TITLE: Protoplast isolation and PEG-mediated transformation
AUTHORS: Diep R Ganguly, rebeccah.tyrrell  , Taj          Arndell

[DESCRIPTION]
Adapted from Yoo S-D, Cho Y-H, & Sheen J (2007) Nature Protocols and Arndell T et al (2019) BMC Biotechnology. Originally designed for the isolation of Arabidopsis mesophyll protoplasts to monitor RNA stability using transcriptional inhibitors (e.g. cordycepin) under oxidative stresses such as high-light (Crisp PA et al 2017 Plant Cell). The current version represents modifications to study the influence of 5' UTR sequence composition on gene expression in Arabidopsis and wheat.

[BEFORE_START]
It is recommended to protoplasts plants kept in complete darkness prior to isolation in order to deplete their starch reserves, making for a cleaner extract.
 
Ensure that you have high quality supercoiled plasmid DNA (1 μg/μL) for transformation - Check out Plasmid DNA on Agarose Gel: The Secret of the 3 Bands (bitesizebio.com). We perform Midipreps on 200 mL of culture grown until OD600 > 2 (approx. 24 hrs) using the NucleoBond® Xtra Midi Plus EF (including NucleoBond® Finalizers) kit (Machery-Nagel). 

[GUIDELINES]
Adapted from Yoo S-D, Cho Y-H, & Sheen J (2007) Nature Protocols and Arndell T et al (2019) BMC Biotechnology. Also check out https://molbio.mgh.harvard.edu/sheenweb/main_page.html.
 
Protoplasting is ideally performed using fully-expanded leaf tissue (leaves 4-8) of healthy 3 week-old Arabidopsis plants. Ensure the absence of any pests, including gnats and fungi, to maximise protoplast yield, viability, and transfection efficiency. Handle protoplasts gently (it is often recommended to keep on ice and in the dark as much as possible). Use chilled centrifuges with slow stopping (or brake off). Use pipette tips with cut ends or serological pipettes for larger volumes.

As a guide, 30 leaves in 30 mL enzyme solution yields ~2.5x106 protoplasts, from which 3 μg RNA can be isolated with a TRIzol-based extraction. For greater output: Yoo et al  (2007) suggest ~100–150 leaves per 40–60 mL enzyme solution yielding 1x107 protoplasts.

[STEPS]
SECTION: Preparation
2. Grow Arabidopsis or wheat under 12-hour photoperiod at 21 °C and 50% relative humidity, with approx. 100 µmol photons m-2 s-1 light intensity on soil (Premium potting mix, Martins fertilizers) supplemented with fertiliser (1 g/kg Osmocote; Arabidopsis: 3 weeks, wheat: 1 week) or (Arabidopsis only) nutrient-rich media (half-strength Gamborg B5 or Murashige Skoog media supplemented with 1% sucrose; 1-2 weeks). Ensure plants are stress-free, especially from droughts, pathogens, and pests.
SECTION: Preparation
1. Prepare the following solutions. Ensure all buffers are sterile (e.g. filter sterilize using 0.45 µm PVDF membrane).

Stock solutions
100 mM MES-KOH  (pH 5.7) [MW = 195.24 g/mol]
1 M Mannitol [MW = 182.17 g/mol]
100 mM KCl [MW = 74.55 g/mol]
500 mM NaCl [MW = 58.44 g/mol]
500 mM CaCl2.H2O [MW = 147.01 g/mol]
150 mM MgCl2 [MW = 95.211 g/mol]

 

 

 

SECTION: Maceration
4. Prepare enzyme solution.
 
Important steps:
After mixing digestion enzymes with MES, mannitol, KCl, and water; warm solution to 55°C for 10 min with occasional swirling (inactivates nucleases and proteases, and aids solubilising enzyme). 
Allow to cool then supplement with CaCl2 and BSA.
Filter-sterilize digestion medium using a 0.45 µm PVDF membrane.

Prepare 40% PEG solution (need approximately 200 uL per transformation)
 
Add all components and mix until dissolved (1h). Make fresh on the day of transfection.
SECTION: Maceration
8. Vacuum infiltrate for 30 minutes in a desiccator then release pressure gently and evenly.
SECTION: Maceration
9. Incubate in the dark at 23 °C for ≥ 3 hours with gentle rotation (~40 rpm).
SECTION: Resuspension
13. Centrifuge solution for 3 minutes at 80 rcf (soft ramp / brakes off).
SECTION: Resuspension
23. Keep protoplasts in the dark at 23 °C (approx. room temperature) until ready to transfect (<8 hours).
SECTION: Resuspension
12. Filter solution through 50 or 70 µm cell strainer into a falcon tube (or round bottom centrifuge tube). Keep solution on ice and shielded from strong, direct light.
SECTION: Maceration
10. Add equal volume W5 and mix with gentle swirling for 10 seconds. The solution should turn green as protoplasts are released.
SECTION: Preparation
3. Perform protoplast isolation as early in the day as possible to limit starch accumulation, which should result in a cleaner extract (can move plants to dark on the morning of isolation).  Keep plants in the dark as much as possible during protoplast isolation (e.g. turn room lights off, cover digest in foil).
SECTION: Resuspension
20. Determine protoplast by counting cells on a haemocytometer. 

