Protoplast Production

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  Protoplasts being released from hyphae.

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  Protoplasts on a hemocytometer.

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  Protoplasts on a hemocytometer.

1. Inoculate 50 mL PDY or MPDY in a 125 mL flask with 9 plugs of leading edge tissue. Culture at 30C, 70 rpm for 5 days.
2. Prepare 20ml of lysing enzymes (0.04g/ml of Lysing Enzyme from Trichoderma harzanium in 0.6 M KCl, 0.1 M phosphate buffer pH 5.8) and incubate 15min at 37C, 200 rpm (use a 50 ml tube)
3. Collect 5 day old mycelium from the flask using a 40um filter and wash it using 10ml of 0.6 M KCl, 0.1 M phosphate buffer pH 5.8 (use a 50 ml tube and 40 um filter).
   1. A second wash can sometimes improve yield with older cultures: resuspend the mycelia in 10ml of 0.6 M KCl, 0.1 M phosphate buffer pH 5.8 in a 50 mL conical tube, vortex, and then centrifuge at 6k rpm for 5 minutes. Discard the supernatant.
4. Transfer the mycelium into a single 125 ml flask,
5. Filter lysing enzymes (0.2um syringe filter) and add the enzyme to the flask with mycelia. Incubate for 3-4h, 25C, 70 rpm.
6. Collect 10 µL and check for protoplast formation.
7. Filter the protoplasts through sterile cheese cloth stuffed into a 60 mL syringe that is directed into a 40um cell filter over a 50 mL conical tube.
   1. If needed filter through a 5 um MILLEX syringe filter into a clean conical tube.
8. Collect the protoplasts by centrifugation, 3500 rpm, 4C
9. Discard the supernatant and wash the protoplasts using 10ml of TMS
10. Collect the protoplasts by centrifugation, 3500 rpm, 4C
11. Discard the supernatant and resuspend the protoplast into 300ul of TMSC
12. Count the protoplasts
    1. Using microscope at 40x and Neubauer improved 0.1mm 0.0025 mm^2
    2. Image all four large corner squares each composed of 16 small squares.
    3. Using Fiji count protoplasts in all 64 squares.
    4. http://insilico.ehu.eus/counting_chamber/neubauer_improved.php
    5. https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx
13. Adjust the protoplast accordingly using TMSC.

Reagents list and recipe:

Autoclave Store at 4 ° C in 50 ml falcons.

0.6 M KCl / 0.1 M NaP 500 ml 0.6 M KCl + 50 ml 1 M NaP pH 5.8. Autoclaved (1 +2) (1) 0.6 M KCl: 22.37g KCl in 500 ml of H2O. Autoclaved. (2) 1M NaP pH 5.8: 15.8ml 1M Na2HPO4 + 184.2ml 1M NaH2PO4 pH 5.8 (NaOH pellets at the start). Autoclaved.

(3 + 4) (3) 1 M Na2HPO4: in 50 ml H2O. Autoclaved. (4) 1 M NaH2PO4: in 500ml of H2O. Autoclaved.

TMS Sorbitol 1 M MOPS 10 mM 18.2g of Sorbitol + 209 mg MOPS per 100 ml of H2O, pH 6.3 (NaOH). Sterilize by filtration.

TMSC Sorbitol 1 M MOPS 10 mM CaCL2 40 mM 18.2g of Sorbitol + 209 mg MOPS + 588 mg CaCl2 per 100 ml of H2O, pH 6.3 (NaOH). Sterilize by filtration.

MS Sorbitol 0.6 M MOPS 10 mM 10.9g of Sorbitol + 209 mg MOPS per 100 ml of H2O, pH 6.3 (NaOH). Sterilize by filtration.

TE CaCl2 2X, pH 7.5 2 M Tris pH 8 1 ml 0.5 M EDTA 0.4 ml CaCl2, 2H2O 80 mM 1176 mg H2O qs 100 ml. Adjust the pH to 7.5 (HCl). Filter. Aliquot by 10 ml.

PEG 60% Prepare immediately. 1.2 g of PEG6000 + 800 µl of MS (= 2 ml of final volume) in 14ml Falcon tube. Dissolve in the microwave for a few seconds. Let cool to room temperature.

References

1. Antal et al. 2012
2. Liu et al. 2020