Identification of enzymes that are effective for isolating protoplasts from grass leaves [Trichoderma viride, Fungi]

Cellulase C1, cellulase Cx, and xylanase were isolated and purified from a cellulase preparation of Trichoderma viride as enzymes effective in the isolation of protoplasts from oat leaves. Pectin lyase which is specific for methyl-galacturonide linkages was also found to be a useful enzyme for the isolation of protoplasts from the tissues. This suggested that pectic polysaccharides with a high degree of esterification may play an important role in cell walls of Gramineae. It was necessary to use the mixture of cellulase C1, cellulase Cx, xylanase, and pectin lyase for the rapid isolation of protoplasts, while a small amount of protoplasts could be isolated from oat leaves by cellulase C1 plus xylanase or cellulase C1 plus pectin lyase. The mixture of four enzymes also was effective in the isolation of protoplasts from the leaves of wheat, barley, and corn.