Introduction

The term stress refers to any type of external pressure, whether abiotic (such as salinity, heat, water scarcity, etc.) or biotic (such as herbivore action), that limits the rate of photosynthesis and reduces the plants’ ability to convert energy into biomass1,2.

The adverse impact of excess minerals such as sodium (Na+) and/or chloride (Cl-) in plants is known as salt stress3,4,5,6. Salt stress represents one of the most significant factors limiting crop growth and production. There are several sustainable approaches to mitigate this effect, both preventive and proactive, including soil recovery, that can be implemented separately or in combination to enhance plant resistance to salt and improve crop nutrition under conditions affected by this issue7,8.

New research strategies have been exploring the benefits of different organic amendments for plant growth in saline or sodic soils. These strategies report reduction in oxidative and osmotic stress, improvement in conductance and stomatal density, increased seed germination rate, and stimulation of microbial activities7,8, among other benefits.

The application of organic materials has shown promising results, improving the saline soil biome and enriching it with compounds, green manure, poultry manure, and sugarcane residues (pressed lime)9. Cordia verbenacea is a flowering shrub commonly known in Brazil as “Erva baleeira”, belonging to the family of Boraginaceae10,11. It is mainly found throughout the Brazilian Atlantic Forest, as well as in coastal regions and low-lying areas of the Amazon12.

Substantial evidence from the literature indicates that Cordia verbenacea aerial parts are often used in Brazilian folk medicine due to its remarkable antirheumatic, analgesic, and healing properties. The aerial parts are commonly used in the form of herbal extracts, decoctions, and infusions13,14,15.

Allium cepa Linn., a widely cultivated edible bulb from the family Alliaceae, is among the oldest known crops16. The A. cepa assay, a rapid toxicity indicator, evaluates nuclear abnormalities, chromosomal aberrations, mitotic index alterations, and root growth inhibition17,18. In Bangladesh, onion cultivation is widespread but hampered by salinity stress in coastal regions, as it is a glycophytic crop. Salinity affects 1.06 million hectares (32% of coastal land), significantly reducing agricultural productivity19.

Treating saline stress with plant-based organic materials is a promising and relevant approach, considering the increasing impact of soil salinization on agriculture and global food security7,20.Saline soils compromise crop productivity, leading to environmental management and biodiversity loss21.The use of plant organic compounds not only improves soil physical and chemical properties, but also promotes beneficial microbial activity, essential for soil health22,23. Furthermore, many of these materials have bioactive properties that can mitigate the effects of saline stress on plants, favoring physiological and biochemical adaptations that increase crop resilience24. Therefore, this study was undertaken to investigate the effects of Cordia verbenacea essential oil on the emergence of Allium cepa L. Seeds exposed to saline stress. In addition, we investigated the major compounds present in essential oil through molecular analyses, to provide a deeper understanding of its effects.

Material and methods

Reagents used and sowing location

The essential oil of Cordia verbenacea was obtained from a standard supplier. The seeds of A. cepa used for germination were purchased from a local agricultural supply store. Other chemicals, such as acetic acid, distilled water, and NaCl, were obtained from suppliers recognized for their standard quality.

Seed procurement and compliance

The seeds used in the experiment were purchased from a store specialized in agronomy products, which eradicated the need for additional documentation, offering convenience and security in the acquisition process. Baia Periforme onion seeds (071—Allium cepa) from the brand A Super Semente (ISLA), lot number 163245–009 S2, were utilized. These seeds demonstrated a germination rate of 94% and a purity of 100%, ensuring their quality and reliability for the experiment. Harvested in 2022/2023 and valid until May 2025, these seeds guarantee viability throughout the experimental period. In tests involving onions, compliance with the IUNC (International Union For Nature Conservation) policy declaration on research involving endangered species or the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) is not required because onions (Allium cepa) are not classified as endangered species. They are widely cultivated agricultural crops and are not subject to the regulations and protections that apply to endangered or threatened species. Therefore, research on onions does not necessitate adherence to these specific international conservation policies.

GC–MS analysis

Oil analysis was performed using a Shimadzu GC MS – QP2010 series (GC/MS system): Rtx-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness); helium carrier gas at 1.5 mL/min; injector temperature 250 ºC; detector temperature 290º C; column temperature 60º C – 180º C at 5º C/min, then 180º – 280º C at 10º C/min (10 min). Scanning speed was 0.5 scan/sec from m/z 40 to 350. Split ratio (1:200). Injected volume: 1 µL of [25 µL (essential oil) / 5 mL CHCl3] (1: 200). Solvent cut time = 2.5 min. The mass spectrometer was operated using 70 eV of ionization energy. Identification of individual components was based on their mass spectral fragmentation based on Mass spectral library NIST 08, retention indices, and comparison with published data.

