PROTOPLAST CULTURE AND PLANT-REGENERATION OF DIFFERENT AGRONOMICALLY IMPORTANT BRASSICA SPECIES AND VARIETIES

Protoplast cultures were prepared from 6-day-old hypocotyls of six spr ing, seven winter cultivars of Brassica napus L. and one line of Brass ica campestris L. The molarity of enzyme solution was raised to 0,714 M mannitol resulting in well manipulable, cytoplasm dense protoplasts. In the protoplast purification procedure density gradient centrifugat ion was used to minimize physical damage of protoplasts. Three differe nt protoplast culture systems -(1) liquid, (2) 2nd day embedded, (3) d irectly embedded in low melting agarose were compared. The two differe nt protoplast embedding techniques resulted in the same efficiency of cell division as the liquid culture method and over this fact the colo ny browning was avoided. Using protoplast agarose-embedding and cultur e techniques, healthy calli were obtained for plant regeneration exper iments. Incorporation of silver nitrate into the regeneration medium i mproved the efficiency of plant regeneration in responsive genotypes a nd the regeneration was induced in three nonresponsive (without silver nitrate) genotypes, too. The supplement of silver nitrate in regenera tion medium was especially advantageous in plant regeneration of B. ca mpestris. Out of fourteen commercial cultivars of Brassica napus and B . campestris, there is only one recalcitrant genotype in obtaining pla ntlets from protoplast-derived calli.