Plant cell protoplast isolation
Grow plants of Arabidopsis thaliana (ecotype Col0) on a 3:1 (v/v) compost to vermiculite mixture at 20oC, under short day conditions (12h light:12h dark) and low light intensities (50-100µE m-2 s-1).
Prepare fresh enzyme solution and transfer to glass petridish.
Remove 20-40 leaves from 4 week old Arabidopsis thaliana rosettes.
Pile up 5-10 leaves at a time, cut into thin (~2mm) strips across the leaf using a sharp scapel blade dipped into ethanol.
Place strips into enzyme solution.
Repeat until all leaves have been cut.
Vacuum infiltrate the enzyme solution.
Leave cells in the petri dish at 20oC without shaking.
Twice filter cells and enzyme solution through a 125μm nylon mesh to remove residual debris into a pointed bottom, glass test tube.
Leave cells to settle to the bottom of the tube.
Carefully aspirate out the liquid solution leaving the cell pellet intact.
Add Incubation Buffer.
Gently resuspend protoplast in incubation solution by light swirling of the test tube.
Check protoplast integrity and determine yield using a disposable hemocytomer.
