A novel method for the isolation and purification of protoplasts from friable,embryogenic corn (Zea Mays L.) callus

A method is described for the isolation of protoplasts from rapidly-growing, friable embryogenic and organogenic cell cultures of corn. A Sepharose 6MB cyanogen-bromide-activated macrobead column coupled with Cellulase RS was used to separate contaminating cells from protoplasts. The column consists of layering 1.5 cm of the coupled-macrobeads into a 2.2-cm diameter column. Contamination of protoplasts by cells possessing partial or complete walls was reduced from 25% to near zero after a single passage through the column. The column was capable of retaining in excess of 30 million cells and recovering 99% cell-free preparations from culture material consisting of less than 1% protoplasts. Coupled-macrobeads were easily recovered, washed free of cells and stored for repeated use. Corn protoplasts appeared undamaged by the column and rapeseed (Brassica napus) protoplasts which were passed through the column have divided and formed colonies in culture. Uncoupled macrobeads were not as efficient as coupled macrobeads in reducing cellular contamination.