Somatic embryogenesis (SE) is an important method for the vegetative propagation of trees. SE is the developmental in vitro process in which embryos are produced from somatic cells. This method can be integrated with other biotechnological techniques, genomic breeding and cryopreservation, which enables commercial-scale sapling production of selected high-yielding genotypes in wood production combined with fast breeding cycles. The SE is potential tool to improve plant stock in comparison with seed orchards. It can be useful for ecologically and economically important species, such as Norway spruce (Picea abies L. Karst.) and Scots pine (Pinus sylvestris L.), ensuring stable production in the era of climate change and biodiversity crisis. In this review, we summarize the current state of research on problems associated with somatic embryogenesis in P. abies and P. sylvestris.

Key message: Malus sylvestris(L.) Mill.,Pyrus communis(L.),Sorbus aucuparia(L.),Prunus avium(L.),Prunus padus(L.), andCornus sanguinea(L.) are related, co-occurring species producingorthodoxseeds. However, we observed differences in their response to storage conditions, such as storage at different seed moisture contents (5%, 8%, and 11%) and/or temperatures (− 3 °C, − 18 °C, and − 196 °C). Severe desiccation to ca. 5% of MC negatively affected seeds ofM. sylvestris. Seeds ofP. aviumwere sensitive to storage in LN or at − 18 °C.S. aucupariaseeds are best stored at − 3 °C, whereasC. sanguineaseeds tolerate desiccation and storage in LN. In general, species with deeper physiological dormancy (S. aucuparia,P. padus, andC. sanguinea) tended to be more tolerant to desiccation and low temperatures. For all species, storage conditions did not affect seedling growth. Context: Wild fruit woody species face many threats such as genetic loss, population fragmentation, and alien species; thus, their genetic variability should be preserved. Aims: To examine the effect of storage conditions on seed viability and the initial growth of seedlings of six European wild fruit species: Malus sylvestris (L.) Mill., Pyrus communis (L.), Sorbus aucuparia (L.), Prunus avium (L.), Prunus padus (L.), and Cornus sanguinea (L.). Methods: Seeds were desiccated to three different levels of moisture content (ca. 5, 8, or 11%) and stored at three different temperatures (− 3°, − 18°, or − 196 °C; liquid nitrogen, LN) for up to 3 years. Germination and seedling emergence tests were performed as well as measurement of seedling growth. Results: Desiccation of M. sylvestris seeds from 10.7 to 4.9% significantly lowered germination from 91 to 77% and seedling emergence from 88 to 74%. In P. avium, LN storage significantly inhibited seedling emergence, both in the laboratory and the greenhouse, but did not affect total seed germination. In P. communis, P. padus, and C. sanguinea, neither germination nor seedling emergence was affected by seed storage conditions. There were small or no differences in stem height and root collar diameter in the first year of seedling growth of stored seeds. Conclusion: Species with deeper physiological dormancy (S. aucuparia, P. padus, C. sanguinea) tended to be more tolerant of various storage conditions. Seeds of P. padus and C. sanguinea can be stored long term at − 18 °C or in LN at 5–8% MC without losing viability. M. sylvestris and P. avium seeds are sensitive to desiccation below 6% MC or low temperature of storage at − 18 °C or − 196 °C, respectively. We observed that storage conditions had significant influence on germination and seedling emergence but had no effect on seedling growth after the first growing season.

In the present study, we examined the utility of proline usage as a biochemical indicator of metabolic changes caused by climate change (mean temperature and precipitation) during seed development of two Acer species differing in desiccation tolerance: Norway maple (Acer platanoides L.—desiccation tolerant—orthodox) and sycamore (Acer pseudoplatanus L.—desiccation sensitive—recalcitrant). In plants, proline is an element of the antioxidant system, which has a role in response to water loss and high temperatures. Our study considered whether proline could be treated as an indicator of tree seed viability, crucial for genetic resources conservation. Proline content was measured biweekly in developing seeds (between 11 and 23 weeks after flowering) collected in consecutive years (2017, 2018, and 2019). We showed that proline concentrations in recalcitrant seeds were positively correlated with mean two-week temperature. In contrast, in orthodox seeds no such relationship was found. Proline content proved to be sensitive to thermal-moisture conditions changes, which makes it a promising biochemical marker of seed desiccation tolerance in different climatic conditions.