Physicochemical analysis of the soil

The soil samples from the study area were collected at depths of 20 and 40 cm. They were then sent to the laboratory, where the concentrations of pH, phosphorus, potassium, sodium, calcium, magnesium, aluminum, hydrogen, aluminum, and cation exchange capacity (CEC) were measured following the methodology proposed by Teixeira et al. (2017)25.

Cultivation of Allium cepa plants and experimental treatments

The cultivation of Allium cepa was conducted in a greenhouse with shade netting, following the protocol outlined by Fiskesjo (1985)26 with minor revisions. The seeds (120) of Allium cepa were planted at a depth of 2 cm in the soil and were watered daily as follow: Control (distilled water at pH 7), NaCl (150 mM) diluted in distilled water, and NaCl (150 mM) in combination with different concentrations of the essential oil of Cordia verbenacea (100, 300, and 500 µg/mL). Readings were taken on the 21st day after each treatment was applied. The mean effective concentrations (IC50 and IC20) were determined using linear regression. Additionally, the number of germinated seeds was counted, and the relative inhibition of seed growth was calculated and expressed as a percentage.

Phytotoxicity index analysis

Following a modified protocol by Li et al. (2022)27, Allium cepa seedlings were assessed on the 21th day post-sowing, using a phytotoxicity classification system. The data were then statistically analyzed, and the phytotoxicity damage index was calculated with the following formula:

$${\varvec{P}}{\varvec{I}}\boldsymbol{ }\left(\boldsymbol{\%}\right)={\varvec{\Sigma}}\boldsymbol{ }\frac{{\varvec{P}}{\varvec{G}}\boldsymbol{ }{\varvec{x}}\boldsymbol{ }{\varvec{N}}{\varvec{P}}}{{\varvec{H}}{\varvec{L}}\boldsymbol{ }{\varvec{x}}\boldsymbol{ }{\varvec{N}}{\varvec{P}}}\boldsymbol{ }{\varvec{x}}\boldsymbol{ }100$$

where, PI (%) represents the phytotoxicity index (%), PG is the phytotoxicity score, HL denotes the highest level of observed phytotoxicity, and NP is the number of plants evaluated.

Leaf area estimation

To estimate leaf area, we used methods based on linear measurements, specifically leaf length and width. This procedure was adapted from previous studies on vineyards and fruit trees28, peppers 29 and tomatoes30, which provided a foundation for calculating the leaf area of Allium cepa (onion). We applied the ellipse formula, which allows for greater accuracy by considering the elliptical shape of onion leaves. The formula used is:

$${\varvec{L}}{\varvec{e}}{\varvec{a}}{\varvec{f}}\boldsymbol{ }{\varvec{a}}{\varvec{r}}{\varvec{e}}{\varvec{a}}=\frac{{\varvec{\pi}}\boldsymbol{ }{\varvec{x}}\boldsymbol{ }{\varvec{L}}\boldsymbol{ }{\varvec{x}}\boldsymbol{ }{\varvec{C}}}{4}$$

where, L represents the leaf length, C is the maximum leaf width, and π is a constant approximately equal to 3.14159. The division by 4 is necessary because the area formula for an ellipse is based on the semi-axes, whereas L and C represent the full axes. By dividing by 4, we adjust the formula to reflect the semi-axes, making it especially suitable for elongated leaves, such as those of Allium cepa.

Root length measurements and sampling

For a detailed assessment of root growth rates, a precisely calibrated digital caliper was used to measure root length. Twenty plants were randomly selected for each treatment. Measurements were taken 42 days after germination began to ensure reliable and representative data collection. This process was repeated three times to ensure the consistency and robustness of the results obtained.

Recipient treatment

The target of interest chosen from the literature review was subjected to molecular docking. The target protein (PDB id: 5GTE) with its respective ligand was obtained from the Protein Data Bank (PDB). The PDB is a repository of protein data and their three-dimensional structures. Various types of information are associated with each PDB file entry, including atomic coordinates in three-dimensional space, polymer sequence, and metadata (Berman, 2000)31. The removal of the protein inhibitor and water molecules from the receptor structure was performed using the Discovery Studio 2021 Client software.

