Protoplast isolation and PEG-mediated transformation

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 CaCl_2.H₂O [MW = 147.01 g/mol]
• 150 mM MgCl₂[MW = 95.211 g/mol]

A	B	C
Component	Final [C]	Amount (per 100 mL)
100 mM MES-KOH (pH 5.7)	2 mM	2 mL
500 mM NaCl	154 mM	30.8 mL
500 mM CaCl2	125 mM	25 mL
100 mM KCl	5 mM	5 mL
dd-H2O		37.2 mL

W5 solution (per 100 mL)

A	B	C
Component	Final [C]	Amount (per 100 mL)
100 mM MES-KOH (pH 5.7)	4 mM	4 mL
1 M Mannitol	400 mM	40 mL
150 MgCl2	15 mM	10 mL
dd-H2O		46 mL

Mannitol-MgCl2 solution (per 100 mL)

A	B	C
Component	Final [C]	Amount (per 100 mL)
100 mM MES-KOH (pH 5.7)	4 mM	4 mL
1 M Mannitol	500 mM	50 mL
100 mM KCl	20 mM	20 mL
dd-H2O		26 mL

WI solution (per 100 mL)

Grow Arabidopsis or wheat under 12-hour photoperiod at 21 °C and 50% relative humidity, with approx. 100 µmol photons m^-2 s^-1light 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.

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).

Prepare enzyme solution.

A	B	C	D	E
Component	Arabidopsis		Wheat
	Final [C]	Amount	Final [C]	Amount
100 mM MES-KOH (pH 5.7)	20 mM	2 mL	20 mM	2 mL
1 M Mannitol	600 mM	6 mL	600 mM	6 mL
100 mM KCl	20 mM	2 mL	10 mM	1 mL
cellulase R-10	1.5 % (w/v)	150 mg
cellulase RS			1.5 % (w/v)	150 mg
macerozyme R-10	0.4 % (w/v)	40 mg	0.75 % (w/v)	75 mg
500 mM CaCl2	10 mM	200 uL	10 mM	200 uL
BSA	0.1 % (w/v)	10 mg	0.1 % (w/v)	10 mg
dd-water				800 uL

Cell wall digestion composition for Arabidopsis and wheat (per 10 mL). 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)

A	B	C
Component	Final [C]	Amount (per mL)
PEG-4000	40% (w/v)	400 mg
1 M Mannitol	200 mM	200 uL
500 mM CaCl2	100 mM	200 uL
dd-H2O		600 uL

40% PEG-calcium transfection solution Add all components and mix until dissolved (1h). Make fresh on the day of transfection.

Prepare a clean cutting board, tweezers, and a fresh, sharp razor or scalpel.

Arabidopsis leaves

1. Excise fully-expanded Arabidopsis rosette leaves (typically, leaves 4-7 from well-watered 3 week-old plants).
2. 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.
3. 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.

Wheat leaves

1. Excise the primary leaf of approximately 1-week old wheat plants.
2. 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).
3. Place tissue strips mesophyll side down in 0.6 M mannitol for 15 minutes (dark).
4. Continue to make strips until you have sufficient material (mesophyll strips from 1 leaf should yield at least 1 million protoplasts).
5. Transfer peels to enzyme solution.

Vacuum infiltrate for 30 minutes in a desiccator then release pressure gently and evenly.

Incubate in the dark at 23 °C for ≥ 3 hours with gentle rotation (~40 rpm).

Add equal volume W5 and mix with gentle swirling for 10 seconds. The solution should turn green as protoplasts are released.

Check for protoplast release under light microscope ( Arabidopsis mesophyll protoplasts are ~30-50 µm in diameter).

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.

Centrifuge solution for 3 minutes at 80 rcf (soft ramp / brakes off).

Remove majority of supernatant then resuspend protoplasts with gentle swirling (intact protoplasts will resuspend quickly whereas broken cells and free chlorophylls will not).

Wash once more with equal volume W5 solution, centrifuge, and remove supernatant (resuspend protoplasts by swirling before moving on).

Add half-volume W5 solution and rest protoplasts on ice for ≤ 30 minutes. Protoplasts will settle faster to the bottom.

Remove as much supernatant as possible without disturbing the pellet and add half-volume MMg solution.

Centrifuge solution for 3 minutes at 80 rcf (soft ramp / brakes off).

Remove supernatant then resuspend protoplast in 1-2 mL MMg solution.

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.

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)

Adjust protoplast density to 3x10⁵ protoplasts mL^-1 MMg.

Keep protoplasts in the dark at 23 °C (approx. room temperature) until ready to transfect (<8 hours).

Prepare clean 2 mL microcentrifuge tubes and determine plasmids to be transformed.

Add 10-20 μg of plasmid DNA into all tubes (10-20 μL).

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.

Incubate for at least 5 minutes at 23 °C (up to 15 minutes).

Add 2x volume of W5 solution (~840 μL) and mix by gentle inversion.

Centrifuge for 3 minutes at 80 rcf (soft ramp/ brakes off).

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).

Incubate protoplasts in the dark at 23 °C for 16-48 h.

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.

Measure fluorescence using a flow cytometer (e.g. BD FACS Aria II, Invitrogen Attune NxT).