STUDIES OF COTYLEDON PROTOPLAST CULTURES FROM BRASSICA-NAPUS, BRASSICA-CAMPESTRIS AND BRASSICA-OLERACEA .2. CALLUS FORMATION AND PLANT-REGENERATION

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka , Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The form ation of cell colonies and callus from protoplast cultures were signif icantly influenced by the light conditions of seed germination. All tw elve cultivars showed callus formation from protoplast cultures derive d from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modi fied B and modified NN. In contrast, only six cultivars exhibited call us formation from the protoplasts isolated from cotyledons of seedling s germinated under light conditions for 7 days (light-grown) and in on ly three media: modified K8P(1), modified MS, modified B. Callus, deri ved from protoplast cultures isolated from dark/dim light-grown cotyle dons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration m edia. All five cultivars of B. napus, three of the four cultivars of B . campestris (Arlo, Sonja and Bunyip) and one of the three cultivars o f B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplas t cultures within 2-3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1-22.5%. A high degree of reproduc ibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed call us in protoplast cultures derived from light-grown cotyledons, only th ree cultivars from B. napus (Westar, Eureka, Global) exhibited shoot r egeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induc ed to root on rooting media, survived in soil and grew to produce sili que and set seeds.