Enzymatic Isolation of Protoplasts from the Rice Leaves and Callus Cultures

Abstract

The isolation of rice protoplasts from leaves, roots and callus cultures has been examined following the inoculation in cell wall-degrading enzyme solution. It was achieved within 2 hours at 35°C with the materials cut into small pieces using the mixed solution containing macerozyme, cellulase and mannitol etc. The suitable concentration of enzymes and mannitol to isolate the leaf, root and callus protoplasts was ascertained. The size of protoplasts was measured in their diameter and the frequency distribution was acquired. The size was large in the callus protoplasts and small in the leaf ones. Structure of the protoplasts was investigated with the semithin sections by light microscope. Nature of nuclei, nucleoli and lamellar structure in chloroplasts are illustrated. The spontaneous fusion of protoplasts was assumed from the presence of polynuclear protoplasts. In addition, the protoplast isolation from flower petals was performed successfully using carnation and Freesia. We gratefully acknowledges Dr. T. Katayama and Dr. T. Omura, Faculty of Agriculture, Kyushu University, for encouraging the development of this work. Technical assistance was rendered by Mrs. K. Maeda, to whom we are also very grateful. © 1974, CROP SCIENCE SOCIETY OF JAPAN. All rights reserved.