Callus formation and plant regeneration from mesophyll protoplasts of rape plants ( Brassica napus L. cv. Zephyr)

Abstract

Protoplasts from mesophyll cells of rape plants ( Brassica napus L. cv. Zephyr) were isolated by enzymatic removal of cell walls in an osmoticum consisting of sorbitol and mannitol. A two-step enzyme treatment involving 0.5% each of Onozuka P1500 cellulase and Rhozyme HP150 hemicellulase followed by 0.5% each of Driselase and hemicellulase at pH 6.2 resulted in the production of viable protoplasts in 80–90% yield. The protoplasts were cultured in droplets of B5 medium in petri dishes at a light intensity of 100 lux and 26°. They were able to regenerate cell walls. Cell division was apparent after 30 h of culturing and by 60 h up to 38% of the protoplasts had divided once. Cell clusters were formed within 15 days of culturing. The calli originating from cell clusters were induced to differentiate and to form complete plants.