PREPARATION AND FUSION PROPERTIES OF PROTOPLASTS FROM MATURE POLLEN OF NICOTIANA-TABACUM

A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm.kg(-1) H2O)of pollen protoplasts req uired a special maceration medium, 4% KCl (w/v). Action of an enzyme s olution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave ris e to viable protoplasts within 4 hours. When cultured in a tobacco mes ophyll protoplast culture medium, the pollen protoplasts underwent reg eneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/C a2+ method pollen protoplasts were fused with diploid mesophyll protop lasts. Evidence of transfer of chloroplasts into the pollen protoplast s was observed after one day of culture.