Jatropha curcas Seed Germination: Effect of Seed Imbibition, Aging, Storage, and Salinity

Abstract

Renewable energy sources are essential to mitigating climate change, with biofuels offering a sustainable alternative to fossil fuels by reducing greenhouse gas emissions. Jatropha curcas, the best, non-edible, high-oil-yielding species, is a leading candidate for biodiesel production. However, ensuring a stable seed supply through effective storage is critical for biodiesel markets stability. This study evaluated the physiological and biochemical viability of J. curcas seeds stored at 4 °C with controlled humidity using 1.5 g of silica gel per gram of seed over 12 months. The results demonstrated that low-temperature, low-humidity storage significantly reduced metabolic activity, embryo respiration, and seed deterioration, preserving high germinability and oil quality. Despite a slight increase in mean germination time, seeds retained resilience in germination potential and viability. Additionally, preliminary assessments of salt tolerance revealed the potential of J. curcas seeds to germinate under saline conditions, supported by analyses of mineral nutrition and salt tolerance-related gene expression. These findings underscore the practicality of optimized storage conditions for maintaining seed quality and economic value, ensuring a consistent supply chain for biodiesel production. This study highlights the importance of integrating storage strategies into biodiesel systems to enhance sustainability and market resilience in the face of fluctuating production demands.

1. Introduction

The global demand for sustainable and renewable energy sources has intensified interest in biodiesel as a key alternative to fossil fuels. Among the promising biofuel crops, Jatropha curcas L. (Euphorbiaceae) has gained significant attention due to its ability to grow in arid and semi-arid environments, and its non-competitiveness with food crops [1,2]. The seeds of J. curcas are particularly valued for their high oil content, comprising approximately 79% unsaturated fatty acids, which are essential for biodiesel production [3]. J. curcas is crucial in soil conservation, and its ability to thrive in poor-quality and marginal soils.

The impermeability of J. curcas seeds has been the subject of a remarkable debate. Several authors suggest that the rigid seed coat prevents water and gas exchange, leading to dormancy. Gopinathan and Babu [4] argued that macrosclereids contribute to this impermeability. Similarly, Islam et al. [5] reinforced the idea that the hardened seed coat forms a physical barrier that limits seed germination by restricting water uptake. However, recent studies have challenged this view, showing that the macrosclereids in J. seeds do not entirely prevent water passage, as seeds exhibit rapid water imbibition during germination [6].

Some scholars [3,7,8,9] describe that J. curcas’s seeds have 8–30% water, depending on maturation time [10] and storage conditions. Pompelli et al. [6] describe an increase of 241% in seed respiration on J. curcas seeds imbibed for 120 h. The respiration rate is directly proportional to water content, consequently depleting seed reserves. However, a low-humidity container could reduce seed respiration, reserve depletion, and seed viability. Also, the germination of J. curcas seeds is significantly influenced by the initial water content and the duration of imbibition. Seeds with lower initial moisture content, around 8%, experience imbibition damage due to uncontrolled tissue hydration, affecting cell structure reconstruction and reducing germination rates. Moncaleano-Escandon [11] describes the process of seed storage and aging in J. curcas. These authors describe the seed deterioration and the loss of viability over 12 months of storage at room temperature (25 °C) and that only 2% of the seeds retained their ability to germinate. In contrast, 7% of seeds stored under refrigerated conditions (4 °C) could still germinate. This refrigerated condition highlights a significant drop in germinability in high-temperature environments, such as tropical regions, where high relative humidity accelerates seed deterioration [12]. Moncaleano-Escandon et al. [11] showed that seed germinability drastically decreases under room temperature storage, supported by other findings indicating that reducing moisture in the seed environment through desiccant agents like silica gel also helps lower the osmotic potential and thus reduces the metabolic activity within the seed. According to Lozano-Isla et al. [13], seeds stored without desiccants lose viability faster due to uncontrolled respiration, depleting energy reserves needed for germination. Respiration rates decrease significantly in controlled conditions, reducing CO₂ release, which helps preserve seed reserves and supports germination synchronization after extended storage [14,15].

Another problem suffered by J. curcas is the event caused by its salt sensitivity [16,17,18,19,20,21,22,23]. When examining the effects of salinity on seed germination, J. curcas shows moderate tolerance, but increasing salinity significantly delays the germination process and affects overall germination synchrony [13]. At higher concentrations of NaCl (150 mM L⁻¹), germination rates dropped dramatically to nearly zero. In comparison, at lower concentrations (50 mM L⁻¹), a decline in germination rate and delay in mean germination time (MGT) were observed [13]. The osmotic effect, caused by NaCl, limits water absorption and interferes with seed metabolism by inhibiting the mobilization of stored nutrients essential for germination [13,17,24]. Salinity also increased germination uncertainty and disrupted the synchrony of the process, demonstrating that high salt levels cause physiological stress during seedling development, as reported by Munns and Tester [25].

