The Spanish fir (Abies pinsapo Boiss.) is a species endemic to southern Spain. Its distribution area is very limited, with three main localizations in the Sierra de las Nieves, Sierra de Grazalema, and Sierra Bermeja (Alba-Sánchez et al., 2019).

Different factors threat A. pinsapo populations, such as the fire, agriculture, grazing, timber harvesting, insects, like Dioryctria aulloi, Mindarus abietinus, Cryphalus numidicus, Cinara confinis and Cinara pectinatae, and fungi, like Heterobasidion annosum and Armillaria mellea (Esteban et al., 2010). Although evidences of demographic genetic erosion among cohorts have not been found; bottlenecks, genetic drift, and a low among-populations genetic differentiation have been reported in this species (Cobo-Simón, 2020). Moreover, increased drought and warming induced by climate change can lead to a decrease of its ecological niche, thus worsening its future prospects (Gutierrez Hernández et al., 2017). These circumstances make A. pinsapo a highly vulnerable species and currently it is listed in different regional, national and international conservation catalogues, including the International Union for Conservation of Nature’s Red List of Threatened Species, where it is classified as endangered under criteria B1ab(i,ii,iii)+2ab(i,ii,iii) (IUCN, 2024).

Spanish fir stands, relict tree formations descended from ancient tertiary forests, are also protected, being included in the EU Habitats Directive (Council Directive 92/43/EEC on the conservation of natural habitats and of wild fauna and flora) and in the Natura 2000 Network (Gómez-Zotano et al., 2023).

In conifers, different strategies have been utilized for the management and conservation of genetic resources, including the application of biotechnological tools, such as somatic embryogenesis and cryopreservation.

Somatic embryogenesis consists of the development of embryos from somatic cells. This in vitro culture technique presents multiple applications, some of which can be used for the restoration and conservation of threatened species (Sánchez-Romero, 2019, 2021). In fact, it has been applied for conservation purposes in different endangered forest trees, such as Castanea dentata, Tsuga canadensis, Tsuga caroliniana, Chamaecyparis thyoides, Fraxinus pennsylvanica, Fraxinus americana (Merkle et al., 2017), and Abies nebrodensis (Jouini et al., 2023). Somatic embryogenesis is an efficient regeneration system, with a high multiplication capacity (Salaj et al., 2011), which can be exploited for the rapid propagation of high value genotypes for reforestation (Gupta et al., 1993). Moreover, somatic embryogenesis can be used as a complement in breeding programs aiming the generation of genotypes resistant to the main stressors threatening the species, as the possibility of propagating trees vegetatively provides significant advantages for capturing and enhancing genetic gain in plant improvement (Lambardi et al., 2018). Besides, as an in vitro culture technique capable of regenerating a whole plant from a single cell, somatic embryogenesis allows the application of biotechnological tools, such as genetic transformation, somaclonal variation, somatic hybridization, in vitro mutagenesis, production of haploid and double-haploid plants, and in vitro selection, greatly increasing plant breeding results. An important attribute of embryogenic cultures is their amenability to cryopreservation (Lambardi et al., 2018). Cryopreservation, i.e., storage at –196°C, the temperature of liquid nitrogen, is considered the best method for the long-term conservation of plant genetic resources (Engelmann, 2011). It requires a small space and limited maintenance, and decreases the risk of culture loss by contamination or human error (Engelmann, 2004; Winkelmann et al., 2004). Furthermore, cryopreservation allows the integration of somatic embryogenesis within practical forestry (Carson, 1986), as it avoids the occurrence of somaclonal variation and loss of regeneration capacity in cultures (Lambardi et al., 2008), and contributes to maintain the juvenility of donor tissues (Krajňáková et al., 2013).

The combined use of somatic embryogenesis and cryopreservation improves the management and conservation of interesting genotypes. Moreover, they are significant components of advanced breeding programs (Cyr, 1999), considerably enhancing the ability to obtain superior genotypes (Martínez et al., 2003), and contributing to preserve conventional bred material or biotechnological products until the results of field and progeny testing are available (Krajňáková et al., 2013).

Although somatic embryogenesis and cryopreservation constitute very valuable biotechnological tools, which can be incorporated to in situ and ex situ conservation strategies, they have not been reported in Spanish fir. The objective of this work was to develop somatic embryogenesis and cryopreservation protocols for A. pinsapo conservation. For this purpose, the slow-cooling technique was applied, as it is the cryopreservation method most commonly used for storage in liquid nitrogen of conifer embryogenic masses (Häggman et al., 2000; Ballesteros et al., 2024). As indicated by Engelmann (Engelmann, 2011), the steps to carry out the slow-freezing protocols are preconditioning of tissues, cryoprotection, slow cooling to a temperature close to -40°C, rapid immersion in liquid nitrogen, and thawing. In the present investigation, different treatments related to the preconditioning and cryoprotection steps were tested. Growth dynamics of A. pinsapo embryogenic cultures was also investigated as the culture growth phase at which explants are sampled has a critical effect on their subsequent cryopreservation response (Bradaï et al., 2017), due to its influence on tissue cell composition and physiological state. In general, in conifer embryogenic cultures, higher cryotolerance has been reported with tissues taken during the exponential or early-exponential growth phases (Salaj et al., 2010, 2022). At this stage, cells have small vacuoles and reduced water content, which improves freezing survival (Reinhoud et al., 2000). Active and vigorous growth observed at these phases is also considered decisive to withstand cryopreservation (Häggman et al., 2000; Salaj et al., 2010).

