Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs.

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advantages such as mass propagation, genetic transformation, application of synthetic seed, and cryopreservation of elite genotypes. In this report, the state of the art of somatic embryogenesis in holm oak is reviewed. Factors affecting the induction (plant donor age, type of explant, or plant growth regulators) and maintenance and proliferation of the embryogenic cultures are summarized. Advances on the conversion of somatic embryos into plants and on the acclimatization of these plantlets, as well as the results obtained on the application of the genetic transformation and the cryopreservation procedures to holm oak embryogenic cultures, are also presented.

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