If J. curcas is to be considered a model plant for global biodiesel production [26,27,28], this commodity [29] will need harvesting, storage, and transportation techniques. With techniques that optimize harvesting and storage, the producer could hold onto seeds and sell them when the trade balance is more favorable, which could be a major insider for the foreign trade of J. curcas seeds. Based on strategies for J. curcas seed germination, this study describes the challenges in J. curcas seed germination, particularly concerning seed storage conditions, natural or artificial aging, water permeability, and salinity. Despite its potential for biodiesel production and ability to thrive in poor soil, low germination rates and seed dormancy hinder its commercial viability. This study addresses these limitations by evaluating the combined effects of storage conditions, seed aging, and salinity stress on the germination performance of J. curcas. Specifically, it investigates germination rates, synchronization, and physiological responses under controlled conditions, with the aim of identifying strategies to improve seed viability under storage. By advancing the understanding of the physiological and molecular mechanisms underlying seed responses to salt stress, this research seeks to establish J. curcas as a reliable model crop for biodiesel production while contributing to the optimization of harvesting, storage, and transportation practices.

2. Materials and Methods

2.1. Plant Material

The experiment was conducted with Jatropha curcas seeds, sampled from a commercial plantation from the Atlantic rainforest region (09°28′ S; 35°51′ W; 130 m.a.s.l.). Seeds were provided by the Federal University of Alagoas (UFAL), Alagoas, AL, Brazil. The plantation consisted of plants at least 20 years of age, and the spacing between plants was 2 m × 2 m. Fruits and seeds were randomly collected during the rainy season (May to Aug). Fresh seeds presented 92% viability and were stored at 4 °C until their use, following recommendations from previous studies that demonstrated optimal preservation at this temperature [11].

2.2. Germination of J. curcas Seeds After Imbibition Process

This study focused on evaluating the germination pattern of J. curcas seeds under different imbibition times to understand how water affects seed germination and vigor. This experiment was conducted in a controlled environment to replicate field conditions.

Seeds of J. curcas were collected from a commercial plantation described in Section 2.1. Following the storage protocol described by Moncaleano-Escandon et al. [11], the seeds presented an initial viability rate of 89%. The seeds were distributed across 52 glass flasks (400 mL capacity), with 25 in each flask representing the different treatments. The treatments consisted of varying imbibition times (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h). Each flask was filled with 100 mL of deionized water and placed in a controlled chamber maintained at 25 °C. To evaluate seed water content (SWC), 50 seeds were weighed before imbibition (fresh weight, SFW) and after imbibition (turgid weight, STW). The seeds were then oven-dried at 105 °C for 24 h to determine their dry weight (SDW). The seed moisture content (SMT) and water content were calculated using the following formulas:

$$SMT \left(\%\right)=1-\left({\displaystyle \frac{SDW}{SFW}}\right) \times 100$$

$$SWC \left(\%\right)={\displaystyle \frac{\left(STW-SFW\right)}{SFW}} \times 100$$

where SMT, seed moisture; SWC, seed water content; SDW, seed dry weight; SFW (g), seed fresh weight and STW (g), seed turgid weight.

The seeds’ imbibition weight was recorded for each time interval, and the pH and electrical conductivity (EC) of the imbibition solution were measured using a W3B pH meter (Bel Engineering, Monza, Italy) and a CD-4306 conductivity meter (Lutron, Taiwan), respectively.

Following imbibition, the seeds were sown in polyethylene trays (450 × 250 × 70 mm) filled with 1200 g of river sand. The trays were placed in a greenhouse with an average temperature of 27.5 °C and 78% relative humidity. For each replication, 25 seeds were uniformly distributed and covered with ~1 cm of river sand. Germination was monitored daily for 25 days, and seed emergence at least 10 mm above the soil surface was considered germinated. The experiment followed the methodology described by Moncaleano-Escandon et al. [11]. The dataset was processed with one-way ANOVA in accordance with Hastie et al. [30].

2.3. Artificial Seed Aging and Seed Storage

This study investigated the effects of storage and artificial aging on the seed germination and MGT of J. curcas seeds. The experiment evaluated seed viability under natural and artificial aging. For this experiment, fruits were harvested randomly during the rainy seasons, representing the population’s genetic diversity. After collection, the fruits were immediately transported to the laboratory, where seeds were manually separated and air-dried for 2–3 days before storage [11]. Accelerated aging (AA), saturated salt accelerated aging (SSAA), and controlled deterioration (CD) tests were then conducted. The AA test involved placing 20 g of seeds on wire mesh screens suspended over 40 mL of water inside sealed plastic boxes (110 mm × 110 mm × 35 mm) at 42 °C and 100% relative humidity for 48 or 72 h [31]. A saturated salt accelerated aging (SSAA) test was also performed by placing seeds in a box with 40 mL of a saturated NaCl solution (40%) at 42 °C and 76% relative humidity for 48 or 72 h [32]. The CD test was performed by adjusting the seed moisture content to 18% and incubating the seeds in aluminum foil bags placed in a water bath at 41 °C for 12 or 24 h [33]. The collected seeds were germinated immediately (fresh seeds) or stored in paper bags for one year (1-year-old seeds) at room temperature (25 ± 2 °C). The relative humidity (RH) at seed level during storage was 86 ± 5%.

To evaluate seed germination, four replicates of 25 seeds per treatment were placed in germination boxes (110 mm × 110 mm × 35 mm) lined with three sheets of Whatman No. 1 filter paper moistened with 20 mL of distilled water, along with 500 U of Mycostatin solution (100 mg L⁻¹) to prevent fungal growth. The boxes were sealed and placed in a growth chamber (NT 708, New Technical Instruments, Piracicaba, SP, Brazil) with a 12 h photoperiod and 25 ± 0.5 °C temperature. Germination was monitored daily and defined as a radicle > 2 mm emergence through the external integument [34].

