Abstract:The exine which contains chemically resistant sporopollenin makes a severe obstacle for obtaining pollen protoplasts A technique was established for protoplast isolation in large number from mature pollen grains in three plant species i. e. Iris tectorum, Zephyranthes grandiflora and Hemerocallis fulva. Dehiscence of exine by the force of pollen hydration and consequentely exposure of intine to the direct action of enzymatic maceration led to successful release of protoplasts from pollen grains The highest isolation rate up to 90% protoplasts was obtained from mature pollen grains of Z. grandiflora. Pollen protoplasts from tetrads to mature pollen, except early uninucleate pollen stage, were prepared in H. fulva Purified pollen protoplasts were obtained by repeated centrifugation and density gradient centrifugation and proved to be viable by fluorochromatic reaction with FDA. When cultured in vitro, the pollen protoplasts of H. fulva underwent regeneration of cell wall, formation of various tube-, nodule-, bead- and dumbbell-shaped structures and sometimes division of the generative nucleus into 2-4 nuclei. Exogenous hormone showed promotive effect on the growth of above mentioned structures.