Ligand treatment

The compounds ( ±)-α-Pinene, (-)-β-caryophyllene and alloaromadendrene were chosen for in silico evaluation via molecular docking. The ligand selected for this investigation was crafted in 3D using ACD/ChemSketch software, while the 2D model was obtained from ChemSpider ( ±)-α-Pinene ID: 6402, (-)-β-caryophyllene ID: 4,444,848, and alloaromadendrene ID: 10,478,311). Employing the Autodock VINA system within the PyRx software32, the compounds underwent ‘rigid protein-flexible ligand’ docking. Following the docking process, ligands adopting the most stable conformations were scrutinized using Discovery software.

Grid calculation and docking

The grid calculation involved processing 100 conformations using the Autodock VINA system within the PyRx software. For the ligand–protein docking procedure, the grid dimensions were set at 40 × 40x40 Å along the X, Y, and Z axes, with a grid spacing of 0.375 Å. Center coordinates for the grid were specified as 16.819, 71.967, and 31.192 Å for the X, Y, and Z axes respectively. These coordinates were determined based on the binding site location derived from known ligands previously co-crystallized with the protein and archived in the Protein Data Bank (Fig. 1). Following this, the interaction energy between the ligands and the amino acids of the 5GTE protein was calculated using the Discovery Studio software. This software enables the computation of free binding energy by analyzing its underlying energetic components, such as van der Waals forces, electrostatic bonds, and hydrogen bonds33.

Fig. 1
figure 1

Crystal structure of onion lachrymatory factor synthase (LFS), solute-free form (PDB: 5GTE).

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Statistical analysis

Analysis of variance (ANOVA) was performed among different treatments. Significant differences between treatments were assessed by ANOVA, followed by Tukey’s post hoc test (p < 0.05), and the data are expressed as the mean values ± SEM of 3 triplicate.

Results

Chemical composition of Cordia verbenacea essential oil

Table 1 shows the chemical composition of the essential of Cordia verbenacea. The most abundant compounds were α-pinene, caryophyllene and tricyclo 2.2.1 (2,6) heptane representing respectively 33.81%, 15.61% and 14.55%. Similarly, the minor components were α-thujene (1.65%), nerolidol (2.38%) and tetradecane (3.19%) (Table 1). This result suggest that Cordia verbenacea essential oil is mostly composed of compounds from the class of terpene (α-pinene, caryophyllene).

Table 1 Chemical composition (%) of the essential oil of Cordia verbenacea.
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Characteristics of the soil used

When characterizing the soil, we analyzed the presence of parameters at two distinct depths, 0–10 cm and 20–40 cm, and did not identify significant differences between the levels of coarse sand, fine sand, silt, clay, and natural clay. However, upon examining the chemical compounds, we observed slightly differences, but not significant. For instance, in the assortment complex, the Ca2+ content was 14.00 cmol/kg at the depth of 0–10 cm and 13.10 cmol/kg at the depth of 20–40 cm. K+ had a concentration of 3.35 cmol/kg at the surface (0–10 cm) and 2.80 cmol/kg at the depth of 20–40 cm, revealing a difference, although not significant (Table 2). The concentration of Na+ was 0.90 cmol/kg at the surface, showing a higher rate compared to the analysis conducted at 20–40 cm depth (0.75 cmol/kg). However, the organic matter (OM) content showed a considerable difference, being 50.08 g/kg in the 0–10 cm layer and 40.51 g/kg in the 20–40 cm layer, indicating a significant variation.

Table 2 Initial characteristics of the soil used for the germination experiment in trays 0–10 and 20–40 cm.
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Effect of Cordia verbenacea essential oil (EOCv) on seed germination

As depicted in Fig. 2A, the treatment of seeds with NaCl (150 mM) caused a significant reduction of about 38% in the emergence of Allium cepa compared to the control group, with an effective concentration of 37 µg/mL (EC50). However, co-treatment of NaCl (150 mM) with different concentrations of EOCv (100–500 µg/mL) did not show any effect compared to NaCl (150 mM) alone (p < 0.05). Similar result was obtained in the inhibition of emergence of Allium cepa seeds (Fig. 2B) and in the number of rootlets (Table 3). This result demonstrates some protection against the adverse effects of NaCl on the number of roots, and suggest a potential benefit of EOCv in promoting root development, even under conditions of NaCl-induced salt stress.