Pedunculate oak (Quercus robur L.) is an economically important forest-forming species in Poland that produces seeds that are sensitive to desiccation; therefore, short-lived seeds are classified as recalcitrant. Such seeds display active metabolism throughout storage. Acorns stored under controlled conditions (moisture content of 40%, temperature −3 °C) maintain viability for up to 1.5–2 years. Meanwhile, oaks only produce large numbers of seeds every few years during so-called mast years. This results in a scarcity of good-quality seeds for continuous nursery production and restoration. The recalcitrant storage behavior and the requirements of foresters make it necessary to develop a new protocol for longer acorn storage at lower temperatures. Two storage temperatures were tested: −3 °C (currently used in forest practice) and −7 °C. Our results showed that acorns stored for six months exhibited deterioration and reduced germination capacity, as well as reduced seedling performance, particularly when acorns were stored at −7 °C. To elucidate the decrease in quality during storage, an untargeted metabolomics study was performed for the first time and supported with the analysis of carbohydrates and percentages of carbon (C) and nitrogen (N). Embryonic axes were characterized by a lower C:N ratio and higher hydration. A total of 1985 metabolites were detected, and 303 were successfully identified and quantified, revealing 44 known metabolites that displayed significantly up- or downregulated abundance. We demonstrated for the first time that the significant deterioration of seed germination potential, particularly in seeds stored at −7 °C, was accompanied by an increased abundance of phenolic compounds and carbohydrates but also amino acids and phosphorylated monosaccharides, particularly in the embryonic axes. The increased abundance of defense-related metabolites (1,2,4-Benzenetriol; BTO), products of ascorbic acid degradation (threonic and isothreonic acid), as well as antifreezing compounds (sugar alcohols, predominantly threitol), was reported in seed stored at −7 °C. We hypothesize that seed deterioration was caused by freezing stress experienced during six months of storage at −7 °C, a decline in antioxidative potential and the unsuccessful rerouting of the energy-production pathways. Additionally, our data are a good example of the application of high-throughput metabolomic tools in forest management.

Key message: Decreasing acorns moisture content does not significantly increase the frost resistance of pedunculate oak seeds. Slight reduction in acorn moisture content below the relatively high, optimal level decreased seed survival at temperatures below − 5 °C. The limiting temperature for pedunculate oak’s acorns below which they lose their ability to germinate is about − 10 °C. Context: Seed moisture content plays an important role in successful seed storage of many species, as desiccation increases frost resistance; however, oak seeds tolerate desiccation only to a very small extent. Aims: In our study, we examined the impact of decreasing moisture content in acorns of pedunculate oak (Quercus robur L.) on their frost resistance (below − 3 °C) and the growth of seedling derived from frozen seeds. Methods: Germination and seedling emergence of individual seeds, as well as the dry mass of their 3-month-old seedlings, were measured after acorn desiccation (24–40%, fresh weight basis) and desiccation followed by freezing at temperatures from − 3 °C to − 18 °C for 2 weeks. Results: Decreasing acorns moisture content did not significantly increase the frost resistance of pedunculate oak seeds. The lowest temperature at which at least half seeds remain viable was − 10 °C. Slight acorns desiccation had only a small positive effect on seeds frozen below − 11 °C (down to − 13 °C), but in this case (acorn moisture content of 33%), low germinability after freezing made storage uneconomic because of the high mortality of seeds. Germinated seeds after desiccation and freezing showed no significant difference in later growth. Conclusion: Fresh pedunculate oak seed can survive freezing temperature down to − 10 °C and produce good quality seedlings. Temperatures around − 11° to − 13 °C are near lethal to acorns and significantly reduce their viability. Overall, desiccation does not increase their frost resistance; therefore, in practice, it is important to keep acorns during a cold storage in the highly hydrated state.

Somatic embryo quality is still a problem for many researchers. To improve the efficiency of germination, special procedures are used, such as partial drying of somatic embryos at high relative humidity or desiccation in the presence of supersaturated solutions of salt. In this work, cotyledonary somatic embryos of Norway spruce (Picea abies) and Serbian spruce (P. omorika) were placed on culture media (ME or BM-5) to germinate. We found that after 4 weeks of incubation on these media, hypocotyl and radicle growth of control (non-dried) somatic embryos of both species was not adequate to yield seedlings able to acclimatize to greenhouse conditions. Therefore, somatic embryos were partly dried at relative humidity of 97 % or desiccated at relative humidity of 79 %, for 2 or 3 weeks, and then placed on the Margara (ME) medium. Partial drying of somatic embryos at the higher relative humidity (97 %) enabled an improvement of radicle growth of germinating somatic embryos in both species. The highest conversion rate (45 %) was obtained for embryos of Norway spruce maintained for 2 weeks at relative humidity of 97 %. This treatment contributed to the improvement of germination and conversion efficiency of somatic embryos of Norway spruce, regardless of the drying period. Improved radicle growth facilitated development of better quality seedlings of this spruce species. In Serbian spruce, we did not obtain seedlings of sufficient quality, due to poor hypocotyl growth. Desiccation at humidity of 79 % for 3 weeks proved to be lethal to somatic embryos of both species.