PROTOPLASTS FROM PHYTOLACCA-DODECANDRA LHERIT (ENDOD) AND PILULARIA-AMERICANA L (POKEWEED)

Pokeweed (Phytolacca americana L.) and ended (P. dodecandra L'Herit) p roduce ribosome-inactivating proteins which are sequestered in leaf ce ll walls. These proteins display strong antiviral activity. To aid in studying the antiviral mechanism, we developed protocols to isolate pr otoplasts from suspension culture cells and leaves. Ninety-five percen t of pokeweed or ended culture cells were converted to protoplasts usi ng 2% cellulase, 0.25% pectinase, 0.2 M mannitol, 2% sucrose, 15 mM Ca Cl2, Murashige and Skoog salts, pH 5.7. Viability was >85% after 24 h. Culture-derived protoplasts were purified by centrifugation through a 15% sucrose pad. Protoplasts collected from the supernatant were then pelleted in 0.3 M mannitol. Pokeweed leaves provided respectable yiel ds (4 x 10(6) protoplasts/g f w) of partially-purified viable protopla sts when digested in solution containing 1% cellulase, 0.2% Pectolyase , 0.4 M mannitol, CPW salts, 0.5 mM MES, pH 5.6. We were unable to com pletely separate cell debris from mesophyll protoplasts, which were sm all and easily damaged by centrifugation. Ended leaves were found to b e resilient to several digestion enzymes tested.