Enhanced regeneration of haploid plantlets from microspores of Brassica napus L. using bleomycin, PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mu g ml(-1)), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l(-1), on microspore embryogenesis of rapeseed cv. 'Amica' were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 mu g ml(-1) bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish(-1)) was observed with 24 h treatment of 4 mg l(-1) PCIB. The highest percentage of secondary embryogenesis was observed on B-5 medium containing 0.15 mg l(-1) of gibberellic acid (GA(3)) and 0.2 mg l(-1) 6-benzyladenine (BA) in 4-6 mm microspore-derived embryos (MDEs). Most callus formed on B-5 medium containing 0.15 mg l(-1) GA(3), 0.1 mg l(-1) BA and 0.1 mg l(-1) indole-3-acetic acid (IAA) when 4-6 mm embryos were used. Regeneration was highest on B-5 medium containing 0.05 mg l(-1) GA(3) or 0.1 mg l(-1) BA and 0.2 mg l(-1) IAA with 2-4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.