3.2 Transformation
of Fusarium fujikuroi
Protoplasts
The transformation protocols are based on the ones described by
[23, 24].
1. Inoculate 50 ml of Darken medium with approximately a
4-mm
2 mycelium plug in a 250-ml flask. Incubate for 3 days,
at 30
C in a shaker at 200 rpm.
2. Take 1 ml of the culture to inoculate 100 ml of fresh ICI
medium. Incubate it overnight at 30
C, and 150 rpm.
3. Filter the young mycelium by vacuum through a sterile filter
paper, wash the filtered mycelium with sterile osmostabilization
buffer and filter again until there is no liquid in the mycelium.
Remove carefully the mycelium from the filter with a sterile
spatula, resuspend it in 25 ml of enzyme solution (see materials,
see Note 1) and incubate it for 3–4 h at 30
C at 100–150 rpm
(see Note 2).
4. Separate protoplasts from mycelia filtering through two borosilicate crucibles filters, first use a pore size 0 filter
(160–250 μm), collect the filtrate in a sterile Petri dish and
pass through pore size 1 filter (100–160 μm). Collect the
resulting filtrate in a 50-ml tube.
5. Fill the tube with osmostabilization buffer up to 50 ml and
centrifuge it for 15 min at 2000 Â g.
6. Discard the supernatant and fill again up to 50 ml with osmostabilization buffer. Resuspend the protoplasts and centrifuge
them again for 15 min at 2000 Â g (see Note 3).
7. Repeat the previous step.
8. Discard the supernatant and resuspend the protoplasts in
500 μl of osmostabilization buffer (see Note 3).
9. Determine concentration of protoplasts using a Neubauer
counting chamber under a microscope.
10. Use 3 Â 10
7 protoplasts for each transformation. Protoplasts
can be stored at À80
C (see Note 4). Before storing protoplasts, add 2 μl of DMSO and add PEG to a final concentration
of 0.1% per 100 μl of aliquots.
11. Add 10 μg of linear DNA per transformation. In this case, we
added 10 μg of pPO5 (plasmid containing the Tet-on cassette)
and 7 μg of PAN7–1 [25], which contained a hygromycinresistance cassette (see Note 5).
12. Prepare two microcentrifuge tubes with 50 μl STC 2Â buffer.
One of them will be used both as a control for regeneration and
as a control for the selective medium. Add 50 μl of sterile
distilled H 2 O to the control. Add to the other tube 50 μl of
the DNA to transform.
13. Add 100 μl with 3 Â 10
7 protoplasts to each of the tubes.
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
351
of Fusarium fujikuroi
Protoplasts
The transformation protocols are based on the ones described by
[23, 24].
1. Inoculate 50 ml of Darken medium with approximately a
4-mm
2 mycelium plug in a 250-ml flask. Incubate for 3 days,
at 30
C in a shaker at 200 rpm.
2. Take 1 ml of the culture to inoculate 100 ml of fresh ICI
medium. Incubate it overnight at 30
C, and 150 rpm.
3. Filter the young mycelium by vacuum through a sterile filter
paper, wash the filtered mycelium with sterile osmostabilization
buffer and filter again until there is no liquid in the mycelium.
Remove carefully the mycelium from the filter with a sterile
spatula, resuspend it in 25 ml of enzyme solution (see materials,
see Note 1) and incubate it for 3–4 h at 30
C at 100–150 rpm
(see Note 2).
4. Separate protoplasts from mycelia filtering through two borosilicate crucibles filters, first use a pore size 0 filter
(160–250 μm), collect the filtrate in a sterile Petri dish and
pass through pore size 1 filter (100–160 μm). Collect the
resulting filtrate in a 50-ml tube.
5. Fill the tube with osmostabilization buffer up to 50 ml and
centrifuge it for 15 min at 2000 Â g.
6. Discard the supernatant and fill again up to 50 ml with osmostabilization buffer. Resuspend the protoplasts and centrifuge
them again for 15 min at 2000 Â g (see Note 3).
7. Repeat the previous step.
8. Discard the supernatant and resuspend the protoplasts in
500 μl of osmostabilization buffer (see Note 3).
9. Determine concentration of protoplasts using a Neubauer
counting chamber under a microscope.
10. Use 3 Â 10
7 protoplasts for each transformation. Protoplasts
can be stored at À80
C (see Note 4). Before storing protoplasts, add 2 μl of DMSO and add PEG to a final concentration
of 0.1% per 100 μl of aliquots.
11. Add 10 μg of linear DNA per transformation. In this case, we
added 10 μg of pPO5 (plasmid containing the Tet-on cassette)
and 7 μg of PAN7–1 [25], which contained a hygromycinresistance cassette (see Note 5).
12. Prepare two microcentrifuge tubes with 50 μl STC 2Â buffer.
One of them will be used both as a control for regeneration and
as a control for the selective medium. Add 50 μl of sterile
distilled H 2 O to the control. Add to the other tube 50 μl of
the DNA to transform.
13. Add 100 μl with 3 Â 10
7 protoplasts to each of the tubes.
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
351