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Morphological and Biochemical Responses of Pinus radiata Embryogenic Cultures to Elicitors, Salinity and Heat Stress

A peer-reviewed version of this preprint was published in:
Forests 2026, 17(8), 898. https://doi.org/10.3390/f17080898

Submitted:

08 July 2026

Posted:

10 July 2026

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Abstract
Pinus radiata is a commercially valuable forest species whose productivity is increasingly threatened by climate change-related biotic and abiotic stresses such as diseases, soil salinization and rising temperatures. Taking as a starting point a sufficiently optimized somatic embryogenesis propagation protocol for radiata pine, this study aimed to evaluate the morphological and biochemical responses embryogenic cell lines subjected to different elicitors such as methyl jasmonate and tagatose during proliferation or sodium butyrate at maturation stage. The effect of various stresses such as salinity and high temperatures was evaluated at proliferation and germination stages. Then, biochemical analyses (total protein, hydrogene peroxide and malondialdehide contents) were carried out in embryogenic tissues and plants and the growth was also assessed in these tissues and in somatic plants. This latter growth assessment led to a classification of plants depending on their suitability to be planted ex vitro. Several embryogenic cell lines were tested and the response to elicitation or stress was found highly genotype-dependent however some trends could be observed particularly when assessing growth. These results suggest that somatic embryogenesis combined with stress priming or elicitor application may be a viable strategy to enhance biotic and abiotic stress tolerance in radiata pine.
Keywords: 
heat
;  
methyl jasmonate
;  
sodium butyrate
;  
sodium chloride
;  
tagatose
Subject: 
Biology and Life Sciences  -   Biology and Biotechnology

1. Introduction

Climate change is exposing the necessity of adapting agriculture and forestry sectors to new challenges by implementing scientific knowledge to produce more resilient plants. To this aim, fundamental research is the first step on the path; uncovering the physiological and biochemical mechanisms underlying plant stress response and adaptation is crucial to developing cutting edge biotechnological approaches.
In this regard, thanks to the basic knowledge generated during the last 50 years, alternative plant improvement approaches such as priming for enhanced defence are gaining special attention. This phenomenon is based on the biochemical mechanism in which plants pre-exposed to a certain stressor or stimulus, activate faster and/or stronger immune and stress response in the future [1]. Some molecules, commonly known as elicitors or priming agents, have the potential to mimic the presence of a stress factor or pathogen, triggering plant signalling and an advanced “warning” physiological state as well as activating the systemic acquired resistance (Tripathi et al. 2019). Well documented examples of elicitors include some phytohormones such as methyl jasmonate (MeJa) [2,3] and salycilic acid [4] or some rare sugars such as D-tagatose (TAG) [5]. Multiple studies have demonstrated that priming entails complex epigenetic modifications [6,7] ensuring the maintenance of a mid-to-long-term stress memory. These findings have also promoted research about the use of epigenetic modulators (e.g., azacitidine, sodium butyrate, trichostatin A to improve the behaviour of plants under different stress factors [8].
Somatic embryogenesis (SE) is a powerful and versatile micropropagation technique. Not only it is useful for producing a high number of genetically uniform, disease-free plants, but it can also be combined with long-term cryopreservation protocols [9] and genome-editing technologies [10] or just be used as a research model for the study of plant stress response [11] and the implementation of priming procedures under controlled and sterile environmental conditions [12]. Furthermore, as demonstrated by [13,14], and afterwards corroborated in our laboratory [15], the environmental conditions during plant embryo development (e.g., temperature, water availability) can determine the behaviour of the resulting plants years later.
In our region, the Basque Country (Spain), radiata pine (Pinus radiata D. Don) is a conifer species of great economic importance. Native to the Central Coast of California and Mexico, its cultivation has spread to numerous countries, including Spain, Australia, Chile, New Zealand, and South Africa [16]. It is considered the most widely planted exotic forest species worldwide, with over four million hectares planted globally [17,18], thanks to its rapid growth, adaptability, ease of plantation establishment, forest management and versatile wood properties [19]. However, this species is vulnerable to several biotic and abiotic stressors, which have been intensified by global climate change [20]. For instance, salinity due to poor soil management and is leading to an increase in barren land (about 40% of the terrestrial area), negatively impacting forest plantations [21]. Also, high salt concentrations cause physiological drought by lowering the osmotic potential of the soil [22].
One of the main consequences of climate change, the increase in temperatures, also provokes adverse effects on plant growth and development [23]. Elevated temperatures have negative effects on various physiological processes such as photosynthesis, primary and secondary metabolism, water relations and lipid metabolism [24]. Specifically, heat stress generates damage to the cell membrane due to the overproduction of reactive oxygen species (ROS), causing senescence and cell death and leading to altered plant growth and reproduction [25,26].
In this context, the aim of this study was to exploit the advantages that SE offers as a research tool and study 1) the in vitro response of P. radiata embryonal masses (EMs) and somatic plants (SPs) to high sodium chloride (NaCl) concentrations, 2) the effect of exposing EMs to two different priming agents (MeJa and tagatose), and 3) the long-term effect of a histone deacetylase inhibitor [sodium butyrate (NaB)] applied during the proliferation of EMs on the performance of the resulting SPs under heat stress. This first approach has been based on the measurement of different biochemical parameters [total protein, hydrogen peroxide and malondialdehyde (MDA) contents] and morphological parameters.

