Protoplast preparation (from plant tissue)

PROTOPLAST PREPARATION (FROM PLANT TISSUE)

Last Update: December 2006

PREPARE SOLUTIONS

1. GM 15.4% sucrose (1 L):

Mix 800 mL of dH₂O, 3.2 g of Gamborg's B-5 (G5893), and 154 g of sucrose. pH to 5.7 with KOH and add dH₂O to 1 L. Filter through a 0.45m filter (Autoclave)

2. Protoplast Solution (in GM 15.4% sucrose) (1 L):

Mix 800 mL of dH₂O, 3.2 g of Gamborg's B-5 (G5893), 200 mg of Cellulase (O -R10), 154 g of sucrose, and 50 mg of Macerozyme (O -R10). pH to 5.7 with KOH and add dH₂O to 1 L. Filter through a 0.45m filter (Autoclave)

3. Betaine (1 L):

Mix 800 mL of dH₂O, 54 g of Betaine-H₂O (B2754), 0.58 g of Mes (M8250), and 1.47 g of CaCl₂-2H₂O. pH to 5.7 with KOH. Add dH₂O to 1 L (Autoclave)

PROCEDURE

1. Sterilize a 80m mesh, a large (1 L) and a small (100 mL) beaker, a funnel, 150 mm/100 mm petri plates (weigh them before use), a spatula and babcock bottles by spraying with EtOH. Leave in the hood until they dry: >20 mins

2. Place the funnel on top of the beaker and the mesh on top of the funnel

3. Pour Arabidopsis cell suspension cells over the mesh, letting most of the old media filter through the mesh into the beaker.optional: can rinse cells with some GM 15.4% sucrose

4. Scoop cells into an appropriate container for digestion:

4.1 If using a 150 mm glass pettri plate, add ~20 g of cells (close the plate before taking it outside the hood to weigh it)

4.2 If using a 100 mm glass pettri plate, add ~10 g of cells

NOTE: the amount of cells used will depend on the amount of protoplasts needed and the age of the cells

5. Add 25 mL of Protoplast Solution, swirl to mix and add ~20-30 mL of GM 15.4% sucrose solution

The idea is to have the mixture dilute enough that the swirling and movement of cells is evident when shacking: this will expose the most cells to the enzymes. GM 15.4% sucrose solution should be added accordingly

6. Incubate in the dark for 1.5 - 2.5 hours on a rotary shacker at 50-80 rpms (again, shake so that a clear swirling of the media is evident. The speed will vary depending on the amount of cells and the volume of the solution)

NOTE: longer incubations lead to unhealthy non-viable cells

7. Pour digested cells into a 100 mL beaker and from the beaker transfer the cells to a babcock bottle. Add enough GM 15.4% sucrose solution to the 100 mL beaker and use this to fill the babcock bottle

Optional: if cells clump, pippet the cells up and down using a 10 mL pippet (4 times up and down) to break clumps before tranferring to babcock bottles.

8. Float protoplasts by centrifugation at 1,100 rpms (~50g) in an IEC HNSII clinical centrifuge swinging bucket rotor for 5-10 mins at room temperature

9. Collect floated protoplasts (in the neck of the bottle) using a sterile long Pasteur pippet into a 50 mL conical tube containing ~20 mL betaine solution

10. Pellet protoplasts in a rotor at 50g (600 rpms in a Sorvall H100B rotor) for 5-10 mins at room temperature

11. Discard the supernatant by aspiration and resuspend protoplasts in an appropriate buffer (10 mL betaine)

NOTE: if a large volume of protoplats is being handled and/or the betaine is not clear after the first wash, repeat wash one more time

NOTE: 1 mL of packed cell volume of protoplats (1 pcv) is approximately equal to ~2.0 x 106 Arabidopsis protoplasts

NOTE: to prevent protoplast lysis, handle cells carefully all the time, specially when changing solutions