Wheat Protoplast Preparation and Transformation

¾ fill a 10cm diameter pot with medium-grain potting mix without fertiliser1. Sprinkle 10 to 20 seeds on top and fill pot to just below rim

1. Move to growth cabinet set at 21°C with 16 hr days
2. Place pots in shallow black tray and dampen mix with water mixed with complete soluble fertiliser (Hortico brand, complete mix – 1 scoop/1L)
3. Add the fertiliser water to the tray, filling to approx 3cm below rim.
4. Check the plant at day 5 and top-up with plain water. Keep well-watered as health of plant is essential to protoplast success.
5. Plants are ready to use after 7-9 days (2nd leaf just emerging) Some wheat cultivars may emerge more slowly so adjust as necessary

Prepare fresh on day of use & don't reuse:

A	B	C	D
	Volume	Directions	Final Concentration
MES-KOH (0.2 M)	0.5 mL		20 mM
Mannitol (0.8 M)	3.75. mL		0.6 M
KCI (2 M)	25 μL		10 mM
		55ºC for 5 min (water bath)
Cellulase 'Onozuka' R-10 or RS	75 mg		1.5 % w/v
Macerozyme R-10	37.5 mg		0.75 % w/v
		55ºC for 10 min
		cool to RT
CaCl2 (2 M)	25 μL		10 mM
BSA (10% w/v)	50 μL		0.1 % w/v
H2O (Milli-Q)	to 5 mL	mix gently
		filter 0.45 μm to ensure enzyme is completely dissolved

Add 10 mL of 0.6 M Mannitol (filter sterilised) to a 7 cm petri-dish1. Using a blunt razor, make a shallow cut in the abaxial or adaxial epidermis, and carefully peel epidermal layer off.

1. Place the leaf, peeled-side down in the Mannitol for 10 mins (any more than 20 mins and the leaves get too floppy) – try to fill the petri dish with as many leaves as possible, but be careful not to overlap them
2. Add 10 mL of the enzyme solution (freshly made) to another 7 cm petri-dish
3. Transfer the leaf peels from the mannitol to the enzyme solution using tweezers – being careful to touch them as little as possible
4. Wrap the petri-dish in foil and incubate in the dark at room temperature with gentle rotation on orbital shaker (horizontal movement at 60 RPM) for three hours. Gently tap/swirl dish to release protoplasts, leaves will appear transparent.

Prepare PEG-Ca solution (shake vigorously to dissolve PEG) Make freah each day, don't reuse| A | B | C | | --- | --- | --- | | | Volume | Final Concentration | | PEG 4000 | 1200 mg | 40 % w/v | | Mannitol (0.8 M) | 750 μL | 0.2 M | | CaCls (2 M) | 150 μL | 100 mM | | H2O | to 3 mL | |

1. Dispense amount of W5 and PEG required for transfection according to setup
2. Thaw 5% BSA and label culture plates for incubation

Prepare Plasmid DNA for Transfection step:

1. Set up number of round-bottom tubes required according to setup sheet
2. Thaw plasmid DNA, flick and spin tubes to ensure homogenous mixture. Combine aliquots where needed to avoid variation between plasmid DNA batches. Freeze/thaw each aliquot three times only.
3. If plasmid appears viscous, heat at 65^oC for 10 min. This is due to high concentration of plasmid DNA (this may also affect concentration reading).
4. Dispense required amount into bottom of tubes (usually 20 µg reporter plasmid and 10-15 µg of Avr/R plasmid). Use a new tip each time to prevent cross-contamination.

Add 5 mL of W5 solution to petri dish1. After three hours, remove the leaves from the enzyme solution using tweezers, gently lift up and down to release trapped protoplasts

1. Place a cell strainer in the lid of the petri-dish, add 1 mL of W5 to wet the strainer, then filter the protoplasts solution (make sure not to drop them from a height)
2. Using a funnel, transfer the filtered protoplasts to a 30 mL plastic, round bottomed tube, and add another 5 mL of W5
3. Gently swirl the protoplasts to mix, until just homogenous
4. Pellet at 100 x g for 3 mins
5. Carefully remove the supernatant (make sure not to suck up any of the protoplasts, and if you do, don’t pipette them back into the solution – once they’re gone, they’re gone, pipetting will destroy them)
6. Add 15 mL of W5
7. Estimate the concentration using a haemocytometer (at this stage there may be some chloroplasts/debris visible still)
8. Calculation: total number of cells = (average cells of 4 squares counted) x 10,000 x initial volume x dilution factor (Recommended: 5 µL protoplasts and 20 µL W5, (4x dilution) add 8 µL to each side of the haemocytometer)
9. incubate on ice in the dark for 40 mins or until the protoplasts have settled to the bottom of the tube (up to 1 hr)
10. Remove the supernatant, and adjust to 300,000-350,000 protoplasts/mL using MMG