2. Materials and Methods

2.1. Plant Material

Embryonal masses (EMs) and somatic plants (SPs) of P. radiata were obtained following the protocols outlined by Montalbán and Moncaleán [27,28]. Briefly, one-year-old green female cones, enclosing immature zygotic embryos at precotyledonary stage were collected from genetically distinct mother trees. Megagametophytes were isolated from seeds and cultured on embryo development medium (EDM) [29] for 4 to 8 weeks. After that, the resulting EMs were separated from the megagametophytes and fortnightly subcultured onto EDM proliferation medium [28]. After four subcultures, the EMs growing actively were considered as embryogenic cell lines (ECLs).
For experiments with SPs, EMs at the proliferation stage were suspended in liquid EDM, filtered and cultured on EDM maturation medium [30] to promote somatic embryo development. After 15 weeks on maturation medium, somatic embryos (ses) were transferred to Petri dishes containing RG medium [28] for 6 weeks, followed by additional 6 weeks in OV80+OVD80 microbox (SacO2) culture boxes with the same medium to produce SPs. Unless stated otherwise, initiation, proliferation and maturation were carried out in darkness at 22ºC. Germination was carried out at the same temperature but under a 16 h photoperiod provided by cool white fluorescent tubes (TLD 58 W/33; Philips, France) at 120 μmolm− 2s−1.

2.2. Experiment 1: Salt Stress Response in EMs and SPs

For experiments carried out with EMs, five ECLs (R101, R330, R55, R156 and R4) at proliferation stage were used as initial material. The EMs were transferred to EDM proliferation media supplemented with different concentrations of NaCl (0, 50 or 100 mM). Each treatment comprised 5 Petri dishes and 4 calli were cultured on each plate.
To assess growth, photographs of each EM were taken on days 1 and 14 of proliferation. Then, with the program ImageJ V. 1.54g, the diameter of each callus was determined, and growth was estimated as the difference between the final and initial measure.
For the determination of dry weight, 0.2 g of EM was collected and oven dried at 70 °C for 48 h; 3 replicates per ECL and salt concentration were performed. The percentage of dry weight was calculated by applying the formula: Dry weight percentage (DW%) = dry weight (DW; g)/fresh weight (FW; g*100). The remaining material was crushed into liquid nitrogen and stored at -80 °C until the biochemical analyses were performed.
Total protein content was determined colorimetrically using the Bradford dye-binding assay with bovine serum albumin (BSA) following the protocol described in [31]. Three technical replicates for each ECL and salt concentration were measured.
For hydrogen peroxide (H2O2) determination, the protocols described by Alexieva et al. [32] were used as a reference. Three technical replicates of 0.5 g were processed for each treatment and ECL (except for R55 due to lack of material enough for the analyses in 100 mM treatment).
For experiments with SPs, three different ECLs (R23-94, R23-11 and R23-125), previously cultured for six weeks in Petri dishes containing RG germination medium, were transferred to OV80+OVD80 culture boxes (SacO2) with 80 mL of RG medium and supplemented with different treatments of NaCl (0, 25 and 50 mM). Eight SPs per microbox and three microboxes per treatment and ECL were cultivated under 16 h photoperiod at 120 μmols-1m-2 and 22 °C for seven weeks.
Photographs were taken of SPs from every treatment and embryogenic cell line (ECL) at the start and at the end of the culture in microboxes. Root and shoot lengths were measured using ImageJ V. 1.54 g. Then, growth was estimated as the difference between the final and initial length measurements. According to final root and shoot lengths SPs were classified into different categories as described in Table 1.
DW (two plants per replicate), total protein content and H2O2 (0.2 g per replicate) were calculated as previously described.
MDA content was measured following the protocol reported by Morcillo et al. [33], using 0.2 g tissue per replicate.
Chlorophyll a was quantified following the procedure described by López-Hidalgo et al. [34]. The absorbance was measured at 649 and 664 nm and a total of three technical replicates per treatment and ECL were analyzed. The concentration of chlorophyll a (mgDW-1) was calculated as 13.36*A664–5.19 *A649.

