NaH2PO4, 2% Onozuka RS cellulase, 2% Macerase, at pH
5.6. Seal dishes with Nescofilm and incubate overnight on
a shaker slowly rotating at 20 rpm, either in low light or
darkness.
For the leaf parent donor, fully expanded but not completely
hardened off leaves should be taken from plants
grown either in a growth chamber or heavily shaded
greenhouse. Alternatively, in vitro grown leaves are
desirable because they do not require decontamination
prior to protoplast isolation. Leaf material can be decontaminated
by immersion in 1 N HCl for a few seconds
followed by a 12–15 min immersion in 10–15% commercial
bleach (6% sodium hypochlorite) containing 3 drops of
Liquinox soap or another suitable surfactant, followed by a
5-min rinse and two 10-min rinses in double-distilled H2O.
Damaged vascular tissue and the leaf midveins should be
removed with a sharp scalpel. Remaining leaf material is