Germination was monitored daily, and seeds were considered germinated when the radicle emerged at least 0.5 mm from the seed coat [34]. The experiment continued for 25 days, and germination was considered complete when no further germination occurred for five consecutive days, as described by Lozano-Isla et al. [35]. Germination parameters, including seed germination and MGT, were calculated according to the methods described by Lozano-Isla et al. [35] and the formulas below:

$$Germination rate: \left({\displaystyle \frac{{\sum }_i^k{n}_i}{N}}\right) \times 100$$

$$hMGT={\displaystyle \frac{{\sum }_i^k{n}_i{t}_i}{{\sum }_i^k{n}_i}}$$

where n_i is the number of seeds germinated in the ith time; N is the total number of seeds in each experimental unit; k is the last day of germination evaluation; t_i is the time from the beginning of the experiment to the ith observation. The dataset was processed with two-way ANOVA in accordance with Hastie et al. [30].

2.4. Effect of Controlled Air Humidity and Cooler Temperatures on Stored J. curcas Germination

2.4.1. Seed Germination

This study evaluated the effects of controlled air humidity and cooler temperatures on stored J. curcas seeds. The experiments were conducted using seeds collected as described in Section 2.1. In addition, the air humidity in the storage boxes was carefully controlled during storage using desiccants to reduce water content in the seed interstices, as described by Chidananda et al. [36]. Each experimental unit consisted of 50 seeds stored in germination boxes containing silica gel (Sigma-Aldrich, St. Louis, MO, USA, Part number 1.07734) at a ratio of 1.5 g of silica gel per gram of seed. The experimental design was designed so that the start of storage coincided with removing a batch for germination analysis every three months. Thus, the seeds were inserted into the boxes on different dates so that in the end, we had seeds stored for 12 months (storage started in December 2022), 9 months (storage started in March 2023), 6 months (storage started in June 2023), 3 months (storage started in September 2023) and freshly harvested seeds (non-storage). In order to guarantee low relative humidity inside the containers, new silica gel was added to the containers every 15 days under sterilized ambient. In order to prevent the quality of light from influencing storage, each box was wrapped in four layers of aluminum foil in complete darkness.

After each storage period, the seeds were selected, and seeds less than 15 mm [8] in length were discarded. Tiny seeds have poor germination, as previously reported by our team [37]. Also discarded were non-homogeneous seeds. After that, the seeds were germinated as described in Section 2.3. Germination was computed daily for 25 days. Using the germination percentage, the seed germination and MGT were computed. The germination in time was also determined, which shows how each treatment progressed within the time frame. All calculations followed the criteria described and recommended by Lozano-Isla et al. [35] and the formulas above.

2.4.2. Biochemical and Metabolic Analysis in Stored J. curcas Seeds

The biochemical analyses were conducted to evaluate the metabolic changes in seeds due to storage and temperature. Three grams of seeds from each experimental unit were ground in liquid nitrogen and stored at −80 °C until analysis. Using the findings from Fernie et al. [38], these samples were used to compute sucrose, fructose, and glucose. Methanolic soluble fractions were analyzed for their total amino acids [39], malate and fumarate [40], and total proteins [41]. The content of other metabolites was assessed via gas chromatography-mass spectrometry (GC-MS), as described in the study by Lisec et al. [42]. Starch was quantified as an insoluble ethanolic fraction [15]. Carbohydrates were measured following the procedure described by Dubois et al. [43]. The seeds’ relative water content (RWC) was calculated using the method described by Moncaleano-Escandon et al. [11]. Seed water potential was measured with a dew point water potential meter (WP4C, Decagon Devices, Pullman, WA, USA), with seeds lightly cracked to allow water exchange. Respiratory rate (RPR) was assessed using a 120 mm diameter CO₂ flow chamber (6400-09, LiCOR, Lincoln, NE, USA), with measurements recorded every 102 s for each seed lot containing 15 seeds per repetition (n = 4). Before this analysis, seeds were removed from controlled air humidity containers and rapidly inserted in a 300 mm-glass desiccator with 100 g of silica gel coupled to a vacuum pump until transference to CO₂ flow chamber. The net respiration rate was expressed in μmol CO₂ h⁻¹ g⁻¹ seed. The dataset was processed with one- or two-way Anova in accordance with Hastie et al. [30].

2.5. Effect of Salinity on J. curcas Germination

This study evaluated the effects of salinity on the germination of J. curcas seeds. The experiments were conducted with the same seeds described in Section 2.4. Following storage for three months, the seeds were subjected to germination tests under different salinity conditions to determine the effects of sodium chloride (NaCl) on germination. Five NaCl concentrations (0-, 50-, 75-, 100-, and 150 mM L⁻¹) were evaluated. Before the germination tests, the seeds were disinfected with a 2% sodium hypochlorite (NaOCl) solution for 10 min and thoroughly rinsed with distilled water to remove probable contaminants. The seeds were then placed in polypropylene germination boxes containing 2 cm of washed river sand and irrigated daily with Hoagland [44] nutrient solution at 25% concentration to prevent salt accumulation. A 1 cm sand river covered each box containing 25 seeds. Seeds were considered an experimental unit, and the experiment was replicated five times for each NaCl concentration. Germination was monitored daily for 25 days, and seed emergence at least 10 mm above the soil surface was considered germinated [34]. The dataset was processed with one-way Anova in accordance with Hastie et al. [30].