Fig. 2
figure 2

Effects of Cordia verbenacea essential oil (EOCv) on seed germination in the presence of NaCl (A) and inhibition of seed germination in the presence of NaCl (B). Seed germination rates were recorded 21 days after planting under various treatments: control; treatment with NaCl 150 mM, 100, 300, and 500 µg/mL. Data are expressed as mean ± SEM for 3 replicates. * denotes significant difference compared to the control.

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Table 3 Effect of different concentrations of NaCl + EOCv on the number of rootlets. Data are mean ± SD.
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Phytotoxicity

In the phytotoxicity analysis, NaCl (150 mM) caused a significant increase in the phytotoxicity index (~ 27%) compared to control (p < 0.05). Treatment with EOCv (100–500 µg/mL) did not show any significant effect compared to NaCl (150 mM) alone (p > 0.05) (Fig. 3), indicating that EOCv did not provide protection against saline stress damage (Fig. 3).

Fig. 3
figure 3

Effects of Cordia verbenacea essential oil (EOCv) on seed phytotoxicity in the presence of NaCl. Phytotoxicity rates were recorded on day 21 after planting under the treatments: control; NaCl treatment 150 mM, 100, 300 and 500 µg/mL. Data are expressed as mean ± SEM for 3 replicates. * denotes significant difference compared to control.

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Leaf length and leaf area estimation

It was observed that the effect of different concentrations of NaCl combined with Cordia Verbenacea essential oil (EOCv) on the leaf length of Allium cepa resulted in a significant reduction compared to the control group (p < 0.05). However, concentrations of 300 µg/mL and 500 µg/mL showed a significant increase both compared to NaCl 150 mM and between each other (Fig. 4A). This effect was observed in leaf area (cm2), where NaCl resulted in a reduced leaf area compared to the control. However, EOCv treatment caused an increase in the leaf area in a concentration dependent manner, with 300 and 500 µg/mL exhibiting significant improvements compared to NaCl (Fig. 4B and 5). These results suggest a possible positive influence of higher concentrations of EOCv on leaf length, partially counteracting the negative effects of NaCl.

Fig. 4
figure 4

Effects of different concentrations of NaCl + EOCv on leaf length in Allium cepa (A) and effect of different concentrations of NaCl + EOCv on leaf area of Allium cepa (B). Vertical bars denote SEM, n = 8. * indicates significant difference compared to control; # indicates significant difference compared to NaCl; & indicates significant difference compared to NaCl + EOCv (100 µg/mL) (p < 0.05).

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Fig. 5
figure 5

Photo of onion seedlings after 42 days of exposure, control (A), 150 mM NaCl (B), NaCl + EOCv (100 µg/mL) (C), NaCl + EOCv (300 µg/mL) (D) and NaCl + EOCv (500 µg/mL) (E).

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Root length

Figure 6 shows roots length of Allium cepa after 42 days of treatment with NaCl (150 mM) alone or in combination with EOCv (100–500 µg/mL). NaCl 150 mM caused a significant reduction of roots length compared to the control group (p < 0.05). However, concentrations of 300 µg/mL and 500 µg/mL of EOCv in combination with 150 mM NaCl showed a significant increase in the roots length when compared to NaCl (150 mM, Fig. 6). These results suggest that higher concentrations of EOCv may have a positive effect on root length, counteracting the negative effects induced by NaCl. (Fig. 6. (A).

Fig. 6
figure 6

Effects of different concentrations of NaCl + EOCv on root length in Allium cepa, with 42 days after emergence. Vertical bars denote SEM, n = 8. * denotes significant difference compared to control; # indicates significant difference compared to NaCl (p < 0.05). Root length calculation was obtained from measurements of 20 seedlings.

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Molecular analysis

Trehalose showed an affinity of −6.9 kcal/mol, with binding at sites GLN B:161, LEU B:28, THR B:157, GLN B:153, and ALA B:27, where sites GLN B:161, THR B:157, and GLN B:153 were of the conventional hydrogen bond type. LEU B:28 was a carbon hydrogen bond, and ALA B:27 was of the alkyl type (Fig. 7G). The docking results highlighted several key interactions and molecular properties. Hydrogen bond donors were observed in interactions at the LEU B:28 site (Fig. 7A), while the solvent-accessible surface (SAS) predominantly ranged between 22.5 and 25 Å2 (Fig. 7B). Aromatic interactions were characterized as weak, with a lever-to-edge geometry (Fig. 7C). The interpolated charge was close to 0, indicating minimal polarization of the ligand (Fig. 7D). Conventional hydrogen bonds exhibited hydrophobicity values between −1 and −2 (Fig. 7E), and the ionizability remained stable with minimal variation (Fig. 7F).

