Somatic embryogenesis (SE) in the tree fern Cyathea delgadii was first described in 2015 and since then has been used to exploration of this phenomenon in cryptogamic plants. To deepen the knowledge about the hormonal control of SE, stipe explants were cultured on media supplemented with hormone biosynthesis and transport inhibitors (HBTIs). In the presence of 30 µM 2,3,5-triiodobenzoic acid (TIBA), or 40 µM fluridone or 125 µM salicylic acid (SA), somatic embryo production was totally inhibited. The quantitative analysis of the changes in endogenous hormone and sugar contents was conducted every 2 days within 10-day-long initial culture. The results showed that the concentrations of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA), cytokinins (CKs) and soluble sugars were strongly modified either by TIBA and fluridone. Under their influence, the contents of cytokinins such as c-Z, c-ZR, t-Z, t-ZR, KinR were reduced to barely detectable levels. Treatment with SA results in the changes in endogenous IAA and sugar contents. It also modifies the IAA/CKs ratio; however, excluding the first 2 days of culture, the concentrations of ABA and cytokinins were kept on the control level. All HBTIs significantly increased the kinetin (Kin) content. Our work sheds new light on the relationships between the biosynthetic inhibitors and phytohormones and sugars in the process of early SE. It can be helpful to study the role of hormones in acquisition of embryogenic competence.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.

In plants, the embryogenic transition of somatic cells requires the reprogramming of the cell transcriptome, which is under the control of genetic and epigenetic factors. Correspondingly, the extensive modulation of genes encoding transcription factors and miRNAs has been indicated as controlling the induction of somatic embryogenesis in Arabidopsis and other plants. Among the MIRNAs that have a differential expression during somatic embryogenesis, members of the MIRNA172 gene family have been identified, which implies a role of miR172 in controlling the embryogenic transition in Arabidopsis. In the present study, we found a disturbed expression of both MIRNA172 and candidate miR172-target genes, including AP2, TOE1, TOE2, TOE3, SMZ and SNZ, that negatively affected the embryogenic response of transgenic explants. Next, we examined the role of AP2 in the miR172-mediated mechanism that controls the embryogenic response. We found some evidence that by controlling AP2, miR172 might repress the WUS that has an important function in embryogenic induction. We showed that the mechanism of the miR172-AP2-controlled repression of WUS involves histone acetylation. We observed the upregulation of the WUS transcripts in an embryogenic culture that was overexpressing AP2 and treated with trichostatin A (TSA), which is an inhibitor of HDAC histone deacetylases. The increased expression of the WUS gene in the embryogenic culture of the hdac mutants further confirmed the role of histone acetylation in WUS control during somatic embryogenesis. A chromatin-immunoprecipitation analysis provided evidence about the contribution of HDA6/19-mediated histone deacetylation to AP2-controlled WUS repression during embryogenic induction. The upstream regulatory elements of the miR172-AP2-WUS pathway might involve the miR156-controlled SPL9/SPL10, which control the level of mature miR172 in an embryogenic culture.

The effect of explant type (etiolation, age, length), physical (photoperiod, darkness) and chemical (sucrose and mineral salt concentrations) factors on somatic embryogenesis efficiency and sporophyte development in Cyathea delgadii was evaluated. Five-month-old cultures of sporophytes, which had developed 4–5 leaves, were used as a source of explants. The percentage response of stipes and the average number of somatic embryos were calculated after 2 months of culture. Etiolation of sporophytes used for culture initiation, was a critical factor in SE induction. Other factors studied significantly affected the production of somatic embryos. Optimum results were obtained when explants measuring 2.5 mm in length were excised from the youngest leaf of an etiolated sporophyte. Maximum SE efficiency (84.0 % response in explants; 42.3 somatic embryos per explant) was obtained on half-strength Murashige and Skoog medium supplemented with 1 % sucrose, under photoperiod conditions. However, light stimulated two different morphogenetic responses in explant cells: SE and apospory. In darkness, the number of somatic embryos was reduced to 32.9 per explant, but this was the only morphogenetic response observed. The presence of light alone was sufficient for somatic embryos growing on plain agar gel to reach maturity, but further development stopped shortly thereafter. The presence of both light and sucrose was necessary for the development of an embryonic leaf and root. For further sporophyte growth, mineral salts were essential. The micropropagation system described here is the first investigation into factors responsible for SE in ferns.

Cyathea delgadii is the first fern species for which somatic embryogenesis (SE) has recently been described. With this discovery, a new experimental model for exploration of SE was found. In this study, the effect of explant type (stipe and internode), length and diameter of the explant-donor frond, and stress treatment (by sucrose and air desiccation) on somatic embryo origin and SE efficiency was studied. In control culture, somatic embryos originated from single cells of stipe explants, whereas those induced on internodes were of multicellular origin. Although the activation of cell divisions was more abundant and the formation of somatic embryos occurred earlier in a culture of internodes than of stipe explants, the morphogenic capacity of internodes was much smaller. On their surface, the groups of competent cells formed protuberances that give rise to only three somatic embryos per internode. In contrast, almost 92% of stipe explants that were excised from the first frond, measuring 10 mm in length and less than 300 µm in diameter, produced an average of 21 somatic embryos. Stipes treated with sucrose were not able to SE. More than fourfold increase in SE efficiency was obtained on internodes by their treatment with 0.4 M sucrose for 45 min. It was achieved by changing the pathway of somatic embryo differentiation from multicellular to unicellular. These results provide an excellent basis for further research on the mechanism of SE induction associated with a single- and multi-cell proliferation, especially that both types of the embryogenic pathway can occur on the same hormone-free medium.