Subcellular changes during isolation and culture of tobacco mesophyll protoplasts

Summary

Mesophyll protoplasts isolated by treating tobacco leaves with macerozyme and cellulase were cultured in a modifiedMurashige, andSkoog medium (1962) containing 1% agar. Changes occurring in the cells during isolation and culture were followed by light and electron microscopy. During protoplast preparation, pseudocrystals appeared in the chloroplasts and spherical portions of the chloroplast stroma became surrounded by membranes and cut out into the cytoplasm. During culture, the chloroplasts lost three quarters of their volume, thylacoids were reduced and pseudocrystals disappeared. The cytoplasm at first became vacuolated and poor in ribosomes but, by the third day of culture, it was enriched in membranes and polyribosomes, indicating high metabolic activity. In the cells as a whole there were two types of change; first, a shock phase consequent upon protoplast isolation and second, a phase of very rapid dedifferentiation to a meristematic state.