Protoplasts are now ready for the transformation step (they should be used ASAP)

Choose either option A for manual pipette or option B for electronic multichannel pipette (e.g. Integra Viaflo Voyager 8 tip 1250 µL pipette with adjustable spacing)

Transfection: Option A, manual pipette

1. Make sure all the PEG solution is dissolved
2. Pre-load a pipette with 300 µL of PEG (use wide bore tips or cut tips for all protoplast handling steps)
3. Gently swirl the protoplasts to resuspend, and make sure the suspension is homogenous - protoplasts will settle to the bottom of the tube very quickly
4. Add 300 µL of protoplasts to the DNA (in prepared tubes), swirl to mix and quickly add the 300 µL of PEG. Gently swirl to homogenise (PEG is highly viscous so be sure there is no clear layer at bottom of tube)
5. Repeat for all transformations, and incubate for 15 minutes
6. Add 900 µL of W5 to stop the transformation
7. Pellet the protoplasts at 100 x g for 2 mins
8. Remove as much of the supernatant as possible, without losing any of the protoplasts (it’s better to keep the protoplasts than get all of the supernatant)
9. Coat the wells of a sterile 12 or 24 well cell culture plate with 5% BSA (filter sterilised) by adding 1 mL to the first well, and transferring to the next, leaving a coating behind
10. Resuspend in 500 µL of modified W5 (W5 + 1 mM glucose), then add to the culture plate using wide bore tips (or cut tips)

Incubate for 4 to 16 hours (depending on reporter expression) in light, at room temp

Transfection: Option B, multichannel electronic pipette

1. Setup prepared plasmid DNA in tubes in groups of 8. Alignment can be tricky as pipette is on maximum spacing, so manually move them to the same side of each well. Alternatively, use every 2nd tip to accommodate larger tubes.
2. Dispense protoplasts, PEG and W5 into reservoirs (add 10% extra volume, 1 extra rep per 10 samples). Protoplasts will settle very quickly so agitate gently by lifting one side of the reservoir before pipetting
3. Set electronic pipette to ‘protoplasts300’ program as follows:
4. Pipette 300 µL (protoplasts) and mix immediately. Eject tips.
5. Pipette 300 µL (PEG) and mix immediately (Pipette up and down 3x slowly)
6. Pipette according to program. Check tubes are mixed thoroughly.
7. Incubate 15 mins
8. Add 900 µL W5 to stop reaction
9. Pellet the protoplasts at 100 x g for 2 mins
10. Remove as much of the supernatant as possible with a manual pipette, without losing any of the protoplasts (it’s better to keep the protoplasts than get all of the supernatant)
11. Coat the wells of a sterile 12 or 24 well cell culture plate with 5% BSA (filter sterilised) by adding 1 mL to the first well, and transferring to the next, leaving a coating behind
12. Resuspend protoplasts in 500 µL of modified W5 (W5 + 1 mM glucose), then add to the culture plate by manual pipette.

Incubate for 4 to 16 hours (depending on reporter expression) in light, at room temp

Transfer well contents to 2 mL tubes1. Centrifuge 300 x g for 3 min at RT

1. Add 200 µL 1x lysis buffer to each tube
2. Vortex, incubate for 15 min
3. While waiting, prepare PCR tube strip of Luciferase assay substrate for multi-channel pipette
4. Centrifuge 300 x g for 3 min at RT
5. Add 50 µL lysate into white, opaque bottom 96 well plate

Go to plate reader for final addition of substrate:

1. Set up plate reader for dual luciferase reading, check that white, opaque bottom plate type is selected (e.g. Corning flat white)
2. Add 50 µL luciferin (Promega Steady-Glo) with multichannel pipette - pipette in/out 2 times to mix
3. Incubate 5 mins
4. Read using luminometer (1000 ms integration, 0 ms settle)

For ratiometric assay: (normalisation of ELuc readings with pRedF) select red and green filters to run immediately following total luminecesence measurement. Some plate readers have presets for the Chroma-Glo™ Luciferase Assay System. Otherwise select green and red filters.

Perform deconvolution calculations using measurements from red and green filters, using spreadsheet template or Python script (also includes plotting). Note that deconvolution constants may vary between plate readers, requiring calibration measurements as described in Promega Chroma-Glo™ Luciferase Assay System Technical Manual (page 9).

Spreadsheet template for deconvolution calculations

Protoplast luminescence deconvolution and plotting.docx

Template for results input

Results_Template.xlsx

Instructions for plotting script

RatiometricAssayDeconvolutionCalculations.csv

Script

ADA_plotting_script.py