Somatic embryogenesis has been initiated in a large number of conifer species belonging to the genera Abies, Picea, Pinus, Pseudotsuga, Taxus and Araucaria (Attree and Fowke, 1993; Lelu-Walter et al., 2013; Salaj et al., 2015; Klimaszewska et al., 2016; Jouini et al., 2023). However, this developmental process has not been previously reported in A. pinsapo, a biologically very important species.

Induction of somatic embryogenesis normally requires explants culture on media supplemented with auxin, although cytokinins are also frequently needed (Klimaszewska and Cyr, 2002; von Arnold, 2008). However, in different species of the genus Abies, such as A. fraseri (Guevin and Kirby, 1997), A. normanniana, A. alba, A. cephalonica (Krajňáková et al., 2008), and A. nebrodensis (Jouini et al., 2023), single application of cytokinin was sufficient to induce somatic embryogenesis (Nørgaard and Krogstrup, 1995; Kim et al., 2008; Krajňáková et al., 2008; Salaj et al., 2020). Although diverse cytokinins, such as BA, kinetin, 6-(γ,γ-dimethylallylamino)purine (2iP) and thidiazuron (TDZ), have been tested in different investigations (Nørgaard and Krogstrup, 1991; Kim et al., 2008), BA is the plant growth regulator more frequently used for somatic embryogenesis induction in species of the genus Abies (Salajova et al., 1996; Krajňáková et al., 2013; Salaj et al., 2019). In A. pinsapo, induction of somatic embryogenesis was achieved in the presence of 5 mg L^-1 BA, at a 4% induction rate. Similar induction percentages have been reported in other conifer species like A. balsamea (3.5%) (Guevin et al., 1994), A. fraseri (3.5-18%) (Guevin and Kirby, 1997), Picea abies (8%) (von Arnold, 1987), Abies alba (0.83–29.4%) (Szczygiel and Kowalczyk, 2001; Krajňáková et al., 2013; Salaj et al., 2020) or Pinus korainesis (1.67%) (Gao et al., 2020). Proliferation of embryogenic tissues successfully occurred on culture medium of the same composition supplemented with 500 mg L^-1 L-glutamine, 1 g L^-1 casein hydrolysate, and 1 mg L^-1 BA. Therefore, as found in other Abies species (Nørgaard and Krogstrup, 1995; Kim et al., 2008), in A. pinsapo, auxins do not play an essential role in somatic embryogenesis, and induction and proliferation of embryogenic cultures can be executed using cytokinins as the sole plant growth regulator.

In this work, successful cryopreservation of Spanish fir embryogenic cultures is also reported. Long-term conservation of plant genetic resources using cryopreservation relies on freezing (Gonzalez-Arnao et al., 2008). Formation of lethal intracellular ice crystals is the main cause of cell damage when exposing biological materials to subzero temperatures (Panis, 1995). Different preconditioning and cryoprotective treatments contribute to protect tissues before freezing. The most common preconditioning treatments include cold hardening and preculture with osmotic agents (Ballesteros et al., 2024). In the present investigation, both types of treatments were applied, with a different influence on the results obtained.

Exposition to low but non-freezing temperatures can induce freezing tolerance, as different processes occur during cold acclimation (Buchanan et al., 2015). Changes in the membrane lipid composition, such as modifications in the proportion of phospholipids, glucocerebrosides or sterol lipids, play a relevant role in the maintenance of the integrity of the plasma membrane, the primary site of injury during freezing (Ramon et al., 2002). Altered lipid-lipid or lipid-protein interactions induced by cold contribute to stabilize the plasma membrane during freeze-thaw cycles. Compatible solutes, such as soluble sugars (like sucrose, glucose, fructose, raffinose, and stachyose) or low molecular weight nitrogen-containing substances (like proline and glycine betaine) also accumulate during cold acclimation. Apart from a role directly related to freezing, compatible solutes also play an important function during osmotic and dehydration stresses, which also may occur during the execution of cryopreservation protocols (Buchanan et al., 2015; Taiz et al., 2015). Proteins involved in very diverse processes, such as phospholipases, dehydrins, antifreeze proteins, stress-related proteins, membrane-trafficking proteins and proteolysis-associated proteins also change in response to low temperatures (Buchanan et al., 2015; Taiz et al., 2015). Most of changes occurring during cold acclimation are mediated by alterations in gene expression. Different cold-responsive pathways appear to be involved in activating cold-induced reactions, thus suggesting complex regulatory systems for genes associated with freezing tolerance (Buchanan et al., 2015).

