TITLE: Botrytis cinerea transformation protocol
AUTHORS: Paulo  Canessa

[DESCRIPTION]
Botrytis cinerea transformation protocol. Please, acknowledge the work of others.

[STEPS]
SECTION: Day 1: Fungal growth
1. Add 10 ml sterile MiliQ water on top of a well-sporulated Botrytis cinerea plate (7-day old PDA-bean plate).
SECTION: Day 1: Fungal growth
2. Disaggregate conidia.
SECTION: Day 1: Fungal growth
3. Collect conidia and filter using Nytex attached to a flask. You can use the spatula to speed up the process.
SECTION: Day 1: Fungal growth
4. Inoculate all collected spores in one flask containing 100 ml of maltose medium (1.5%) for 18 hours.
SECTION: Day 1: Fungal growth
5. Incubate at 20º Celsius and 120 rpm for 18 hours.
SECTION: Day 2: Protoplast generation
6. Filtrate de ON culture using Nytex and one 250 ml flask. You can use a sterile spoon to speed up the process.
SECTION: Day 2: Protoplast generation
7. Wash 4-5 times with KC buffer until completing 50 ml (at room temperature). It is important to remove the media. Collect approximately 2.0 gr. of half-wet mycelium or 1.5 gr. if it is really dry. Transfer to a 100 ml sterile flask in order to prepare protoplasts as indicated below.
SECTION: Day 2: Protoplast generation
8. Place KC buffer on ice and add filtered-sterilize enzyme mixture solution described below to the “half-wet mycelium”.

SECTION: Day 2: Protoplast generation
9. Incubate for 2 hours at 28 Celsius and 95 RPM.
SECTION: Day 2: Protoplast generation
10. Filtrate using Nytex on top of a small 100 ml flask. Wash with KC buffer (on ice). For this procedure, use a total of 50 ml KC buffer.
SECTION: Day 2: Protoplast generation
11. Centrifuge the filtrate at 4000 rpm (50 ml swinging bucket rotor; Eppendorf 5804 R centrifuge) and 5 min, 4 Celsius. Adjust the speed-up and the speed-down setting of the centrifuge to the minimum.

SECTION: Day 2: Protoplast generation
12. Wash with KC buffer (50 ml) and centrifuge again as mentioned. Resuspend protoplast in the backflow. Alternatively, remove all KC and then add 500 μl KC.

SECTION: Day 2: Protoplast generation
13. Take a 1:50 aliquot (4μl:200μl) in order to quantify protoplasts in a Thoma chamber. Adjust the protoplasts to the desire 1x108/ml stock solution.
SECTION: Day 2: Transformation
14. Add 70 μl KC to the genetic construct (three 50 μl purified PCRs in 30 μl total).

SECTION: Day 2: Transformation
15. Add 100 μl protoplasts to each transformation (from the solution prepared in step 13). Incubate 5 min in ice.
SECTION: Day 2: Transformation
16. Add 100 μl PEG solution and incubate at RT for 20 min.
SECTION: Day 2: Transformation
17. Add 500 μl PEG solution and incubate on ice for 10 min.
SECTION: Day 2: Transformation
18. Add 200 μl KC solution for a final volume of 1ml.

SECTION: Day 2: Transformation
19. Add 500 μl of protoplasts to 50 ml 45 Celsius SH agar (liquid; using 50 ml sterile disposable tubes). Transfer 10 ml to five Petri dishes. Repeat with the remaining 500 μl of protoplasts to end up with 10 plates per transformation.
SECTION: Day 2: Transformation
20. Incubate the plates at 20-22 Celsius (12:12 h. photoperiod) for 20 hours.
SECTION: Day 3: Selection
21. Add 10 ml SH agar with the selection marker: Hygromycin B (70 μg/ml) or Nourseothricin (140 μg/ml).
SECTION: Day 3: Selection
22. Approximately, between 3-7 days, thin (and flat) growing colonies will emerge. Transfer each individual colony to B5-medium + 2% Glucose Petri plates with selection: Hygromycin B 70 μg/ml.