2.6. RT-PCR Analysis

This study builds upon prior work evaluating the effects of salinity on Jatropha curcas seed germination. Using seeds collected as described in Section 2.4 and stored for three months, germination tests were conducted under five salinity conditions (0, 20, 40, 60, 80, and 100 mM L⁻¹ NaCl). Seeds were disinfected with a 2% NaOCl solution for 10 min, rinsed with distilled water, and germinated in polypropylene boxes with washed river sand, irrigated daily with Hoagland nutrient solution at 25% concentration. Germination was monitored for 25 days, and seedlings with at least 10 mm emergence above the sand surface were recorded as germinated. After 25 days, root tissues were harvested from seedlings for molecular analysis. RNA was extracted using TRIzol™ reagent (ThermoScientific™, Missouri City, TX, USA), and first-strand cDNA synthesis was performed with the PrimeScript™ RT reagent kit (Takara Bio, San Jose, CA, USA). RT-qPCR was conducted to analyze the expression of differentially expressed genes (DEGs) associated with salinity tolerance. Gene-specific primers were designed using Primer3, with actin and β-tubulin used as reference genes for normalization. RT-qPCR reactions were carried out in a LineGene 9600 PCR thermal cycler with three biological replicates per treatment, each analyzed in technical triplicates. The 2⁻ΔΔCt method was used to calculate relative expression levels, and one-way ANOVA with Tukey’s post hoc test (p < 0.05) was applied to determine significant differences among treatments.

2.7. Statistical Analysis

All experiments were arranged in a completely randomized design with four repetitions. Data were analyzed using R statistical software, version 4.4.2 with an analysis of variance (ANOVA) to assess differences between treatments. Means were compared using the Student-Newman-Keuls test at a significance level of p < 0.05. Germination parameters, including seed germination and mean germination time (MGT), were calculated using the GerminaR package [45]. Principal component analysis (PCA) was performed using Minitab^® version 17.0 software (Minitab Ltd. Coventry, UK). Heatmaps were used to compare the mean of each treatment, using non-stored seeds as a reference. After the log₂ transformation, the false color method was used, including a color scale. The heatmaps were constructed using Microsoft^® Office 360 (Microsoft Corporation, Redmond, WA, USA) and CorelDRAW Graphics Suite X8 (Corel Corporation, Ottawa, ON, Canada).

3. Results

3.1. Germination of Jatropha curcas Seed After Imbibition

3.1.1. pH and Electrical Conductivity of the Imbibition Water

The pH of the imbibition water exhibited a marked decrease as the seeds absorbed water over time. At the beginning of the experiment, the measured pH was 7.7, but after 24 h of imbibition, it had dropped to 7.02. The pH drop may indicate cellular or metabolic processes leading to leakage of organic acids or other acidic compounds from the seeds. The reduction in pH was strongly correlated with the duration of imbibition (r = −0.88, p < 0.05; Figure 1A). This process reflects potential deterioration in seed quality over time as prolonged water exposure may cause damage to cell membranes, allowing the release of intracellular compounds into the imbibition water.

The imbibition water’s electrical conductivity (EC) increased significantly as the seeds imbibed water. Initial EC values were as low as 0.021 dS m⁻¹, but after 24 h, the EC had risen to 0.69 dS m⁻¹ (Figure 1B), reflecting the leakage of electrolytes from the seeds into the water, as postulated earlier. This increase was positively correlated with the duration of seed imbibition (r = 0.80, p < 0.05; Figure 1B). The rise in EC indicates damage to seed cell membranes, which allows the release of ions and other solutes from the seed into the surrounding solution. The loss of membrane integrity is often associated with reduced seed vigor and viability (Section 3.1.3). The relationship between EC and seed vigor also demonstrated a strong negative correlation (r = −0.74, p < 0.05), suggesting that seeds become increasingly damaged and lose more electrolytes. The use of EC as a vigor test is supported by this finding, as higher EC values typically indicate poorer seed quality.

3.1.2. Effect of Seed Imbibition on Seed Water Content and Seed Moisture

The initial seed moisture was 7.9%, increasing slightly to 9.5% after 24 h of imbibition (Figure 1C). While this increase seems minor, the water content of the seeds (SWC) showed a much more pronounced rise. In the first two hours of imbibition, the SWC rose to 25.7%. By 24 h, it had reached approximately 59.2%, a sixfold increase from the initial value (Figure 1D). This substantial increase in SWC reflects the rapid uptake of water during the early phases of imbibition, confirming that J. curcas seeds are non-waterproof. This process was strongly correlated with the duration of imbibition (r = 0.93, p < 0.05). This significant increase in seed moisture is a critical factor in germination, as seeds need water to activate metabolic processes essential for germination. However, rapid hydration can lead to imbibitional damage if the seeds cannot accommodate the influx of water, leading to cellular damage. The positive correlation between seed moisture and SWC (r = 0.96, p < 0.05) suggests that both measures of water content increase in tandem, further indicating the importance of controlling the rate of water uptake to avoid damage.

