Several olive cultivars, characterized by high-quality olive oil show agronomical issues such as excessive vigor, high susceptibility to biotic and abiotic stresses, and low propagation ability. They are strong candidates for breeding based on new technologies to improve their performance in a short period of time. For this reason, the first step is developing efficient somatic embryogenesis (SE) protocols. Somatic embryogenesis in olive is highly genotype-dependent for both adult tissues and mature embryos as initial explants, requiring the development of specific protocols for each genotype. Trials using cotyledons and radicles as initial explants, isolated from ripe seeds from the Portuguese olive cv. ‘Galega vulgar’, gave more than 95% calli development. Radicles proved to be the most responsive tissue for SE induction, with an average of 2 embryos per callus after callus transfer to expression medium, and 14 embryos per callus after subculture on the olive cyclic embryogenesis medium (ECO). Embryogenic competence could be recovered after several subcultures on ECO medium that maintained cyclic embryogenesis for an indeterminate period of time. Embryo conversion and plant acclimatization were also attained with high success rates. Media management for cyclic embryogenesis maintenance is of general importance for SE protocols in any olive genotype. Somatic embryogenesis was thus attained for the first time in embryo-derived explants of cv. ‘Galega vulgar’.

In vitro micropropagation is already a current multiplication tool for walnut self-rooted cultivars and rootstocks, but walnut grafting is still performed in the field or in greenhouses, mainly using seedlings as rootstocks. The present work describes a new approach to obtain clonal walnut-grafted plants, involving in vitro shoot production of ‘Paradox’ (Juglans hindsi × Juglans regia) cl. ’Vlach’, to be used as rootstock, and J. regia cv. ‘Chandler’, to be used as scion. After completing the in vitro multiplication phase and a seven-day root induction treatment, ‘Vlach’ explants are transferred to ex vitro conditions for root expression while being simultaneously grafted using the in vitro produced ‘Chandler’ scions. The importance of the presence of leaves on both the scion and the rootstock for the success rate of the technique was evaluated. Under optimal conditions, average success rates of 82% for rootstock rooting, 72% for micrografting survival, and 84% for grafted plant acclimatization were achieved. This rooting/grafting combination technique seems able to compete with the traditional techniques of nursery grafting, allowing obtaining high-quality walnut-grafted plants independently of the external weather conditions in a significantly shorter time.

Pinus pinea is widely distributed in the Mediterranean basin. In Portugal, it is predominantly located in the southwestern coastal area. Yet, there are also forest areas of Pinus pinea in inland southern Portugal. Four plots were settled to study the effects of soil chemical characteristics, air temperature and precipitation on Pinus pinea growth. In these plots, stand structure, soil texture, soil organic matter, soil content on macronutrients and micronutrients, air temperature and precipitation were analysed. The results indicate that the greatest growth was achieved in the plots with good availability of nutrients and the highest precipitation, whereas the lowest growth was observed in the plots with the lowest precipitation. There seems to be a trend toward the main limiting factor to Pinus pinea growth being the availability of water.

Somatic embryogenesis (SE) involves the formation of embryo-like structures from somatic cells without fertilization and is widely used for clonal propagation and genetic transformation. However, in olive (Olea europaea sp. europaea), SE remains challenging due to the recalcitrant behavior of adult tissues when used as initial explants. Bioactive molecules released into the culture medium (conditioned medium, CM) by embryogenic cultures have been identified as modulators of the SE response. However, their potential role in enhancing SE efficiency in olive and overcoming tissue recalcitrance remains largely unexplored. To investigate the role of these biomolecules in olive SE, a protocol was established using SE cultures of cv. ‘Galega Vulgar’. Proteins and metabolites were separated by filtration, concentrated through lyophilization, and precipitated using three methods: Acetone, TCA/Acetone, and Methanol/Chloroform. The efficiency of these methods was evaluated through total protein quantification and via SDS-PAGE electrophoresis. LC-MS/MS was employed to analyze secretome composition using the TCA/Acetone precipitation method. Additionally, metabolite profiles were analyzed using ¹H NMR spectroscopy. The results led to the identification of 1096 (526 protein groups) Olea europaea proteins, including well-known SE biomarkers such as kinases and peroxidases. NMR spectroscopy identified several metabolites secreted into the medium or resulting from the metabolic activity of secreted enzymes, confirming the applicability of the procedure. Although extracting secreted biomolecules from the culture medium presents significant challenges, the protocol established in this study successfully enabled the isolation and identification of both proteins and metabolites, revealing a valuable workflow for future in-depth analyses of secreted biomolecules in olive SE.

Somatic embryogenesis (SE) is a powerful biotechnological tool for large-scale clonal plant propagation. However, most woody species exhibit a recalcitrant response. Olea europaea L., a highly valuable tree crop, is among these recalcitrant species. Unravelling the molecular mechanisms underlying SE efficiency in O. europaea is, therefore, essential. Access to embryogenic lines with contrasting capacities for somatic embryo differentiation is a key requirement for such studies. Mature fruits of olive cultivars ‘Galega Vulgar’ and ‘Arbequina’ were collected from open-pollinated orchard-grown plants, and various explants taken from zygotic embryos were used to establish SE. A differentiated response was observed both within each cultivar and between cultivars, with cv. ‘Arbequina’ showing the highest embryogenic induction, particularly when radicles were used. Secondary SE was successfully established for both high- and low-efficiency lines, providing valuable material for future molecular studies. Somatic embryo conversion into plants, a key indicator of embryo quality, was successfully achieved in both cultivars. Flow cytometry analysis revealed a high degree of chromosomal stability. This study presents a reliable procedure to obtain and maintain distinct embryogenic responses in O. europaea, identifying lines with both high and low embryogenic efficiency that can serve as model systems for future molecular investigations.