Fig. 7
figure 7

In silico molecular docking analysis with the target protein, crystal structure of the onion lachrymatory factor synthase (5GTE) and the Trehalose ligand, including analysis of hydrogen bonds (H-Bonds) (A), solvent-accessible surface area (SAS) (B), aromatics (C), interpolated charges (D), hydrophobicity (E), ionizability (F), and 2D binding analysis with the target protein (G).

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( ±)-α-Pinene showed an affinity of −5.0 kcal/mol, with alkyl bonds at sites LEU B:28, LEU D: 28 and ALA B:27 (Fig. 8G). The docking analysis revealed neutral hydrogen bond interactions in the vicinity of the ligands (Fig. 8A). The solvent-accessible surface (SAS) ranged from 22.5 to 25 Å2 at binding sites interacting with the ligand (Fig. 8B). Aromatic interactions were slightly oriented toward an edge geometry (Fig. 8C), and no interpolated charge was detected (Fig. 8D). Hydrophobicity values near the ligand ranged from 1 to 2 (Fig. 8E), while ionizability remained neutral, indicating no significant variations (Fig. 8F).

Fig. 8
figure 8

In silico molecular docking analysis with the target protein, crystal structure of the onion lachrymatory factor synthase (5GTE) and the ( ±)-α-Pinene ligand, including analysis of hydrogen bonds (H-Bonds) (A), solvent-accessible surface area (SAS) (B), aromatics (C), interpolated charges (D), hydrophobicity (E), ionizability (F), and 2D binding analysis with the target protein (G).

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Alloaromadendrene showed an affinity of −6.6 kcal/mol and alkyl bond at sites PRO B:30 and ALA B:27 (Fig. 9G). The docking analysis showed no donor or acceptor reactions in hydrogen bonds (Fig. 9A). The solvent-accessible surface (SAS) was close to 12.5 to 10 Å2 at the ALA B:27 site (Fig. 9B). Aromatic interactions displayed a slight edge orientation (Fig. 9C), and no interpolated charge was detected (Fig. 9D). Hydrophobicity at the ALA B:27 site ranged from 1 to 2, while for other ligands, it varied between −2 and −3 (Fig. 9E). Ionizability near the PRO B:30 site was slightly acidic, suggesting a localized pH effect at these binding sites (Fig. 9F).

Fig. 9
figure 9

In silico molecular docking analysis with the target protein, crystal structure of the onion lachrymatory factor synthase (5GTE) and the Alloaromadendrene ligand, including analysis of hydrogen bonds (H-Bonds) (A), solvent-accessible surface area (SAS) (B), aromatics (C), interpolated charges (D), hydrophobicity (E), ionizability (F), and 2D binding analysis with the target protein (G).

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Caryophyllene showed an affinity of −6.5 kcal/mol, with alkyl bonds at sites ALA D:27 and LEU B:28 (Fig. 10G). The docking results showed no reactions involving hydrogen bonds (Fig. 10A). The solvent-accessible surface area (SASA) was predominantly between 22.5 and 25 Å2 at the binding sites (Fig. 10B). Aromatic interactions were slightly oriented toward an edge geometry (Fig. 10C), and the interpolated charge remained close to 0, indicating minimal electronic polarization (Fig. 10D). Hydrophobicity values ranged from 1 to 2 at the binding sites, while ionizability remained neutral in proximity to the ligand (Fig. 10F).

Fig. 10
figure 10

In silico molecular docking analysis with the target protein, crystal structure of the onion lachrymatory factor synthase (5GTE) and the (-)-β-caryophyllene ligand, including analysis of hydrogen bonds (H-Bonds) (A), solvent-accessible surface area (SAS) (B), aromatics (C), interpolated charges (D), hydrophobicity (E), ionizability (F), and 2D binding analysis with the target protein (G).