2.3. Experiment 2: Elicitation of EMs with MeJa and TAG

Three ECLs (RE22-25, RE22-51 and R23-126) were used as starting material. A total of 0.9 g was placed in a sterile 50 mL tube for each treatment and ECL. In each tube, 15 mL of liquid EDM [29] supplemented with MeJa (0, 5 or 20 µM) or TAG (0, 0.5 or 1.5 gL-1) were added. Cultures were incubated in darkness at 22 °C and 70 rpm for 3 days. After that, 5 mL of the suspension containing 0.3 g of embryogenic tissue were poured on a filter paper (Whatman, nº2) and the liquid medium was drained in a Büchner funnel; then, the filter paper with the attached tissue was transferred onto proliferation medium. Three Petri dishes for each treatment and genotype were cultured for three weeks. The whole procedure (culture of EMs with MeJa or TAG and subsequent proliferation) was repeated two more times to induce three elicitations of plant material. Then, morphological and biochemical analyses were performed.
Tissue growth was calculated indirectly weighing the Petri dish with the proliferating tissue at the start and at the end of the proliferation period.
The same protocol used to determine dry weight in Experiment 1 was followed, but using 0.5 g of EM for each analysis. Total protein, H2O2 and MDA were determined for all treatments and ECLs as described above. Three technical replicates were performed for each treatment and ECL.

2.4. Experiment 3: Culture of EMs on Media with NaB and Heat Stress Application on the Resulting SPs

Actively proliferating EMs from three different ECLs (RD21-27, R20-52, R23-54) were divided into three treatments based on NaB application, following the methodology of Sandoval et al. [35]. In NaB 1 treatment, EMs were suspended in liquid EDM with 0.5 µM NaB for 1 h before transferring to EDM maturation medium. For NaB 2 treatment, EMs were cultured directly on semisolid EDM maturation medium supplemented with 0.5 µM NaB. The control group followed the standard maturation procedure. After 15 weeks, ses were germinated as explained above. Then, for each NaB treatment, seven microboxes per ECL and temperature regime, each containing ten SPs, were subjected to heat stress consisting of exposure to 35 ºC for 3 h daily over 3 days, followed by 42 ºC for 3 h daily over 2 additional days. Control microboxes were kept at 22 ºC for 5 days.
Survival per ECL and treatment was registered weekly for 6 weeks. Initial and final longitude of the root and shoot were measured for each treated somatic plant on day 1 and 42 of the experiment using ImageJ software V. 1.54g. Classification of somatic plants was conducted according to final root and shoot lengths as described above (Table 1).
Six weeks after the start of heat stress application, extraction and quantification of total phenolic compunds and flavonoids was carried out as described by Kim et al. [36] with some modifications. Briefly, 50 mg of the grounded plant material was suspended in 1 mL 80% (v:v) methanol, homogenized and centrifuged at 11000 g for 10 min. The supernatant was collected and evaporated. Afterwards, 1 mL methanol 50% (v:v) was added to the tube and homogenized. For phenol quantification, 100 µL plant extract was diluted in 900 µL distilled water. Subsequently, 100 µL Folin & Ciocalteu’s phenol reagent were added, followed by incubation for 5 minutes. Then, 1 mL 7% (m:v) Na₂CO₃ solution was added. For flavonoid quantification, 100 µL of the extract were mixed with 400 µL distilled water, 30 µL 5% (m:v) NaNO₂, 30 µL 10% (m:v) AlCl₃, 200 µL 1M NaOH, and 240 µL distilled water. Then, total phenol and flavonoid contents were measured spectrophotometrically, and measurements were expressed as mg gallic acid and catechin equivalents (for total phenolic compounds and flavonoids, respectively) per g of fresh plant tissue after construction of standard curves. Three technical replicates for each treatment and ECL were analysed.

2.5. Statistical Analysis

Experiments were conducted in a completely randomized block design. The assumptions of normality and homoscedasticity were determined and linear model or generalized linear model fittings were performed followed by Tukey’s test of multiple comparisons (α = 0.05) to determine significant statistical differences.

