The embryogenic cell suspension culture of Gentiana cruciata, cryopreserved by the encapsulation/dehydration method, survived both short- (48 h) and long-term (1.5 years) cryostorage with more than 80% viability. To assess the influence of cryotreatments on the embryogenic potential, a proembryogenic mass was encapsulated and exposed to the following treatments: (1) osmotic dehydration (OD), (2) OD + air desiccation (AD) and (3) OD + AD + cryostorage (LN). The somatic embryogenesis efficiency increased ten times after osmotic dehydration. The AD and LN cryotreatments did not cause any significant alterations in somatic embryo production. We monitored the (epi)genetic stability of 288 regenerants derived from: non-cryotreated, short-term, and long-term cryostored tissue using metAFLP markers and ten primer combinations. Changes in the sequence and DNA methylation levels were studied by subjecting the DNA to digestion with two pairs of isoschisomer restriction enzymes (KpnI/MseI and Acc65I/MseI). Two new AFLP unique DNA fragments at the DNA sequence level, with no differences at the methylation level, were found between regenerants derived from cryopreserved tissue, compared with the non-cryotreated controls. The Acc65I/MseI methylation levels for the three groups of regenerants were not significantly different. Cluster analysis was capable of identifying a number of sub-clusters. Only one of the sub-clusters comprises almost all regenerants derived from non-cryotreated and short-term cryostored tissue. Plantlets derived from long-term cryostored tissue were grouped into separate clusters. The observed AFLP alterations did not appear to be associated with the use of cryopreservation, but were probably related to the process of in vitro culture.

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.

Key message Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. Abstract We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12–13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

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.

A simple and reliable technique for in vitro multiplication and long-term preservation using liquid nitrogen was developed for gametophytes of Osmunda regalis. The effect of Knop’s medium and various concentrations (1/2, 1/4, 1/8) of mineral salts provided in Murashige and Skoog (MS) basal medium, together in the presence or absence of both ammonium nitrate and a full complement of vitamins on gametophyte proliferation and sporophyte production was determined. Moreover, the effectiveness of gametophyte cryopreservation by vitrification and encapsulation–vitrification techniques was assessed. Maximum gametophyte proliferation (89 %) occurred on the ammonium nitrate- and vitamin-free [(-NH4NO3)(-vit)] MS medium with 1/2 or 1/4 strength mineral salts. The maximum production of sporophytes (30 plantlets per gametophyte clump) required 1/8MS (-NH4NO3)(-vit) medium. The flow cytometric analysis revealed that the sporophytes contained twofold more pg DNA than gametophytes. This confirmed that the sporophytes were obtained by sexual reproduction. The vitrification protocol and PVS2 solution were ineffective for cryopreservation. The greatest survival rate (81.6 %) following cryo-exposure occurred following treatment of encapsulated gametophytes with PVS3 solution for 3 h. This protocol allowed the recovery of gametophyte cultures following 6 weeks after rewarming. Finally, 100 % of sporophytes produced in vitro were successfully acclimated to ex vitro conditions. Application of the in vitro and cryopreservation methods made it possible to improve the number and time of O. regalis sporophyte production. Whole system of micropropagation can be completed in approximately 1 year. The protocols open new avenues for the mass propagation, germplasm conservation and resource management of the species.