Protoplast culture and plant regeneration of different agronomically important Brassica species and varieties

Protoplast cultures were prepared from 6-day-old hypocotyls of six spring, seven winter cultivars of Brassica napus L. and one line of Brassica 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 centrifugation was used to minimize physical damage of protoplasts. Three different protoplast culture systems —(1) liquid, (2) 2nd day embedded, (3) directly embedded in low melting agarose were compared. The two different protoplast embedding techniques resulted in the same efficiency of cell division as the liquid culture method and over this fact the colony browning was avoided. Using protoplast agarose-embedding and culture techniques, healthy calli were obtained for plant regeneration experiments. Incorporation of silver nitrate into the regeneration medium improved the efficiency of plant regeneration in responsive genotypes and the regeneration was induced in three nonresponsive (without silver nitrate) genotypes, too. The supplement of silver nitrate in regeneration medium was especially advantageous in plant regeneration of B. campestris. Out of fourteen commercial cultivars of Brassica napus and B. campestris, there is only one recalcitrant genotype in obtaining plantlets from protoplast-derived calli