3. Results

3.1. Experiment 1: Salt Stress Response in EMs and SPs

After 14 days in proliferation medium, the EMs of each treatment showed significant differences in size from day 1 to 14; the growth decreased significantly as the concentration of salt in the medium increased in all the ECLs assayed (Figure 1 and Figure 2A), although this reduction was most noticeable in those cultured with100 mM NaCl. DW percentage of EMs was significantly higher as the salt concentration in the medium increased (Figure 2B).
Total protein content was significantly lower in EMs cultured at the highest concentration of salt. The amount of protein per dry weight followed a tendency to decrease in the EM from the lowest concentration to the highest of sodium chloride and was significantly lower at 50 mM NaCl (Figure 3A). On the contrary, H2O2 contents did not show a clear pattern, moreover a significant interaction between treatments and ECLs was found. (Figure 3B).
The relative percentage of SPs categorized as 1 from 25 mM NaCl treatment was equal or higher than control treatment for lines R23-11 and R23-125. However, this percentage decreased as NaCl increased in R23-94. Plants from class 2 and 3 showed a different tendency among treatments and ECLs. The relative percentage of class 4 SPs coming from different ECLs was always highest after culture at the highest salt concentration (Figure 4).
The growth depended significantly on the genotype tested; the root growth was not affected by salinity. Whereas at the highest NaCl concentration, plants displayed shorter shoots. (Figure 5A). DW percentage was one of the few parameters analysed not genotype-dependent, showing significantly higher percentages the plants cultured at control conditions (Figure 5B).
A significant interaction between the genotype and the treatments was found for the total protein contents. Within each cell line, SPs cultured at 25 mM NaCl showed significantly higher protein contents than control, whereas culture at the highest salinity concentration led to different results depending on the genotype (Figure 6A). When H2O2 content was analysed, although significant differences were found for the ECLs and treatments, the contents (ranging from 40.6 to 60.8 µg g DW-1) did not follow a clear pattern (Figure 6B).
In the same way, chlorophyll a content did not follow a trend; however, the highest (R23-125, control) and lowest values (R23-94 50 mM NaCl) were observed in the same ECLs and treatments (Figure 7A) in relation to H2O2 accumulation (Figure 6B). The MDA content was similar in line R23-11 regardless of the treatment applied, whereas in the other lines tested it was lower at control conditions (Figure 7B).
Figure 8 shows that most SP from R23-11 and R23-94 exhibited yellowish colors under 25 and 50 mM treatments compared to 0 mM NaCl. In contrast, SP from R23-125 ECL did not show the same trend.

3.2. Experiment 2: Elicitation of EMs with MeJa and TAG

The growth of EMs was similar regardless of the treatment, only significant differences were found depending on the ECL tested (Figure 9). A significant effect of the ECL, elicitation treatment and their interaction was found. Embryonal masses tissue of R23-126 at the end of the experiment in control (a), 5 µM methyl jasmonate (b), 20 µM methyl jasmonate (c), 0.5 g L-1 tagatose (d) and 1.5 g L-1 tagatose (e).
The effect of elicitors on the total protein content was genotype dependent, but no significant interactions were found between them and the treatments assayed. The EMs at control conditions exhibited a significantly higher total protein content than those subjected to MeJa treatments (Figure 10 A). In the case of TAG, significant differences were found between all treatments, the highest protein content was shown in control treatment, followed by TAG at 1.5 and 0.5 g L-1 (Figure 10B).
When MeJa was applied to EMs, the results for H2O2 contents showed a significant interaction without a clear trend (Figure 11A). In relation to TAG treatments, H2O2 content was significantly lower when elicitor was applied (Figure 11B).
Although a significant interaction was found between ECLs and MeJa (Figure 12A) or TAG (Figure 12B) treatments in relation to MDA contents, their concentration appears to be more influenced by the ECL, with higher contents in R22-51, intermediate contents in R22-25 and lower contents in R23-126.

3.3. Experiment 3: Culture of EMs on Media with NaB and Heat Stress Application on the Resulting SPs

The addition of NaB did not imply an improvement in the quality of SPs (Figure 13); however, under thermal stress conditions the lowest number of low-quality somatic plants (class 4) were observed in plants from both NaB treatments (Figure 13).
The biggest SPs with the highest flavonoid content were those from control conditions (Figure 14A, B). The growth (Figure 14A) and total flavonoid content (Figure 14B) were significantly reduced in non-primed SPs subjected to heat stress, these values were similar to those observed in plants from sodium butyrate treatments regardless of their culture conditions during germination.
The total phenol content was significantly affected by all the factors studied separately (Figure 14B). However, no significant effect of the interaction between NaB treatments and temperature stress was observed, ranging from 3.5 and 4.5 mg of gallic acid equivalents per gram of fresh weight plant tissue.

