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Production of Protoplasts from Plant Cells in Liquid Culture Using Purified Commercial Cellulases ¹

Viable protoplasts were obtained from cells of 19 higher plant species grown in liquid shake culture utilizing purified commercial celluiases. Crude commercial cellnlases produced dead protoplasts. Viable protoplasts were obtained after purification by extraction with acidified acetone, fractional precipitation with ethanol, gel-filtration, and charcoal adsorption. Results suggested that, in addition to cellulase, other enzymes present in commercial celluiases may be required for release of protoplasts from the cell walls. Protoplast formation from living unstained cells was observed in microcultures using phase microscopy. Protoplasts of some species survived only a few hours, but others survived for as long as 2 weeks. The routine production of protoplasts from a consistent source of enzyme is considered to be the first step toward the goal of producing somatic hybrids.

Key Words: Plant cell culture • Enzymes • Somatic hybridization

² Research Associate, Department of Plant Pathology, and Professor of Plant Pathology, University of Wisconsin, Madison 53706.

Received for publication March 11, 1969.