3.1.3. Effect of Seed Imbibition on Germination Percentage and Mean Germination Time

The seed germination showed a significant decline as the duration of imbibition increased. Initially, 85% of the seeds germinated without prior imbibition, but this value dropped to 68% after two hours of imbibition. By 24 h, only 44% of the seeds germinated (Figure 2A). The negative correlation between germination percentage and imbibition time (r = −0.721, p ≤ 0.01) indicates that more extended periods of imbibition adversely affect germination performance. This decline in germination is likely due to imbibitional damage, where rapid water uptake causes physical damage to seed tissues, disrupting cellular structures and reducing the seeds’ ability to germinate successfully. Seeds with lower initial moisture content, as seen in this study, are particularly vulnerable to this type of damage because they absorb water too quickly, leading to mechanical stress and potential failure in reactivating metabolic processes needed for germination.

The mean germination time (MGT) increased significantly with the duration of imbibition. The MGT was 4.8 days for non-imbibed seeds. However, after just two h of imbibition, the MGT increased to 6.2 days. By 24 h, it had reached 7.1 days (Figure 2B). This extension in MGT is strongly negatively correlated with germination percentage (r = −0.88, p < 0.05), suggesting that as seeds become damaged during imbibition, not only do fewer seeds germinate, but those that do germinate take longer to do so. This delay in germination may be attributed to the physiological stress caused by rapid water uptake, which can impair the reactivation of metabolic functions and delay radicle emergence. The increased MGT reflects a decrease in seed vigor, as seeds that take longer to germinate generally have lower overall performance and may not develop into robust seedlings.

In general, the findings in this section demonstrate that careful hydration control is crucial to minimizing damage and maintaining seed viability, especially in seeds with low initial moisture content. Electrical conductivity measurements provide a valuable tool for assessing seed vigor and could be employed as a rapid test to predict germination outcomes in J. curcas.

3.2. Germination of Jatropha curcas Seed Under Accelerated Aging (AA), Saturated Salt Accelerated Aging (SSAA), and Controlled Deterioration (CD)

3.2.1. Seed Germination

Fresh and 1-year-old seeds show significant differences in germination parameters depending on the aging treatments applied. Under control conditions, seeds from both seed lots exhibited relatively high germination rates, with fresh seeds performing notably better than those from 1-year-old seeds. The natural aging process and the effects of seed lot are evident in these results. Figure 3A highlights that the 1-year-old seeds significantly reduced germination capacity compared to their fresh seed counterparts. The fresh seeds germinated at least 8.8-fold higher than those naturally aged (1-year-old seeds) (Figure 3A). However, when seeds were exposed to accelerated aging (AA), both seed lots reduced the germination percentage. Moreover, germination was significantly lower in seeds treated with 72 h of AA, especially in 1-year-old seeds, where deterioration was more pronounced (i.e., 66.3% in 72h-AA and 45.5% in 24h-AA).

Saturated salt accelerated aging (SSAA), designed to simulate stress while controlling moisture absorption, resulted in even lower germination percentages than AA. Seeds exposed to 72 h of SSAA (Figure 3A) showed higher reductions in germination, especially in 1-year-old seeds. Comparable results were verified in controlled deterioration (CD), which showed the most severe impact on germination. Both seed lots subjected to CD showed a nearly complete loss of germination capacity, particularly the 1-year-old seeds, which failed to germinate after 24 h of CD treatment (Figure 3A). The germination dropped to nearly zero, indicating irreversible damage caused by the extreme conditions of this test. While in fresh seeds, the germination rates were still significantly compromised under CD treatment, suggesting that controlled deterioration is particularly harmful to seed longevity, regardless of natural age in 1-year-old J. curcas seeds.

3.2.2. Mean Germination Time (MGT)

The different aging treatments also affected the mean germination time (MGT), which is closely linked to seed lot and treatments. In non-treated seeds, the fresh seed lot germinated more uniformly and faster than 1-year-old seeds, which had a slower germination response due to the natural aging effects over time.

Under AA, the MGT increased substantially, especially for seeds subjected to 72 h of treatment. The more prolonged exposure to high temperature and humidity delayed germination, with the MGT of 1-year-old seeds extending considerably compared to fresh seeds. However, 24- or 72 h showed a similar increase in MGT, regardless of time in AA (Figure 3B). SSAA produced similar results, although the delay in MGT was slightly more pronounced than in the AA treatment, particularly for 72 h SSAA, which showed a germination delay 26.2% compared to 2.8% delay in 48 h SSAA. Then, the controlled environment of SSAA, which stresses the seeds while limiting water absorption, led to a more asynchronous germination process, as seen in the 1-year-old seeds, where MGT increased sharply.

The CD treatment had the most substantial effect on MGT. Figure 3B shows that seeds exposed to CD, particularly in 1-year-old seeds, experienced prolonged delays in seed germination, with MGT values reaching significantly high levels. The controlled deterioration process severely impacted the seeds’ ability to germinate quickly and uniformly, indicating that this treatment compromises germination and the synchronization of germination.

3.2.3. Viable Seeds

The viability of seeds, as measured by tetrazolium staining, revealed a stark contrast between control and aged seeds, particularly those exposed to CD treatments (Supplementary Figure S1). Fresh seeds showed the highest viability, with over 90% displaying bright red staining, indicating healthy and viable embryos. In contrast, control seeds from 1-year-old seeds showed reduced viability, with only about 9.5% being viable.

