feathered or cut into thin strips with a sharp scalpel and
incubated in 3 ml of Enzyme Solution (see above) combined
with 8 ml 0.7 M BH3 medium in a 125 ml sidearmed
Erlenmeyer flask (with the side-arm covered with
Miracloth to prevent contamination). Leaf material in the
enzyme cocktail is evacuated for 15 min at 50 kPa to
facilitate enzyme infiltration. Preparations are sealed and
incubated as above. It should be noted that newly initiated
and younger callus/suspension cultures consist of cells with
higher starch content. This can reduce protoplast yields and
viability due to higher levels of protoplast breakage. This
problem diminishes over time with continuous subcultures