However, in embryogenic tissues of Spanish fir, cold hardening delayed cultures recovery after freezing in liquid nitrogen. This negative effect, despite the positive molecular processes triggered by cold treatment, could be due to the decreased growth observed in cooled explants. The growth state of cultures critically affects cryopreservation (Bradaï et al., 2023), due to its influence on explants physiological state, which is considered an important factor determining freezing tolerance (Engelmann et al., 2008). As pointed by Bradaï et al. (2017), high metabolic activity and proliferation capacity can play a decisive role on cryopreservation success as it is not only determined by cell survival after thawing, but also by cell ability to proliferate and continue growing later. In this line, in Pinus caribaea, Laine et al. (1992) found that only embryogenic cell suspensions rapidly growing could be successfully recovered after liquid nitrogen storage.

Incubation in culture media containing high concentrations of osmotically active agents is a preconditioning treatment frequently used to improve cryotolerance in conifer embryogenic cultures (Häggman et al., 2000). Sugars, such as sucrose, maltose and glucose, and sugar alcohols, such as sorbitol or mannitol, are the most commonly used compounds for pretreatment of conifer embryogenic tissues (Salaj et al., 2010, 2022; Ballesteros et al., 2024). Preculture with osmotic agents has a direct effect decreasing explant´s water content and consequently the amount of intracellular freezable water (Panis and Thinh, 2001), thus avoiding detrimental ice crystal formation during freezing. Besides, mild osmotic stress at which tissues are subjected during preculture with sugars or sugar alcohols increased desiccation and freezing tolerance (Touchell et al., 2002).

In the case of sucrose, it has also been identified as an important signaling molecule regulating metabolism and gene expression (Gupta and Kaur, 2005; Carpentier et al., 2010; Wang et al., 2020). Thus, as pointed by Bradaï et al. (2023), it can increase tolerance to dehydration and cooling by inducing the accumulation of compatible solutes (Suzuki et al., 2006; Pociecha et al., 2009), alterations in fatty acids metabolism (Ramon et al., 2002; Zhu et al., 2006), changes in the proteome (Carpentier et al., 2010; Folgado et al., 2015) or a transient increase of abscisic acid levels (Suzuki et al., 2006). Sorbitol´s positive effects have been attributed to stereochemical arrangements. As suggested by Turner et al. (2001), the stereo-orientation of the hydroxyl groups of sugar alcohols may allow better hydrogen bonding and packing around the membrane bilayer, thus increasing desiccation and freezing tolerance. Pritchard et al. (1986) also reported modifications in cell structure leading to a reduction of the vacuolar volume, due to replacement of the large vacuole by several smaller vesicles.

In embryogenic cultures of Spanish fir, the precultures tested differently affected cultures growth rate. While incubation on proliferation medium supplemented with 0.2 M and 0.4 M sucrose for two successive 24 h-periods did not have a significant detrimental effect, a significant growth reduction could be observed in uncooled, sorbitol-precultured tissues. Besides, despite the short sorbitol pretreatment, variations in cultures morphology could be observed over time.

Cryoprotection also plays an important role in two-step freezing protocols, protecting cells from osmotic damage and ice formation during the cooling steps (Ballesteros et al., 2024). Overall, cryoprotectants appears to act in a multimodal way, with cryoprotection arising from a combination of processes, such as modulation of hydrogen bonding, effects on cell membrane properties, dilution of solute effects and increase of solution viscosity at low temperatures, among others (Murray and Gibson, 2022).

The choice of the cryoprotectant is especially important in the cryopreservation of embryogenic cells, as high water content normally present in this type of tissues, highly increases the risks of intracellular ice crystal formation during freezing (Lambardi et al., 2018). Although DMSO is the cryoprotectant mostly used in conifers (Häggman et al., 2000), different mixtures of penetrating and non-penetrating substances have also been utilized (Cyr et al., 1994; Aronen et al., 1999; Krajňáková et al., 2011; Ozudogru and Lambardi, 2016), as they are often more effective than a single component at the same total osmolarity (Ulrich et al., 1979).

In embryogenic cultures of A. pinsapo, the cryoprotectants tested did not have a deleterious effect on cultures recovery. Nevertheless, the best response in terms of survival after freezing was achieved in explants cryopreserved using PGD I. The mixture PGD, including polyethylene glycol, glucose, and DMSO, have been repeatedly utilized in conifers species like Pinus sylvestris (Häggman et al., 1998), Picea abies, Pinus taeda (Gupta et al., 1987), and Abies cephalonica hybrids (Aronen et al., 1999). In general, replacement of DMSO by PGD improved the recovery and/or growth of cultures (Häggman et al., 2000). According to Aronen et al. (1999), the other components of the mixture can probably reduce harmful effects of DMSO and retain good growth potential of cultures.

In this work, somatic embryogenesis has been induced from mature zygotic embryos of A. pinsapo cultured on solid MS medium with macroelements at half-strength and supplemented with 5 mg L^-1 BA. Successful cryopreservation of Spanish fir embryogenic cultures has also been achieved by using the slow-cooling technique. The recovery rates achieved after sucrose preculture and cryoprotection with PGD I (100%) allow secure long-term storage of germplasm of this valuable endangered species. The results obtained allow to establish the bases for the integration of somatic embryogenesis and cryopreservation into in situ and ex situ strategies for the conservation of A. pinsapo.