A Simple Protocol for Regenerating Mesophyll Protoplasts of Vegetable Brassicas

We report here a simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli. Protoplasts from in vitro grown leaf material were cultured in Kao’s medium with a supplementation of 2,4-D, NAA, BAP and glucose, initially in dark for 3d and subsequently in light. Dilution of protoplast cultures was effected on the 7th, 10th and 13th day of culture initiation with Kao’s medium supplemented with sucrose, and reduced 2,4-13 content; NAA was omitted. Micro-colonies were plated on a K3 medium having 2,4-D, BAP and sucrose gelled with agarose. Transfer of calli to another K3 medium with zeatin regenerated shoots from cauliflower protoplast derived calli, whereas a medium with kinetin and zeatin supported shoot regeneration in cabbage and broccoli. Shoot regeneration occurred within 6-6 weeks of culture initiation. Shoots were easily rooted on MS medium without growth regulators.