l of distilled water.
Autoclave.
2. ICI medium: 10 g glucose, 1 g (NH 4 ) 2 SO 4 , 5 g KH 2 PO 4 , 1 g
MgSO 4 ·7H 2 O, and 2 ml of Geissman microelement solution
in 1 l distilled water [21]. Autoclave.
3. Geissman microelement solution: 0.1 g FeSO 4 ·7H 2 O; 0.015 g
CuSO 4 ·5H 2 O; 0.161 g ZnSO 4 ·7H 2 O; 0.01 g MnSO 4 ·7H 2 O;
0.01 g (NH 4 ) 6 Mo 7 O 20 ·4H 2 O in 100 ml of distilled
water [21].
4. Osmostabilization buffer: 1.2 M KCl, 0.66 M CaCl, pH 5.5.
Dissolve 35.97 g of KCl and 2.94 g of CaCl 2 in 400 ml of
distilled water. Adjust pH to 5.5 and autoclave.
5. Enzyme solution: Mix 200 mg of Lysing enzymes from Trichoderma harzianum (Sigma), 150 mg of Driselase from basidiomycetes (Fluca), 15 mg of lyticase (Sigma), and 10 mg of BSA
in 50 ml of osmostabilization buffer. Mix with a magnetic
stirrer bar during at least 30 min. Sterilize through a 0.42 μm
filter.
6. STC 2Â: sorbitol 2.4 M, 20 mM Tris–HCl, pH 8, 100 mM
CaCl 2 .
7. 30% PEG Tris–HCl: 30% PEG 8000, 10 mM Tris–HCl, pH 8,
50 mM CaCl.
8. Selective medium: DG medium with 50 mg/l hygromycin
(Roche).
9. Regeneration medium: Prepare a 2Â regeneration medium
containing 100 mg of yeast extract, 100 mg of NZ amine A,
and 1.2 g of agar in 50 ml of water. Autoclave. Let it cool until
55
C and keep it warm in a heat cabinet until preparation of
the dishes. Then mix with 50 ml of 1.6 M sucrose solution
prewarmed at 42
C.
10. Regeneration top agar: Prepare regeneration 2Â medium
replacing agar by the same amount of agarose. Add the same
amount of 1.6 M sucrose (2Â), mix and maintain the regeneration top agar in a bath at 55
C. Add 40 μl of 50 mg/ml
hygromycin (Roche) per 5 ml of regeneration top agar.
11. Sterile borosilicate filter, porosity 1 and 0, 50-ml volume,
ROBU
® (Glasfilter-Ger€ ate GmbH).
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
347