Load 10 μL of protoplast suspension and use a light microscope. Count the total number of protoplasts across 3-5 squares (count intact protoplasts only, which should be perfectly spherical).

Protoplast density (mL-1 MMg) = (# protoplasts / # squares) x 104 x Dilution Factor

Also check for lysed cells, free chlorophylls, and epidermal debris, which reduces transformation efficiency. 
SECTION: Resuspension
14. Remove majority of supernatant then resuspend protoplasts with gentle swirling (intact protoplasts will resuspend quickly whereas broken cells and free chlorophylls will not).
SECTION: Resuspension
16. Add half-volume W5 solution and rest protoplasts on ice for ≤ 30 minutes. Protoplasts will settle faster to the bottom.
SECTION: Resuspension
17. Remove as much supernatant as possible without disturbing the pellet and add half-volume MMg solution.
32. Supplement protoplast solution with 10 μg/mL propidium iodide (λEx = 535 nm; λEm = 617 nm) to stain for cell integrity. Mix with gentle flicking. Do this as close to measurement as possible.
33. Measure fluorescence using a flow cytometer (e.g. BD FACS Aria II, Invitrogen Attune NxT).
SECTION: Maceration
6. Arabidopsis leaves
Excise fully-expanded Arabidopsis rosette leaves (typically, leaves 4-7 from well-watered 3 week-old plants).
Cut the mid-section of leaves (i.e. remove leaf apex and base) into approximately 1-mm strips (perpendicular to midvein) using a sharp razor blade (on a solid, clean cutting surface). Do not crush or grind the tissue, instead allow the blade to glide across tissue.
Immediately place leaf strips into enzyme solution, ensuring to dip both sides in the digestion medium (to ensure cutting edges are exposed to solution), using clean flat-tip forceps.


SECTION: Maceration
7. Wheat leaves
Excise the primary leaf of approximately 1-week old wheat plants.
Peel away the epidermis of the abaxial layer to expose the mesophyll. This can be achieved by lightly scoring the abaxial side of the leaf with a sharp razor blade, then bending the leaf along that section to create a tab. Pull this tab using forceps to remove the epidermis (Arndell et al 2019 BMC Biotechnology).
Place tissue strips mesophyll side down in 0.6 M mannitol for 15 minutes (dark).
Continue to make strips until you have sufficient material (mesophyll strips from 1 leaf should yield at least 1 million protoplasts). 
Transfer peels to enzyme solution.
SECTION: Maceration
5. Prepare a clean cutting board, tweezers, and a fresh, sharp razor or scalpel.
SECTION: Resuspension
18. Centrifuge solution for 3 minutes at 80 rcf (soft ramp / brakes off).
SECTION: Resuspension
22. Adjust protoplast density to 3x105 protoplasts mL-1 MMg.
SECTION: Transformation
24. Prepare clean 2 mL microcentrifuge tubes and determine plasmids to be transformed.
SECTION: Transformation
27. Incubate for at least 5 minutes at 23 °C (up to 15 minutes).
SECTION: Transformation
25. Add 10-20 μg of plasmid DNA into all tubes (10-20 μL).
SECTION: Transformation
26. Perform the following steps in quick succession: 
(I) Add 30,000 - 60,000 protoplasts (100 - 200 μl of 3x105 protoplasts mL-1 MMg) and mix with gentle flicking.
(II) Add equal volume 40% PEG-calcium solution and mix with gentle flicking.

Notes: 
Minimise incubation time of protoplasts with DNA only to improve transfection efficiency (Arndell et al 2019).
Continually swirl protoplast solution to prevent sedimentation and achieve equal distribution. 
Mass of plasmid DNA to protoplast number is important, make sure to optimise for each experiment. Currently, we are transfecting with a ratio of 1 μg : 3,000 protoplasts.
SECTION: Transformation
28. Add 2x volume of W5 solution (~840 μL) and mix by gentle inversion.
SECTION: Transformation
29. Centrifuge for 3 minutes at 80 rcf (soft ramp/ brakes off).
SECTION: Transformation
30. Discard supernatant and resuspend protoplasts in culture plate wells (e.g.  Sarstedt 83.3921.500) with 1 mL (6-well ), 0.5 mL (12-well), or 0.25 mL (24-well) W5 or WI solution (W5 is preferable for FACS due to lower mannitol, however, be sure to continually swirl protoplasts to avoid sedimentation).
SECTION: Transformation
31. Incubate protoplasts in the dark at 23 °C for 16-48 h.
SECTION: Maceration
11. Check for protoplast release under light microscope (Arabidopsis mesophyll protoplasts are ~30-50 µm in diameter).
SECTION: Resuspension
15. Wash once more with equal volume W5 solution, centrifuge, and remove supernatant (resuspend protoplasts by swirling before moving on). 
SECTION: Resuspension
19. Remove supernatant then resuspend protoplast in 1-2 mL MMg solution.
SECTION: Resuspension
21. Adding cell stains prior to counting on a haemocytometer can help identify broken protoplasts:
0.1% (w/v) trypan blue 
10 μg/mL propidium iodide (λEx = 535 nm; λEm = 617 nm)