14. Add 50 μl of 30% PEG-Tris–HCl and mix carefully by
inversion.
15. Incubate the tubes in ice, for 20–30 min.
16. While the transformation process is in progress, keep two flasks
with 100 ml of 2Â regeneration medium at 50–55
C.
17. Transfer the content of each microcentrifuge tube to a sterile
10-ml tube and add 2 ml of 30% PEG-Tris–HCl to each one.
Mix carefully by inversion and incubate for 10 min in ice.
18. Add 4 ml of 1Â STC to each tube and mix by inversion.
19. Mix the 6 ml of the transformation tube with 120 ml of
regeneration medium (see Note 6) and prepare five plates
spreading approximately 20–25 ml per Petri dish. Similarly,
prepare another two Petri dishes with the corresponding
amount of protoplasts from the control reaction and carry
out appropriate serial dilutions.
20. Check growth on transformation plates on the following day.
When a thin mycelial growth is visible, approximately after 19 h
of incubation, add top agar with hygromycin to the transformation plates and one of the controls. For this purpose, put
5 ml of regeneration top agar to a 10-ml sterile tube, keep it at
55
C in a water bath, and add 40 μl of 50 mg/ml hygromycin
B. Immediately, lay the top agar to the transformation plates,
and incubate at 30
C. Colonies will appear after 2–3 days.
Don’t add the antibiotic to one of the control plates to be used
as a positive growth control.
21. Pick the colonies and transfer them to selective medium.
Transfer again the colonies obtained in the selective medium
to EG sporulation medium. Incubate for 7 days and collect
spores. Filter them through borosilicate filter with a porosity of
1. Streak spores to isolate colonies originated from a single
spore.
22. Repeat twice the single-spore purification procedure (see
Note 7).
23. Keep spores of transformants at À80
C in 20% glycerol for
storage.
3.3 Quantification of
Transcript Levels of
Carotenoid Genes
3.3.1 Cultivation
Conditions
1. Inoculate F. fujikuroi wild type and transformants strains on
EG agar Petri dishes. Use sterile toothpicks to inoculate in
seven symmetrical points on EG medium from a 3-day-old
mycelial colony (see Note 8).
2. Incubate the Petri dishes at 30
C for 7 days under $6 W/m
2
white light, $360 lx. In our case, we used four fluorescent
tubes Philips TL-D 18 W/840 at a distance of ca. 60 cm.
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Julia Marente et al.
inversion.
15. Incubate the tubes in ice, for 20–30 min.
16. While the transformation process is in progress, keep two flasks
with 100 ml of 2Â regeneration medium at 50–55
C.
17. Transfer the content of each microcentrifuge tube to a sterile
10-ml tube and add 2 ml of 30% PEG-Tris–HCl to each one.
Mix carefully by inversion and incubate for 10 min in ice.
18. Add 4 ml of 1Â STC to each tube and mix by inversion.
19. Mix the 6 ml of the transformation tube with 120 ml of
regeneration medium (see Note 6) and prepare five plates
spreading approximately 20–25 ml per Petri dish. Similarly,
prepare another two Petri dishes with the corresponding
amount of protoplasts from the control reaction and carry
out appropriate serial dilutions.
20. Check growth on transformation plates on the following day.
When a thin mycelial growth is visible, approximately after 19 h
of incubation, add top agar with hygromycin to the transformation plates and one of the controls. For this purpose, put
5 ml of regeneration top agar to a 10-ml sterile tube, keep it at
55
C in a water bath, and add 40 μl of 50 mg/ml hygromycin
B. Immediately, lay the top agar to the transformation plates,
and incubate at 30
C. Colonies will appear after 2–3 days.
Don’t add the antibiotic to one of the control plates to be used
as a positive growth control.
21. Pick the colonies and transfer them to selective medium.
Transfer again the colonies obtained in the selective medium
to EG sporulation medium. Incubate for 7 days and collect
spores. Filter them through borosilicate filter with a porosity of
1. Streak spores to isolate colonies originated from a single
spore.
22. Repeat twice the single-spore purification procedure (see
Note 7).
23. Keep spores of transformants at À80
C in 20% glycerol for
storage.
3.3 Quantification of
Transcript Levels of
Carotenoid Genes
3.3.1 Cultivation
Conditions
1. Inoculate F. fujikuroi wild type and transformants strains on
EG agar Petri dishes. Use sterile toothpicks to inoculate in
seven symmetrical points on EG medium from a 3-day-old
mycelial colony (see Note 8).
2. Incubate the Petri dishes at 30
C for 7 days under $6 W/m
2
white light, $360 lx. In our case, we used four fluorescent
tubes Philips TL-D 18 W/840 at a distance of ca. 60 cm.
352
Julia Marente et al.