Fig. 1.

Protoplast isolation from Arabidopsis seedlings. a Ten-day-old seedlings grown under long-day (LD; 16 h light/8 h dark) conditions. b Preplasmolysis of seedlings in 0.5 M mannitol. c Protoplast isolation in enzyme solution. d Microscope image of isolated protoplasts at 12 h post-incubation in enzyme solution. e Overlaying protoplast solution onto 0.6 M sucrose during sucrose density gradient-based protoplast purification. f Viable protoplasts in the middle of the sucrose gradient solution after centrifugation. Arrow indicates the protoplast layer. g Immobilization of protoplasts in Ca²⁺-alginate hydrogel. h Protoplast culture in 4 mL PIM. i Protoplasts in PIM-incubated alginate hydrogel. White scale bars = 1 cm; black scale bars = 50 µm