Improved microspore embryogenesis induction and plantlet regeneration using putrescine, cefotaxime and vancomycin in Brassica napus L.

Abstract

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l⁻¹), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l⁻¹) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l⁻¹ putrescine. Putrescine treatment at 0.5 mg l⁻¹ for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish⁻¹) was possible when induction medium was supplemented with 50 mg l⁻¹ cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l⁻¹ at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l⁻¹ cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l⁻¹ during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish⁻¹) in contrast to untreated cultures (93.6 embryos Petri dish⁻¹) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l⁻¹ for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.