A novel approach to microspore embryogenesis in Brassica napus L.
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The objective of this study was to investigate development of haploid embryos of 'Brassica napus' induced from isolated microspores, and to provide a comparison to the corresponding developmental stages of zygotic embryos. A novel method for induction and culture of microspore-derived (MD) embryos was designed, based on restricted sucrose supply and use of high molecular weight polyethylene glycol (PEG) as an osmoticum. Haploid embryos cultured under these conditions were studied, from the induction phase to the formation of cotyledonary embryos, through their maturation and desiccation. Finally, plantlet formation and their ex vitro acclimatization were assessed. Induction and formation of MD embryos was not affected by limited sucrose supply; embryos proceeded through globular, heart, torpedo and cotyledonary stages within two weeks. After exposure to light, PEG embryos turned dark green and appeared similar to dissected early cotyledonary stage zygotic embryos. Morphological changes during the time-course of microspore embryogenesis were studied using scanning electron microscopy. Early embryo development from embryogenic microspores to the globular stage was irregular and differed from that of zygotic embryos. However, at heart, torpedo and cotyledonary stages, PEG embryos were remarkably similar to their zygotic counterparts in size and shape, with well-developed cotyledons. Sucrose embryos were 2-3 times larger than PEG embryos, but cotyledons were small and poorly differentiated. Numerous large starch grains were observed in cells of sucrose embryos at the early cotyledonary stage, but were almost completely absent in PEG embryos. The characteristic presence of pollen wall remnants suggested an origin of polarity in MD embryos, possibly established in late uninuclear microspores and early bicellular pollen. Application of ABA and desiccation treatment improved the in vitro conversion frequency of both sucrose and PEG embryos. However, PEG plantlets directly transferred to soil had a higher survival rate and enhanced vigor during the acclimatization ex vitro. This novel microspore culture system is suitable for studies of in vitro embryogenesis with a broad range of plant species and should have important potential application in plant biotechnology and breeding programs.