In order to expand gene resources and improve Brassica napus cultivars, protoplasts isolated from hypocotyls of Brassica napus cv. Huayou No. 3 and Eruca sativa were fused by PEG-high Ca2+-high pH. Fusion frequency was up to 18.2% when fusion system contained 5 x 10(5) protoplasts/mL, and when PEG concentration of fusion agents were 35% and when fusion time was 25 min. Then the fused protoplasts were cultured by the method of thin liquid layer at the density of 1 x 10(5) protoplasts/mL in improved KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, 0.5 mg/L 6-BA, 200 mg/L inositol, 300 mg/L protein hydrolysate, and the combinations of 0.1 mol/L sucrose and 0.2 mol/L glucose and 0.2 mol/L mannitol for osmotic regulator, the frequency of callus regeneration was up to 6.8%. When the micro-calli transferred to the proliferation medium that contained B5 salts, 0.087 mol/L sucrose, 0.2 mg/L 2,4-D, 0.5 mg/L NAA, 0.2 mg/L 6-BA and 0.5% Agar, pH 5.8, have grown up to 3-5 mm of diameter, the calli were transferred to the differentiation medium that contained MS salts, 0.087 mol/L sucrose, 0.1 mg/L IAA, 0.8 mg/L 6-BA, 0.8% Agar, pH5.8, the shoots were regenerated in 4 weeks and its frequency was up to 32.8%. Then 2-3 cm shoots were transferred to 1/2 MS medium with 0.5 mg/L IBA+0.2mg/L 6-BA, plantlets were obtained in 14 days and the plantlet frequency was up to 88%. When the protoplasts of Eruca sativa were treated with UV radiation for 2 minutes calli and plantlets have been regenerated, treated for 4 min only calli have been regenerated, and treated for more than 5 min calli have not been regenerated. The callus regeneration and callus proliferation and plant regeneration from symmetric fusion were more than from asymmetric fusion. 16 hybrid plantlets have been regenerated on 21 piece of hybrid calli identified by cytology method.
Brassica ; genetics ; Brassicaceae ; genetics ; Cell Fusion ; Hybrid Cells ; Hybridization, Genetic ; Protoplasts ; Regeneration ; Ultraviolet Rays