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Discussion

Salt-induced stress in plants is known in agriculture and plant sciences to play a major role in soil by negatively impacting ion levels, which results in oxidative damage. Essential oils from plants origin containing a variety of compounds including terpenes and phenols can potentially be helpful in managing saline stress. In the current study, essential oil of Cordia verbenacea (EOCv) exhibited a variety of chemical components including α-pinene as the major component, representing 33.81% of the composition. Other components like Caryophyllene with a proportion of 15.61%, and Tricyclo 2.2.1 (2,6) heptane, with a proportion of 14.55% were also represented. According to Xanthis et al. (2021)34, α-pinene demonstrated moderate activity in the scavenging of DPPH and ABTS radicals, with 47.9 ± 2.78% and 49.28 ± 3.55% inhibition, respectively. Also, β-Caryophyllene was defined as an antioxidant by Ames-Sibin et al. (2018)35. Several studies have highlighted the beneficial role of the cell wall in response of the plant to salt stress36,37,38.

The study by Ugras et al. (2024)39 demonstrated the protective effects of essential oil (EO) components from Origanum onites on Allium cepa root cells subjected to lead nitrate-induced toxicity. Among the evaluated EO constituents, α-pinene exhibited the strongest protective activity. Similarly, EO from Campomanesia xanthocarpa, with β-caryophyllene as its major constituent (8.87%), displayed genotoxic and antiproliferative properties when tested in infusions using the in vivo root tip assay of Allium cepa40.

It is important to note that the study of essential oils in plant protection under saline conditions is still an emerging field, with much of the existing research focusing on how salinity affects essential oil production rather than their potential as biostimulants or stress protectants. In a study by Ben Saad et al. (2024)41, the authors demonstrated that Rosmarinus officinalis essential oil enhanced germination and biomass accumulation under salinity, likely due to its α-pinene content (10.1 ± 0.02%). In comparison, in the current study, Cordia verbenacea exhibited a higher α-pinene concentration (33.81%), suggesting potential advantages in stress tolerance. Additionally, research has shown that essential oils from Baccharis dracunculifolia, Schinus terebinthifolius, and Porophyllum ruderale effectively control phytopathogenic fungi, supporting their protective roles in plant health42.

Although limited studies have specifically examined C. verbenacea under saline conditions, its essential oil composition is known to vary based on environmental factors such as seasonal changes, which could influence its bioactivity in salt-stressed environments43,44. For instance, Day (2016) reported that essential oils extracted from different tissues of safflower (Carthamus tinctorius) exhibited differential effects on the germination of wheat, barley, sunflower, and chickpea. Similarly, growing evidence supports the biostimulant role of phenolic compounds and plant extracts in seed germination, rooting, and seedling growth, whether applied as seed treatments, foliar sprays, or soil amendments41,45. Given these findings, further comparative studies on the efficacy of various essential oils under saline conditions are necessary to better assess their potential applications in plant stress management.

The protective characteristics of Cordia verbenacea essential oil (CVE) were observed in leaf and root development, but were not evident during germination, where the treatments showed similar effects. When Allium cepa seeds were exposed to a concentration of 150 mM NaCl, there was a significant change compared to the control group, evidencing the negative impact of NaCl on seed development. This salt stress resulted in reduced root growth, reflecting lower elongation and cell division. Salt stress, such as that caused by high NaCl concentrations, interferes with osmosis and water uptake, leading to suppression of cell expansion and, consequently, impaired plant growth6,46,47.

Particularly, increased salt-concentration, can cause water absorption issues due to the osmotic stress that it creates, thereby restricting root elongation and inhibiting cell division. This results in shorter roots and reduced root biomass, impairing the plant’s ability to absorb water and nutrients. Additionally, salt stress leads to the accumulation of reactive oxygen species (ROS), which damage cellular structures and disrupt metabolic processes48,49,50,51. In this context, antioxidants compounds like terpenes found in Cordia verbenacea essential oil may help to maintain the cellular integrity of A. cepa and consequently prevent damage to vital structures of the plant.

The cell wall plays a vital role in determining the shape and function of the cell, serving as the first line of defense against salt stress. Furthermore, salt stress induces water deficiency in plant cells, causing changes in cell turgor pressure. The cell wall provides mechanical resistance to cope with these changes in turgor pressure25. The cell wall acts as a vital support system for plants, helping them endure changes in their environment and cope with osmotic stress37,52.Alterations in key physiological and metabolic mechanisms have adverse repercussions on seed germination and seedling growth, resulting in delayed development of both shoot and root systems, as well as reductions in fresh and dry weight, chlorophyll content, and its synthesis38 .However, our results demonstrated that EOCv did not protect against delayed seed development, with an inhibition similar to that of NaCl, but with a significant difference in relation to the control. This effect was also noted in seed germination.