4. Discussion

After analyzing the results of salt stress experiment, the growth of both EMs and SPs was negatively affected by increasing salt concentrations. This effect was particularly evident at concentrations above 50 mM NaCl and reached its maximum at 100 mM, where the growth of EMs was almost completely suppressed. The results are consistent with previous studies on SE in Medicago truncatula [37], in which a slight reduction in cell growth was observed at 50 mM NaCl when compared with the control treatment (0 mM NaCl), which was attributed to osmotic stress. At higher concentrations (100 and 150 mM NaCl), the authors reported a reduction in cell size, which was associated with plasmolysis. Similar responses have also been described in embryogenic cultures of other plant species; for example, callus and cell suspension cultures of Stevia rebaudiana exposed to salt stress exhibited lower growth rates than control cultures [38]. In Psidium guajava SPs, shoot growth decreased when 50 and 100 mM NaCl treatments were applied, which was attributed to limitations in water uptake and detrimental ionic effects on biochemical processes [39].
Regarding dry weight, EMs presented a tendency to increase their dry matter content at high NaCl concentrations. This pattern indicates that all ECLs were affected by the osmolarity of the culture medium [40]. In SPs, however, the opposite behavior was detected. This trend is in accordance with other studies conducted at the whole-plant level, where salinity reduces dry weight presumably because of water deficit, nutritional imbalance and osmotic stress, ultimately leading to stomatal closure [41]. Since the present study was performed in vitro, where plants rely largely on heterotrophic nutrition, the reduction in dry weight may be more closely related to impaired water uptake, and consequently, reduced carbohydrate absorption from the culture medium rather than stomatal dysfunction. Nonetheless, total dry weight alone may provide a limited view of plant responses to salinity, as some species tend to allocate a greater proportion of biomass to the root system under stress conditions [43].
Total protein content in EMs showed a general decreasing trend with increasing NaCl concentrations. This reduction can be attributed to the detrimental effect of salinity on mRNA biosynthesis and nutrient uptake required for protein synthesis, leading to alterations in this metabolic process [41]. Similarly, Jasim et al. [44], reported that increasing NaCl concentrations in the culture medium resulted in lower protein contents in the embryogenic calli of Phoenix dactylifera. On the other hand, the total protein content of SPs increased after culture at 25 mM NaCl for all ECLs, and for two of the three ECLs at 50 mM NaCl. It is well-known that moderate salinity can promote the synthesis of specific stress response proteins [45]. For example, in a study carried out by Azizi et al. [46], an increase in the total protein of all Rosa damascena seedling genotypes treated with 25 mM NaCl was observed, whereas only a few genotypes showed that response at 50 mM treatment.
The levels of H2O2, chlorophyll a and MDA appeared to be genotype-dependent under salt stress. In the case of H2O2, this behavior is consistent with Hasanuzzaman et al. [47] and Wang et al. [48], who reported that the degree of oxidative stress caused by ROS like H2O2 in plants under saline conditions is highly dependent on the characteristics of each ECL and their genetic background. Despite the absence of a clear pattern, combining phenotypic observations with biochemical analyses enabled the differentiation of salt tolerance levels among the different ECLs. Two of the three genotypes presented a yellowish color under salt stress, suggesting higher susceptibility to elevated NaCl concentrations. These ECLs exhibited reduced chlorophyll content under stress conditions, following trends observed in other species [49,50]. Interestingly, the most susceptible genotype presented almost double the malondialdehyde content when growth under stress conditions, indicating increased lipid peroxidation and cellular damage, as observed in Solanum melongena seedlings at 30 mM NaCl [51]. Together, these findings support the usefulness of in vitro systems as effective tools for the early selection and propagation of stress-tolerant plant material [52].
