STUDIES ON PLANT-REGENERATION FROM COTYLEDONARY PROTOPLASTS IN BRASSICA-CAMPESTRIS

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dar k/dim light-grown cotyledons of four Brassica campestris varieties (Ar lo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: m odified K8P, modified MS and modified Pelletier's B to compare the cap acities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyle dons of four varieties showed rapid division and high frequency of cel l division compared with those isolated from light-grown cotyledons. T he frequencies of cell division were significantly influenced by varie ties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7-14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli su bsequently grown on a solid medium developed shoots when transferred o nto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2-3 months after protopl ast isolation, with a high degree of reproducibility in Arlo and Bunyi p. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l-1 IBA, survived in soil and grew to produce siliq ues and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.