Conifer somatic embryogenesis (SE) is a process driven by exogenously supplied plant growth regulators (PGRs). Exogenous PGRs and endogenous phytohormones trigger particular ontogenetic events. Complex mechanisms involving a number of endogenous phytohormones control the differentiation of cells and tissues, as well as the establishment of structures and organs. Most of the mechanisms and hormonal functions in the SE of conifers have not yet been described. With the aim to better understand these mechanisms, we provided detailed analysis of the spectrum of endogenous phytohormones over the course of SE in Norway spruce (Picea abies). Concentrations of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates, and salicylic acid (SA) in somatic P. abies embryos were analyzed by HPLC-ESI-MS/MS. The results revealed that the concentrations of particular phytohormone classes varied substantially between proliferation, maturation, desiccation, and germination. Endogenous ABA showed a maximum concentration at the maturation stage, which reflected the presence of exogenous ABA in the medium and demonstrated its efficient perception by the embryos as a prerequisite for their further development. Auxins also had concentration maxima at the maturation stage, suggesting a role in embryo polarization. Endogenous jasmonates were detected in conifer somatic embryos for the first time, and reached maxima at germination. According to our knowledge, we have presented evidence for the involvement of the non-indole auxin phenylacetic acid, cis-zeatin- and dihydrozeatin-type CKs and SA in SE for the first time. The presented results represent the currently most comprehensive overview of plant hormone levels in embryos throughout the whole process of conifer SE. The differences in concentrations of various classes of phytohormones over the proliferation, maturation, desiccation, and germination in somatic P. abies embryos clearly indicate correlations between endogenous phytohormone profiles and particular developmental stages of the SE of conifers.

The somatic embryogenesis of conifers is a process susceptible to exogenous phytohormonal treatments. We report the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the auxin inhibitor p-chlorophenoxyisobutyric acid (PCIB) on the endogenous level of the auxin indole-3-acetic acid (IAA) and on the anatomical composition of early somatic embryos of Abies alba (European silver fir). The embryogenic suspensor mass (ESM) of Abies alba proliferated on a medium supplemented by 2,4-D as well as on an auxin-free medium. The endogenous level of IAA was significantly higher in the ESM cultivated on a medium supplemented by 2,4-D. The decrease in the endogenous level of IAA in the first week of maturation is one of the most important stimuli responsible for the subsequent development of embryos. However, suppression of IAA synthesis by an auxin inhibitor did not stimulate the development of embryos. The maturation of somatic embryos from the globular to the cotyledonary stage occurs when the concentration of endogenous auxin in the ESM (including the embryos) increases. Early somatic embryos proliferating on a medium supplemented by auxin had an increased probability of maturing successfully. Exogenous auxin treatment during maturation did not compensate for the auxin deficiency during proliferation.

The polyamine (PA) contents and activities of PA biosynthetic enzymes in Norway spruce somatic embryos [Picea abies L. (Karst.), genotype AFO 541] were studied in relation to anatomical changes during their development, from proliferation to germination, and changes in these variables associated with the germination of mature somatic and zygotic embryos were compared. Activities of PA biosynthetic enzymes steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages. In these stages, the spermidine (Spd) level was significantly higher than the putrescine (Put) level, and the increases coincided with the sharp increases in S-adenosylmethionine decarboxylase activity in the embryos. The biosynthetic enzyme activity subsequently declined in mature cotyledonary embryos, accompanied by sharp reductions in PA contents, especially in cellular Put contents in embryos from 6 weeks old through the desiccation phase (although the spermine level significantly increased during the desiccation phase), resulting in a shift in the Spd/Put ratio from ca. 2 in early cotyledonary embryos to around 10 after 3 weeks of desiccation. In mature zygotic embryos, Spd contents were twofold lower, but Put levels were higher, than in mature somatic embryos, hence their Spd/Put ratio was substantially lower (ca. 2, in both embryos and megagametophytes). In addition, the PA synthesis activity profiles in the embryos differed (ornithine decarboxylase and arginine decarboxylase activities predominating in mature somatic and zygotic embryos, respectively). The start of germination was associated with a rise in PA biosynthetic activity in the embryos of both origins, which was accompanied by a marked increase in Put contents in somatic embryos, resulting in the decline of Spd/Put ratio to about 2, similar to the ratio in mature and germinating zygotic embryos. The accumulation of high levels of PAs in somatic embryos may be causally linked to their lower germinability than in zygotic embryos.

Maturation of Norway spruce (Picea abies L.) somatic embryos is induced by abscisic acid (ABA). Several proteins were proven to be involved in ABA sensing including ABI3/VP1 transcription factors and their orthologue PaVP1 was characterized in spruce. To evaluate the role of PaVP1 both in embryogenic potential and in the process of embryo maturation, we studied PaVP1 expression in lines with contrast embryogenic capacities in parallel with detailed anatomical characterization. PaVP1 expression was determined by northern blot hybridisation, which revealed presence of two differentially regulated VP1-like B3-domain transcripts. Full-length PaVP1 transcript level was negligible in all lines on the proliferation media, but it differed strongly on the maturation media containing ABA. In non-embryogenic line, lacking any differentiated structures, the transcript remained undetectable. In contrast, in embryogenic lines with meristematic centres attached to suspensor cells, PaVP1 expression increased strongly after transition onto the maturation media. In highly embryogenic lines, it kept on a high level until the embryos reached cotyledonary stage, while in developmentally arrested line incapable to form mature embryos, the expression dropped down in connection with advanced disintegration of the meristematic centres. Removal of ABA from the maturation media after 2 weeks of maturation resulted in aberrant embryo development and rapid decrease in PaVP1 expression, indicating the impact of exogenously supplemented ABA on both initiation and maintenance of PaVP1 expression and proper embryo development. Since permanently high or increasing PaVP1 transcript levels accompanied proper embryo development in all experiments, it could be regarded as a good marker of this process.

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.