Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Abstract

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×10⁴ ml^-1 in Kao's medium supplemented with 1.0 mgl-12,4-D, 0.1 mgl^-1 NAA and 0.5 mgl^-1 zeatin riboside. After three days of culture in darkness at 25±1౨C, cultures were transferred to light (70 µEm^-2s^-1) in a 16/8 h ligø ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl^-1 benzyladenine. On the fifteenth day, microcalli were plated on K3 medium gelled with 0.5% agarose (Type 1, low EEO, Σ). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8-10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.