Protoplast Isolation and Transformation

Protocol

  1. Prepare D-mannitol Solution
    • Make sure it is fully dissolved; transfer into a sterilized 50 ml flask
  2. Prepare the Enzyme Solution
  3. Prepare plant material
    • Cut healthy and young Tragopogon seedling (2-3 weeks in ½ MS germination medium) leaves into 0.5 mm small pieces
    • Before cutting, blot the leaves on Kimwipes
    • Slice the leaves on a piece of glossy paper (from flyers/magazines) with a blade; do not press the blade vertically
    • To avoid over-dry, transfer the small leaf pieces into the D-mannitol buffer occasionally
  4. Transfer the small leaf pieces into D-mannitol solution, and shake at 30-50 rpm, room temperature for ~15 min; the flask is covered by foil
    • Make sure all leaf pieces are in the solution
  5. Remove the D-mannitol solution using a trimmed 1 ml pipette tip; avoid taking plant materials away
  6. Add enzyme solution from step 2 into the flask
    • As softly as possible using trimmed pipette tip
    • Make sure all leaf pieces are in the solution
  7. Vacuum for ~45 min under dark
  8. Shake (30-40 rpm; 25-26 °C; under dark) for ~5 hours
    • Check the results regularly
  9. Filter the enzyme solution (containing protoplasts) through 40 mm nylon meshes
    • Shake the flask slowly before transfer (avoid the protoplasts sink at the bottom)
    • Using trimmed tips
    • Add W5 Solution, wash the flask first, and then transfer the solution onto the nylon meshes
  10. Wash the protoplast using 21% Sucrose Solution (2.1 g sucrose in 10 ml water)
    • Add 5 ml 21% sucrose into a 15 ml round bottom falcon tube
    • Using trimmed tips, add protoplast solution (~ 5ml) from the petri dish softly on top of the 21% sucrose buffer; therefore, 4 tubes are needed as the volume of the protoplast solution is > 15 ml (15 ml enzyme solution + W5 solution)
    • Swirl the petri dish slowly when transferring protoplast solution (protoplasts are at the bottom); petri dish can be washed by W5 solution
  11. Centrifuge at 250 g for 3 min
  12. Take away the middle layer (containing protoplasts)
    • About 2 ml
    • When pipetting, move the trimmed tip around the tube
    • Before pipetting protoplasts, could take away part of the top layer (~ 2ml) first
  13. Wash the protoplast using W5 solution
    • Add 3 ml W5 solution into a new 15 ml falcon tube
    • Add protoplasts solution (~2 ml) softly into the tube; mix gently and slowly
    • Add 2 ml W5 solution; mix gently and slowly
  14. Centrifuge at 250 g for 3 min
  15. Remove supernatant using trimmed tips
  16. Wash the protoplasts again using W5 solution
    • Add 2 ml W5 solution into the tube; mix gently and slowly
    • Add another 2 ml W5 solution; mix gently and slowly
  17. Centrifuge at 100 g for 2 min
  18. Remove supernatant using trimmed tips
  19. Add 1 ml W5 solution, and put the tube on ice for 30 min (under light)
    • If using 4 tubes at step 10), mix two tubes into one at this step. Therefore, there will have two tubes, with 2 ml W5 solution in each one
  20. Centrifuge at 100 g for 2 min
  21. Remove the supernatant, and add 400 ml MMG Solution in each tube; mix gently and slowly
    • If there are 2 tubes, there will have 800 ml MMG, which would be enough for 3 transformations
  22. For each transformation, in the 2 ml tube (follow the order below)
    • Add 30 ml plasmid (10 µg for each construct)
    • Add 200 ml MMG (containing protoplast), and mix
    • Add 230 ml fresh PEG-4000 Solution, mix gently and slowly
  23. Put the 2 ml tube under dark for 15 min at room temperature
  24. Wash by W5 solution
    • Add 460 µl (1X volume) W5 solution; mix gently and slowly
    • Add 920 µl (2X volume) W5 solution; mix gently and slowly
  25. Centrifuge at 250 g for 3 min
  26. Remove the supernatant
  27. Add 400 ml W5 solution into each 2 ml tube; mix gently and slowly
  28. Prepare the cell culture plate
    • Coat the plate with 5% BSA (~600 µl)
  29. Transfer 400 ml W5 solution (containing protoplasts) into the well
  30. Cover cell culture plate using foil

Solution

D-mannitol Solution

 

For 10 ml

For 15 ml

Final concentration

D-mannitol

1.09 g

1.635 g

0.6 M

H2O

9.5 ml

14.25 ml

 

 

 

 

 

Enzyme Solution-see Figure below

 

 

 

W5 Solution

 

For 50 ml

Final concentration

MES (0.5 M; KOH, pH 5.7)

200 ml

2 mM

NaCl (1 M)

7.7 ml

154 mM

CaCl2 (1 M)

6.25 ml

125 mM

KCl (0.5 M)

0.5 ml

5 mM

H2O

~35.35 ml

 

Store at room temperature

 

 

 

 

 

MMG Solution

 

For 10 ml

Final concentration

MES (0.5 M; KOH, pH 5.7)

80 ml

4 mM

Mannitol (1 M)

4 ml

0.4 M

MgCl2 (1 M)

150 ml

15 mM

H2O

Up to 10 ml

 

Store at room temperature

 

 

 

 

PEG-4000 Solution

 

For 1 ml

For 5 ml

Final concentration

PEG-4000

0.4 g

2 g

 

Mannitol (1 M)

400 ml

2 ml

0.4 M

CaCl2 (1 M)

100 ml

0.5 ml

0.1 M

H2O

140 ml

700 ml

 

 

 

 

Enzyme Solution Details

Figure. Protoplast transient assays of Tragopogon porrifolius (3078). (a,b) Protoplasts transfected with Cas9, sgRNA and GFPm display green florescence at a rate of 6.8%. (c,d) Approximately 42.3% of protoplasts transfected with functional GFP emit green fluorescence. (e,f) No GFP signal is detected in protoplasts transfected with GFPm. Upper panels are from images under fluorescent light, whereas lower panels are from images under visible light.