Regeneration of hybrid plantlets via pollen-hypocotyl protoplast fusion in Brassica spp.

Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8% of cellulase, 0.5% pectinase, 0.1% pectolyase, 13% mannitol, 10% glucose, 0.3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B.napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prelabeled pollen protoplasts. The fusion produets were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8mg/L 2.4-D, 0.25 mg/L NAA, 0.5mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0mg/L BA, 3% sucrose and 0.4% agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0.2 mg/L NAA, 0.5mg/L IBA and 20% potato juice for root fornation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was.....