The AA treatment significantly reduced seed viability, particularly for 1-year-old seeds. Seeds exposed to 72 h of AA showed only 18% viability. The fresh seeds fared better under AA treatment, maintaining a viability of around 54% (Figure 3C). However, this still represents a considerable reduction from the control group’s high viability. SSAA had a more detrimental effect on seed viability. Seeds subjected to 48 and 72 h of SSAA showed significantly lower viability, mainly in a 1-year-old seed lot. Controlled deterioration was the most damaging treatment for seed viability. Seeds from both seed lots subjected to 24 h of CD treatment exhibited almost complete loss of viability (Figure 3C), where nearly 100% of the seeds were non-viable. Even after 12 h of CD treatment, viability was severely reduced, particularly for 1-year-old seeds, which showed only 3.2% viability. These results demonstrate that controlled deterioration leads to extensive damage to the embryo, significantly reducing the seeds’ capacity to remain viable.

3.3. Germination of Jatropha curcas Stored Under Two Different Temperatures for 12 Months

During the 12 months, seeds stored at 25 °C exhibited a marked reduction in germination percentages, with only 2% germinating by the period’s end (Figure 4A). Contrastingly, seeds stored at 4 °C retained slightly better germination capacity, although still diminished over time, reaching about 7% at 12 months. This decline correlates with a significant rise in MGT, indicating that seeds require longer to initiate germination as storage time increases, especially under warmer conditions (Figure 4B). This slower germination rate can probably be attributed to the progressive degradation of biochemical structures necessary for rapid germination, as seeds stored at higher temperatures showed increased signs of deterioration.

Regarding MGT, the study showed that seeds stored at room temperature exhibited significantly delayed germination timing (Figure 4B), which points to deteriorative metabolic changes accelerated by the higher temperature. For seeds stored at 4 °C, while MGT did increase over time, the rate was considerably less severe than for those at room temperature. Then, we observed that lower storage temperatures are crucial in maintaining the viability and germination vigor of J. curcas seeds, as higher temperatures accelerate deterioration processes, causing structural and biochemical degradation that impede effective germination.

3.4. Germination of Jatropha curcas Seeds Stored Under Controlled Air Humidity and Cooler Temperature

3.4.1. Seed Germination and Mean Germination Time

Air-controlled cold storage significantly affects the MGT of J. curcas seeds, showing prolonged germination times (Figure 5B). In contrast, seed germination (Figure 5A) suffers less than in those evaluated in Section 3.2, where the seeds were stored in paper bags. Initial results indicate that J. curcas seeds retain viability and germinability primarily even after 12 months at 4 °C, yet the time required to complete germination progressively extends. The increased MGT with air-controlled stored seeds suggests that less humidity and colder container conditions leads to metabolic deceleration, potentially through reduced enzymatic activity necessary for rapid seed germination. This delayed response implies that metabolic changes accrue over extended cold storage, affecting the seeds’ readiness to transition from dormancy to active growth. The storage-induced slowdown aligns with reductions in seed respiration rate and alterations in reserve mobilization, indicating that while cold storage preserves overall viability, it does so at the cost of germination promptness.

The cumulative germination patterns (Figure 5C) emphasize the lag effect caused by prolonged storage under cold conditions. Non-stored seeds demonstrate a more synchronized germination process, starting within the first few days and reaching completion in approximately 8 days. In contrast, seeds stored for extended periods, significantly beyond 6 months, show delayed onset and germination completion, which occurs approximately 12 days after sowing. Interesting points were observed in seeds stored in an air-controlled container, where the seeds maintained their germinability (20%) even after 12 months of storage.

3.4.2. Water Content, Osmotic Potential, and Respiration Rate

During the seed storage in air-humidity-controlled containers, the seed lost significant water content (Figure 6A). Fresh seeds contained 8.75 ± 0.24% of water, but 6 months in air-humidity-controlled containers, the seed content was reduced by 15.5% and progressively reduced to 6.18 ± 0.28% of water content, a reduction of 29.4% (Figure 6A). As a consequence of a decrease in water content provoked by silica gel, the seed osmotic potential was reduced from −16.4 ± 0.6 MPa to −91.1 ± 1.5 MPa, a substantial reduction of 454% (Figure 6B). In consonance, in the same period, the seed respiration was reduced from 144.1 ± 2.9 to 9.34 ± 1.22, significant seed respiration to 94% (Figure 6C).

3.4.3. Metabolism and Metabolic Pathway Under Storage

Sucrose, oil, starch, and proteins were metabolized during storage to maintain basal embryo respiration. In 12-month-aged seeds, the seed content was drastically reduced by 47.3% in sucrose (Figure 7A) and 26% in oil content (Figure 7B), with moderate reduction (11.4%) in starch (Figure 7C), and milder but significant reduction of 8.7% in protein (Figure 7D) content. Contrarily, the content of glucose (Figure 7E), soluble amino acids (Figure 7F), and total sugars (Figure 7G) increased by 99.8%, 36.5%, and 29%, respectively. Also, the fructose was first increased by 90.1% in 6-month seed storage, reducing to 10.7 mmol kg⁻¹ MS (34.7%) in 12-month seed storage (Figure 7H).

The metabolic pathway shows us that in the first 6 months of storage, some biochemical compounds inside the seeds were metabolized to produce carbon skeletons to drive the tricarboxylic acid cycle (TCA cycle) since citrate, isocitrate, α-ketoglutarate, and succinate were substantially increased by 6.5-, 6.5-, 4.8-, and 3.6-fold. After 12 months, the content of citrate and isocitrate remains higher, i.e., twice compared to non-stored seeds (Figure 8), while α-ketoglutarate and succinate were reduced to 32.1% and 37.8%, respectively.

