Abstract
Exposure of Norway spruce (Picea abies) somatic embryos and those of many other conifers to post-maturation desiccation treatment significantly improves their germination. An integration analysis was conducted to understand the underlying processes induced during the desiccation phase at the molecular level. Carbohydrate, protein and phytohormone assays associated with histological and proteomic studies were performed for the evaluation of markers and actors in this phase. Multivariate comparison of mature somatic embryos with mature desiccated somatic embryos and/or zygotic embryos provided new insights into the processes involved during the desiccation step of somatic embryogenesis. Desiccated embryos were characterized by reduced levels of starch and soluble carbohydrates but elevated levels of raffinose family oligosaccharides. Desiccation treatment decreased the content of abscisic acid and its derivatives but increased total auxins and cytokinins. The content of phytohormones in dry zygotic embryos was lower than in somatic embryos, but their profile was mostly analogous, apart from differences in cytokinin profiles. The biological processes “Acquisition of desiccation tolerance”, “Response to stimulus”, “Response to stress” and “Stored energy” were activated in both the desiccated somatic embryos and zygotic embryos when compared to the proteome of mature somatic embryos before desiccation. Based on the specific biochemical changes of important constituents (abscisic acid, raffinose, stachyose, LEA proteins and cruciferins) induced by the desiccation treatment and observed similarities between somatic and zygotic P. abies embryos, we concluded that the somatic embryos approximated to a state of desiccation tolerance. This physiological change could be responsible for the reorientation of Norway spruce somatic embryos toward a stage suitable for germination.
Highlights
Somatic embryogenesis could play an important role in the commercial breeding of conifers, but many problems have to be solved before its widespread use in forest management, e.g., insufficient quality and yield of mature embryos and their low ability to germinate (Pullman and Bucalo, 2014)
Desiccation of somatic embryo (SE) isolated from clusters was carried out under high relative humidity on dry filter paper in small Petri dishes (3 cm in diameter) that were left open and placed in large Petri dishes (18 cm in diameter) containing several layers of filter paper wetted with sterile water to maintain high relative humidity
Fresh zygotic embryos collected in late summer were already well morphologically developed, to M5 embryos, and almost filled the corrosion cavity inside the megagametophyte (Figures 3A,B)
Summary
Somatic embryogenesis could play an important role in the commercial breeding of conifers, but many problems have to be solved before its widespread use in forest management, e.g., insufficient quality and yield of mature embryos and their low ability to germinate (Pullman and Bucalo, 2014). Eliminating these obstacles would enable enhanced embryogenic culture production, e.g., large-scale cultivation in bioreactors, which offers a promising method for conifer somatic embryogenesis owing to its increased efficiency and lower costs (Välimäki et al, 2020). Subjecting desiccation-sensitive cells to desiccation conditions of < 90% relative humidity can cause the loss of correct protein conformation, membrane phase transitions and RNA/DNA structural rearrangements and fragmentation (Leprince and Buitink, 2015)
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