In relation to Experiment 2, the growth of EMs did not differ significantly among elicitor treatments; this indicates that the treatment neither promoted nor inhibited cell proliferation. These results are consistent with those reported by Yazdanian et al. [53], who observed that elicitors such as MeJa at a concentration of 25 µM did not significantly affect the growth of Allium jesdianum EMs compared with the control treatment. Likewise, these treatments had no effect on the dry biomass of embryogenic cultures. In contrast, both MeJa and TAG treatments negatively affected total protein content, probably because of alterations in protein biosynthesis or enhanced protein degradation, as previously suggested for EMs of P. radiata [54]. When MeJa or TAG treatments were applied to EMs, no increase in H2O2 content was recorded, suggesting that these elicitors did not generate oxidative stress in treated EMs. Similar results were reported in EMs of Quercus ilex, where the lowest H2O2 content was obtained following treatment with 25 µM MeJa [12]. MDA is a product of the secondary degradation of lipid peroxidation and is widely used as an indicator of oxidative damage [55]. In the present study, a highly genotype-dependent response was observed for MDA content. Some studies have suggested that elicitors such as MeJa can increase MDA levels as part of the stress response MeJa [56]. However, Yazdanian et al. [53] reported that treatment with 25 µM MeJa did not produce significant changes in MDA content compared with the control in Allium jesdianum EMs. Similarly, in Rosmarinus officinalis, MeJa treatment resulted in reduced MDA levels, which was attributed to an enhanced capacity to mitigate and repair oxidative damage [57].
In Experiment 3, in which EMs were treated with NaB and the resulting plants were exposed to heat stress, no clear effect of the histone deacetylase inhibitor was observed on either the quality or the growth of the SPs under control conditions. On the other hand, high temperatures caused a noticeable decline in the shoot growth of plants originating from non-treated EMs. It is well documented that heat provokes a decrease in photosynthesis, cell membrane dysfunction, and oxidative damage, impairing plant development [24,26]. The detrimental effect of elevated temperatures could not be observed in NaB derived SPs, suggesting that the elicitor could be playing a determinant role in plant homeostasis under stress conditions [58]. Multiple studies have addressed the positive effects of NaB on different stages of SE. For example, the addition of 0.5 mM NaB to the induction culture medium improved the embryogenic response in Vitis vinifera [59], or concentrations up to 1 mM on tomato and tabaco protoplast derived-calli increased the frequency of adventitious shoot formation [60]. Less information is available about the implications of this histone deacetylase inhibitor in stress-response processes. Some authors have observed that NaB treatments can alter the expression of stress-related genes, modulating the immune response of plants against certain pathogens [61]. In some medicinal plants, such as Platycodon grandiflorus, NaB-induced changes in histone acetylation led to alterations in secondary metabolism, particularly in the accumulation of polyphenolic compounds in the roots of the plants [62].
In this regard, in the present study, elevated temperatures provoked a significant decrease in total flavonoid levels, which, as observed for shoot growth, was not detected in SPs derived from NaB treatments. It is noteworthy that the basal levels of these compounds under control conditions were lower than those of non-treated plants. Under abiotic stress conditions, an increase in the synthesis of certain groups of secondary metabolites is expected, since these molecules play crucial roles in oxidative stress mitigation, osmoprotection, and overall stress adaptation [63]. Specifically, heat stress promotes the production of antioxidant phenolic compounds such as flavonoids, phenylpropanoids, anthocyanins and lignans which are related to the suppression of stress-induced oxidation of cellular molecules [64]. Nonetheless, some authors have also identified a slight reduction in the levels of these metabolites under heat stress, attributed to overall metabolic malfunction [65]. In tomato seedlings, lower flavonoid levels were detected after heat stress, but contrastingly, the plants showed improved antioxidant capacity in antioxidant assays [66]. Similarly, in Pinus sylvestris, elevated temperatures altered the composition of the flavonoid profile, and specific groups such as acylated flavanols and flavan-3-ols were more affected than others [67]. These findings suggest that long-term epigenetic modifications induced by NaB may be implicated in the fine-tuning of certain secondary metabolism pathways, resulting in improved performance of SPs under stress conditions according with Zhao et al. [68].