Figure 8 demonstrates that TCA cycle reactions were more active than glycolysis, possibly due to the activation of the glyoxylate cycle with beta-oxidations of lipid bodies, as shown by the reduction of 26% of the oil content in the seeds (Figure 7B and Figure 8). The catalysis of proteins into amino acids, producing glutamate and α-ketoglutarate, is another possibility of higher activation of the TCA cycle than glycolysis.

3.4.4. Multivariate Analysis

All data for germination, MGT, water content, biochemical, and metabolic analysis were grouped in a multivariate analysis through principal component analysis (PCA). The PCA, composed of PC1 (0.587) and PC2 (0.326), represents at least 91.3% of the possible variation in these datasets that this PCA could represent. All components were distributed in five groups in a cartesian plan following Euclidian distance at 71.2% dissimilarity. Group 1, formed by non-stored seeds, comprehends the features of respiration, germination, starch, oil, sucrose, and fumarate (Figure 9). In group 2, formed by seeds stored for 3 months, the features of osmotic potential, WRC, proteins, and pyruvate were grouped according PCA, while in a group formed by seeds stored for 6 months, the features of fructose, fructose-6-P, glucose-6-P, α-ketoglutarate, succinate and maltose were grouped. Group 4 englobes the features of soluble sugars, glucose, citrate, and isocitrate. Finally, group 5 was formed by malate, amino acids, and MGT. In summary, each group was formed by a specific functional activity predominant in each group of seeds, such as high germination and respiration activity that marked the non-stored seeds group. In contrast, the highest production of amino acids is stamped the group 5 (Figure 9).

3.5. Effect of Salinity on Seed Germination Stored by 3 Months Under Controlled Air Humidity and Cooler Temperature

3.5.1. Seed Germination and Mean Germination Time

The data in Figure 10 show a significant decrease in seed germination with increasing NaCl concentrations. However, the germination in a NaCl-free medium was significantly higher than that of fresh seeds (Figure 4A) or stored for 3 months (Figure 5A). The difference between those experiments was that the last seeds were stored at 4 °C under controlled air humidity while the first was promptly sampled or stored in paper bags. The decrease in seed germination on seeds stored in controlled containers was milder than those described in seeds stored in paper bags without silica gel.

Seeds stored for three months at 4 °C demonstrate reduced germination at NaCl concentrations as low as 60 mM L⁻¹ NaCl (Figure 10A), where germinability declines sharply from approximately 83% (0 mM) to around 37%. Also, seeds germinated under 100 mM L⁻¹ NaCl show a germination of about 24%. This substantial reduction underlines the susceptibility of J. curcas seeds to saline conditions.

Regarding germination timing, Figure 10B shows that NaCl notably affects MGT. While seeds under non-saline conditions germinate within an average of 4–5 days, increased NaCl levels extend this period significantly. At 100 mM L⁻¹ NaCl, germination time extends to an average of 16 days, reflecting a threefold increase in response to saline stress. This delayed germination time can be attributed to the NaCl-induced osmotic pressure that limits water availability, prolonging the dormancy period and energy mobilization within the seed, as demonstrated in Figure 10.

Comparing the seed germination (Figure 10) with those where seeds were stored in paper bags (Figure 5), which depict seed germination and MGT over various storage durations, it becomes evident that storage alone slightly affects the germination metrics, albeit not as severely as saline conditions. Figure 10A shows that J. curcas seeds exhibit stable germinability around 20–25% across storage and exposure to NaCl. These data indicates resilience in dry and refrigerated storage, likely due to controlled water activity and temperature, maintaining seed viability. However, MGT trends upward with an increase in salinity, which can result from metabolic shifts such as reduced water content and respiratory rate, as documented in the physiological analysis. Thus, while NaCl impacts MGT and may delay the onset of germination, saline exposure presents a more immediate and severe challenge, especially at higher NaCl concentrations where MGT extends beyond regular seedling emergence periods.

It should be noted that seed storage at 4 °C was chosen based on the results shown in Figure 4, which show that seed germination was higher when seeds were stored in cooler temperatures compared to ambient ones. Nonetheless, as salinity was introduced as a secondary stressor, the cumulative effects on germination reflect an interplay where low temperatures safeguard essential germination potential. However, high saline conditions compromise cellular and enzymatic functionality required for germination. Therefore, while dry, the cooler temperature in storage periods aids in mitigating germination delays caused by desiccation and low seed respiration. The additional stress from NaCl reveals vulnerabilities in J. curcas, highlighting its limited tolerance to salinity post-storage. This limited tolerance demonstrates a key area for exploring adaptive strategies in J. curcas cultivation, particularly in regions prone to salinization.

3.5.2. Mineral Nutrition on Seedlings Germinated Under NaCl

Table 1. Level of potassium (K), iron (Fe), phosphorus (P), manganese (Mn), magnesium (Mg), sodium (Na), chlorine (Cl), and calcium (Ca) in 5-day Jatropha curcas plants under different concentrations of NaCl during the germination and first days after embryo emergence. All values denote media (± SE) of 6 repetitions. Means followed by different small case letters denote statistical significance (SNK; p ≤ 0.05).