Salinity poses a significant challenge to crop productivity, as it has a negative impact on both seed germination and seedling growth. This phenomenon affects plant development by disrupting osmosis, imbalancing nutrient channels, and causing ionic toxicity38. This toxicity was evidenced in the phytotoxicity analyses, where all treatments showed significant differences in relation to the control. However, the phytotoxicity rate tends to reduce with increasing EOCv concentration.

The plant transporters existing in the roots play an important role in the absorption of minerals from the soil or hydroponic medium. The uptake of minerals and water is closely related, as both involve these transporters. Among them, aquaporins are of particular importance as they regulate the absorption and transport of water and minerals across cell membranes53. Our results showed that NaCl treatment caused a significant reduction in root length compared to the control, but EOCv in combination with NaCl resulted in increased root length at concentrations of 300 and 500 µg/mL to the control root length.

It was observed that treatment with the essential oil of Cordia Verbenacea (EOCv) resulted in a proportional increase in leaf length as the concentration of EOCv increased. This suggests that EOCv attenuated the impact of NaCl on leaf development, with the concentration of 500 µg/mL showing a significant effect compared to NaCl alone. This trend was also observed when analyzing root length, where the highest concentration of EOCv resulted in greater root development. These results indicate that EOCv possibly provides protection against salt stress caused by NaCl. Our results corroborate with that Chatterjee & Majumder (2010)54 that demonstrated that the exposure of Allium cepa root tips to varying concentrations of NaCl significantly decreased root growth rates, especially at higher concentrations (0.24 and 0.48 M NaCl), which led to notable growth inhibition over 72 h and reduced the mitotic index, indicating an impact on cell division. Additionally, another study revealed that NaCl stress caused chromosomal abnormalities, particularly at elevated levels, along with observable DNA fragmentation and cell death in root meristem cells55,56,57,58.

Trehalose, evidenced at sites GLN B:161, THR B:157, and GLN B:153, established conventional hydrogen bonds. This molecule exhibits remarkable hydrophilicity due to its inability to form internal hydrogen bonds59. This characteristic makes it a valuable molecular, membranous, and proteinaceous preservative46. In situations of dehydration or freezing, trehalose interacts through hydrogen bonds with adjacent macromolecules and membranes, replacing water molecules60. After extreme dehydration, it crystallizes, assuming a vitreous appearance, a distinctive characteristic61,62.

In response to saline stress, plants employ an adaptive strategy to regulate cellular osmotic potential, producing osmolytes or compatible osmoprotectants, such as sugars, among which trehalose stands out63,64. In the current study, the compound α-Pinene established a specific interaction with site LEU B:28, with this interaction classified as an alkyl bond. However, upon examining trehalose, we noticed that, although it also showed bonding with the same site, this interaction was identified as a carbon-hydrogen bond, indicating a different nature of molecular interaction. The presence of this distinction suggests potential implications in the properties and biological functions of these compounds56,65,66,67. Additionally, when investigating Alloaromadendrene, we found that it also established a similar alkyl-type bond with site ALA B:27 as trehalose68,69,70 This similarity in the nature of bonding suggests a possible correlation between the structural properties and molecular interactions of these compounds71,72,73.

The formation of alkyl-alkyl bonds is essential in the construction of molecules, and the stereochemistry of the carbons involved plays a crucial role in determining their structures and properties. Therefore, the development of techniques that allow the formation of these bonds with enantioselective control is of great importance in organic synthesis74,75.

Conclusion

The study highlights the significant potential of Cordia verbenacea essential oil (EOCv) to enhance salt tolerance in onion crops by promoting leaf and root growth and mitigating the adverse effects of salt stress on onion growth. In silico analysis identified alkyl interactions between EOCv compounds such as ( ±)-α-pinene, (-)-β-caryophyllene and alloaromadendrene that may suggest a molecular protective mechanism. However, further biochemical and histochemical investigations are required to elucidate its precise biochemical mechanisms, potential interactions with stress-related metabolic pathways, and long-term implications for plant adaptation in saline environments. Such studies will be essential for developing targeted strategies to enhance plant resilience and promote sustainable agricultural practices in salt-affected regions.