5. Conclusions

The growth and weight of EMs and SPs was affected by salinity; on the contrary, elicitated ECLs did not grow differently from the controls. The total protein content was negatively affected by salinity, MeJa or TAG treatments at proliferation stage of the cultures. Other biochemical parameters tested seemed to be more genotype-dependent in both experiments 1 and 2. When elicitation was carried out at maturation stage, the resulting SPs showed lower flavonoid contents after being subjected to heat stress, finally SPs generated at control conditions grew less after stress application that those elicitated with NaB.

Author Contributions

Conceptualization: I.A.M. and P.M.; Funding acquisition: P.M.; Investigation: M.G., D.V., D.B., A.C., I. A. M.; Data curation: M.G., D.V., D.B. Formal analysis: M.G., D.V., D.B., A.C., I.A.M., J.P.; Visualization: A.C., I.A.M., J.P.; Supervision: A.C., I.A.M., P.M.; Writing—original draft: M.G., D.V., D.B., A.C., I.A.M., J.P. and P.M.; Writing—review and editing: A.C. I.A.M., J.P., P.M. Resources: I. A. M., P.M. Project administration: P. M. Funding adqusition: P.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was performed through Project PID2020-112627RB-C32 and Project PID2024-156422OB-C32 funded by MICIU/AEI/10.13039/501100011033. The authors would like to acknowledge COST Action CA21157 “European Network for Innovative Woody Plant Cloning”, www.copytree.eu (accessed on 12 March 2025), supported by COST (European Cooperation in Science and Technology) www.cost.eu (accessed on 12 March 2025).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BSA     Bovine serum albumin
DW     Dry weight
ECL     Embryogenic cell line
ECLs     Embryogenic Cell ines
EMs     Embryonal masses
FW     Fresh weight
H2O2     Hydrogen peroxide
MDA     Malondialdehyde
MeJa     Methyl jasmonate
NaB     Sodium butyrate
NaCl     Sodium chloride
ROS     Reactive oxygen species
SE     Somatic Embryogenesis
Ses     Somatic embryos
SPs     Somatic plants
TAG     D-Tagatose

