Dec 15, 2025
Version 2
Marchantia protoplast V.2
- Eftychis Frangedakis1
- 1University of Cambridge
- Hornworts
Protocol Citation: Eftychis Frangedakis 2025. Marchantia protoplast. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge6z6wl47/v2Version created by Eftychis Frangedakis
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2025
Last Modified: December 15, 2025
Protocol Integer ID: 235090
Keywords: marchantia protoplast protocol for the preparation, marchantia protoplast protocol
Abstract
Protocol for the preparation of Marchantia polymorpha protoplasts
Materials
8% (w/v) mannitol at pH 5.7
Troubleshooting
Solutions/media to prepare before starting the protocol (Day 1 & Day 2):
Day 1
8% (w/v) mannitol at pH 5.7
Liquid Gamborg B5 plus vitamins
- 0.0948 g Gamborg B5 plus vitamins
- 2.4g mannitol
- Adjust pH to 5.7
- dH2O to 29.7 ml
Day 2
Protoplast Regeneration Media Top layer (PRMT)
- 0.0948 g Gamborg B5 plus vitamins
- 1.6 g mannitol
- 0.1 g agar
- dH2O to make up to 19.6 ml
- Adjust pH to 5.7
- Autoclave and keep at 50oC after autoclaving.
Protoplast Regeneration Media Bottom layer (PRMB)
- 1.57 g Gamborg B5 plus vitamins
- 30 g mannitol
- Adjust pH to 5.7
- 4 g agar
- dH2O to make up to 495 ml
- Autoclave - Keep at 50 oC after autoclaving, and add 5ml sterile 50% glucose before use as indicated in the protocol.
Gemmae growth (Day -4)
Grow gemmae on ½ Gamborg B5 plus vitamins media plates (the plates should be covered like Fig. 1A) for 4 days (Fig. 1B).
Figure 1
Day 1
Add 10 ml of 8% (w/v) mannitol at pH 5.7 on the plate and incubate at room temperature 30min.
Day 1
Prepare 2% Driselase solution: add 0.2 g Driselase in 10 ml 8% mannitol solution and incubate in dark for 20 min at RT. Gently invert to mix at intervals. Spin at 3.3k for 3 min. Filter sterilize supernatant using a 0.2 μm filter attached to a syringe.
Replace mannitol solution with 5 ml 2% Driselase and incubate at RT for 4 hours with gentle shaking (50 rpm).
Using a tip gently release protoplasts from gemmae ("dissolve the gemmae") (Fig.1C).
Filter the solution through a 70 μm cell strainer into a 50 ml falcon tube and then transfer in a round bottom falcon tube (Fig. 1D).
Incubate for further 10 min.
Spin at 120 x g for 3min. Remove the supernatant without disturbing the cells.
Wash the cells by re-suspending themin 6ml 8% mannitol (Mix by swirling not shaking) and spin at 120 x g for 3 min. Repeat the wash 2 times.
Add 300 μl 50% glucose in the autoclaved liquid Gamborg B5 plus vitamins + mannitol solution.
Wrap tubes in foil to keep them dark and leave them at 25C o/n.
Day 2
Prepare PRMB and PRMT and autoclave the media. After autoclaving, keep PRMT at 50°C.
Add glucose to PRMB and pour it in 90mm plates.
Spin cells down 120 x g for 4 min. Mix by swirling not shaking.
Remove the supernatant and gently re-suspend each pellet in 0.5 ml 8% mannitol if plating out on 3 plates, or in 0.8 ml if using 4 plates. Use mannitol from the 10ml 8% mannitol that is autoclaved separately.
Add 2.5ml PRMT to each cell suspension (that is sufficient for plating out on 3 plates). Quickly but gently mix by pipetting up and down and plate 1ml on each PRMB plate.