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Figure 1. Pinus radiata embryonal masses of five embryogenic cell lines after 14 days of proliferation in medium with different sodium chloride concentrations. From left to right, sodium chloride concentrations (0 mM, 50 mM and 100 mM); from top to bottom, embryogenic cell lines (R4, R55, R101, R156, R330).
Figure 1. Pinus radiata embryonal masses of five embryogenic cell lines after 14 days of proliferation in medium with different sodium chloride concentrations. From left to right, sodium chloride concentrations (0 mM, 50 mM and 100 mM); from top to bottom, embryogenic cell lines (R4, R55, R101, R156, R330).
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Figure 2. Embryonal mass growth (A) and dry weight (B) in response to sodium chloride treatments (0, 50 and 100 mM) in five radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 2. Embryonal mass growth (A) and dry weight (B) in response to sodium chloride treatments (0, 50 and 100 mM) in five radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 3. Total protein (mg, A) and hydrogen peroxide content (µg, B) per gram of dry weight in response to sodium chloride treatments (0, 50 and 100 mM) in embryogenic cell lines of radiata pine (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 3. Total protein (mg, A) and hydrogen peroxide content (µg, B) per gram of dry weight in response to sodium chloride treatments (0, 50 and 100 mM) in embryogenic cell lines of radiata pine (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 4. Radiata pine somatic plants (%) classified as 1 (root length ≥ 1 cm and shoot length ≥ 2.5 cm), 2 (root length < 1 cm and shoot length ≥ 2.5 cm), 3 (root length ≥ 1 cm and shoot length < 2.5 cm), and 4 (root length < 1 cm and shoot length < 2.5 cm) according to sodium chloride treatments (0, 25 and 50 mM) in somatic plants from three embryogenic cell lines.
Figure 4. Radiata pine somatic plants (%) classified as 1 (root length ≥ 1 cm and shoot length ≥ 2.5 cm), 2 (root length < 1 cm and shoot length ≥ 2.5 cm), 3 (root length ≥ 1 cm and shoot length < 2.5 cm), and 4 (root length < 1 cm and shoot length < 2.5 cm) according to sodium chloride treatments (0, 25 and 50 mM) in somatic plants from three embryogenic cell lines.
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Figure 5. Shoot growth (cm, A) and dry weight (%, B) in somatic plants from three radiata pine embryogenic cell lines in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 5. Shoot growth (cm, A) and dry weight (%, B) in somatic plants from three radiata pine embryogenic cell lines in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 6. Total protein (A) and hydrogen peroxide (B) content per gram of dry weight in Pinus radiata somatic plants in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 6. Total protein (A) and hydrogen peroxide (B) content per gram of dry weight in Pinus radiata somatic plants in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 7. Chlorophyll a (A) and malondialdehyde (MDA, B) contents per gram of dry weight in Pinus radiata somatic plants in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 7. Chlorophyll a (A) and malondialdehyde (MDA, B) contents per gram of dry weight in Pinus radiata somatic plants in response to sodium chloride treatments (0, 25 and 50 mM; M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 8. Pinus radiata somatic plants from three embryogenic cell lines at the end of culture in medium with different sodium chloride concentrations. From up to down (R23-11, R23-125 and R23-94 ECL); from left to right (0 mM, 25 mM and 50 mM NaCl).
Figure 8. Pinus radiata somatic plants from three embryogenic cell lines at the end of culture in medium with different sodium chloride concentrations. From up to down (R23-11, R23-125 and R23-94 ECL); from left to right (0 mM, 25 mM and 50 mM NaCl).
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Figure 9. Embryonal masses tissue of R23-126 at the end of the experiment in control (a), 5 µM methyl jasmonate (b), 20 µM methyl jasmonate (c), 0.5 g L-1 tagatose (d) and 1.5 g L-1 tagatose (e).
Figure 9. Embryonal masses tissue of R23-126 at the end of the experiment in control (a), 5 µM methyl jasmonate (b), 20 µM methyl jasmonate (c), 0.5 g L-1 tagatose (d) and 1.5 g L-1 tagatose (e).
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Figure 10. Total protein content per gram of dry weight in response to methyl jasmonate treatments (0, 5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) in radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 10. Total protein content per gram of dry weight in response to methyl jasmonate treatments (0, 5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) in radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 11. Hydrogen peroxide content per gram of dry weight in response to methyl jasmonate treatments (0, 5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 11. Hydrogen peroxide content per gram of dry weight in response to methyl jasmonate treatments (0, 5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 12. Malondialdehyde (MDA) content per gram of dry weight in response to methyl jasmonate treatments (0,5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) in radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
Figure 12. Malondialdehyde (MDA) content per gram of dry weight in response to methyl jasmonate treatments (0,5 and 20 µM, A) and tagatose treatments (0, 0.5 and 1.5 g L-1, B) in radiata pine embryogenic cell lines (M±S.E.). Different letters indicate significant differences according to Tukey HSD test (α = 0.05).
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Figure 13. Radiata pine somatic plants (%) classified as 1 (root length ≥ 1 cm and shoot length ≥ 2.5 cm), 2 (root length < 1 cm and shoot length ≥ 2.5 cm), 3 (root length ≥ 1 cm and shoot length < 2.5 cm), and 4 (root length < 1 cm and shoot length < 2.5 cm) from control conditions and sodium butyrate treatments (NaB 1 and NaB 2) after 6 weeks of in vitro culture at 22 °C (control) or after exposure to 3h at 35 ºC for 3 days, 3 h at 42 ºC for 2 days and the resting culture period at 22 ºC (stressed).
Figure 13. Radiata pine somatic plants (%) classified as 1 (root length ≥ 1 cm and shoot length ≥ 2.5 cm), 2 (root length < 1 cm and shoot length ≥ 2.5 cm), 3 (root length ≥ 1 cm and shoot length < 2.5 cm), and 4 (root length < 1 cm and shoot length < 2.5 cm) from control conditions and sodium butyrate treatments (NaB 1 and NaB 2) after 6 weeks of in vitro culture at 22 °C (control) or after exposure to 3h at 35 ºC for 3 days, 3 h at 42 ºC for 2 days and the resting culture period at 22 ºC (stressed).
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Figure 14. Growth (cm, A) and total flavonoid content (TFC, mg of catechin equivalents per g of fresh weight, B) in Pinus radiata somatic plantlets from control conditions and sodium butyrate treatments (NaB 1 and NaB 2) after 6 weeks of in vitro culture at 22 °C (control) and or after exposure to 3h at 35 ºC for 3 days, 3 h at 42 ºC for 2 days and the resting culture period at 22 ºC (stressed) (M±S.E.) Different letters indicate statistically significant differences according to Tukey HSD post hoc test (α = 0.05).
Figure 14. Growth (cm, A) and total flavonoid content (TFC, mg of catechin equivalents per g of fresh weight, B) in Pinus radiata somatic plantlets from control conditions and sodium butyrate treatments (NaB 1 and NaB 2) after 6 weeks of in vitro culture at 22 °C (control) and or after exposure to 3h at 35 ºC for 3 days, 3 h at 42 ºC for 2 days and the resting culture period at 22 ºC (stressed) (M±S.E.) Different letters indicate statistically significant differences according to Tukey HSD post hoc test (α = 0.05).
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Table 1. Morphological characteristics of Pinus radiata somatic plants categorized as 1, 2, 3 or 4 according to final shoot and root length (cm).
Table 1. Morphological characteristics of Pinus radiata somatic plants categorized as 1, 2, 3 or 4 according to final shoot and root length (cm).
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