NaCl	Potassium (g kg−1 DW)				Iron (g kg−1 DW)				Phosphorus (g kg−1 DW)				Manganese (mg kg−1 DW)
0	33.5	±	2.7	a	54.8	±	3.2	c	8.6	±	0.5	a	239.7	±	5.4	a
20	20.4	±	1.3	b	58.6	±	2.6	c	7.9	±	0.4	ab	242.7	±	4.6	a
40	11.3	±	0.5	c	62.3	±	2.2	c	7.1	±	0.4	b	245.7	±	8.8	a
60	11.1	±	0.7	c	74.7	±	1.4	b	6.7	±	0.4	b	223.4	±	8.0	ab
80	10.3	±	0.4	c	81.4	±	1.7	ab	6.6	±	0.2	b	205.0	±	7.3	b
100	7.5	±	0.1	c	88.1	±	2.6	a	6.5	±	0.2	b	155.7	±	3.2	c
NaCl	Magnesium (g kg−1 DW)				Sodium (mg kg−1 DW)				Chlorine (mg kg−1 DW)				Calcium (g kg−1 DW)
0	2.3	±	0.1	b	5.1	±	0.0	c	2.7	±	0.1	c	11.3	±	0.4	a
20	2.6	±	0.1	ab	34.3	±	0.7	b	19.1	±	0.7	b	10.3	±	0.4	b
40	2.8	±	0.1	a	45.8	±	1.7	a	26.9	±	2.2	a	9.2	±	0.3	c
60	2.8	±	0.1	a	52.3	±	1.8	a	31.8	±	1.3	a	7.4	±	0.5	d
80	2.9	±	0.1	a	52.5	±	1.5	a	34.2	±	1.3	a	6.1	±	0.3	e
100	2.9	±	0.2	a	52.7	±	1.5	a	33.6	±	1.6	a	4.6	±	0.3	f

shows us the modulation of ions in the first fifteen days after seed germination. In 15-day seedlings, the concentration of potassium (K⁺), phosphorus (P), manganese (Mn⁺²), and calcium (Ca⁺²) decreased, respectively, by 77.5%, 24.3%, 35%, and 59.6% in plantlets exposed to 100 mM L⁻¹ NaCl compared to NaCl-free. Iron (Fe) and magnesium (Mg⁺²) increased by 60.8% and 25.7% in opposite directions. The sodium (Na⁺) and chlorine (Cl⁻) increased by 928% and 1166%, respectively. The decrease in K⁺ and increase in Na⁺ provoked an abrupt rise in the Na⁺/K⁺ relationship from 0.15 in NaCl-free plantlets to 6.98 in plantlets under 100 mM L⁻¹ NaCl. Also, the relationship between Na⁺/Ca⁺² increased by 2442% in plantlets under 100 mM L⁻¹ NaCl.

3.6. RT-PCR

Differential gene expression analysis using RT-qPCR in the studied J. curcas genotype revealed significant transcriptional variations in response to different salt stress levels (0, 20, 40, 60, 80, and 100 mM NaCl;

Table 2. Differential gene expression in the studied genotype under salt stress was analyzed by RT-qPCR across five NaCl treatments.

NaCl Treatment (mM)	SAMe	PAL	SAM	PX	CXE	HD-Zip	NAC	MGL	XTH
0 (control)	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
20 mM	1.45	n.s.	3.20	1.78	1.30	2.40	2.10	2.85	0.90
40 mM	1.85	n.s.	5.80	2.20	1.95	3.40	3.15	4.00	0.65
60 mM	2.00	n.s.	7.00	n.s.	2.10	3.80	3.80	5.10	0.50
80 mM	2.20	n.s.	9.10	n.s.	2.30	4.50	4.60	6.20	0.30
100 mM	2.36	n.s.	10.31	n.s.	2.36	5.10	5.42	7.36	−0.19

Abbreviations: SAMe: S-adenosylmethionine-dependent methyltransferase; PAL: Phenylalanine ammonia-lyase; SAM: S-adenosylmethionine synthase; PX: Peroxidase; CXE: Carboxylesterase; HD-Zip: Zipper homeobox leucine transcription factor; NAC: NAC transcription factor; MGL: Methionine gamma-lyase; XTH: Xyloglucan endotransglucosylase. Values represent relative expression levels (Fold Change, FC) determined using the 2⁻ΔΔCt method, normalized against reference genes. UR: Up-regulation; DR: Down-regulation; n.s.: Not significant. Significant values were considered for FC ≥ 2 and p ≤ 0.05, using ANOVA with Tukey’s test.

). A clear transcriptional response to salinity was observed, with several genes showing differential expression patterns as NaCl concentration increased. The SAM (S-adenosylmethionine synthase) and SAMe (S-adenosylmethionine-dependent methyltransferase) genes exhibited progressive up-regulation, reaching maximum expression levels of 10.31-fold and 2.36-fold, respectively, at 100 mM NaCl. Similarly, the transcription factors HD-Zip and NAC showed significant up-regulation, with peak expression levels of 5.10-fold and 5.42-fold, respectively, under the highest salinity treatment (Table 2). The MGL (methionine gamma-lyase) gene followed a similar trend, increasing its expression to 7.36-fold at 100 mM NaCl. In contrast, the XTH (xyloglucan endotransglucosylase) gene displayed consistent down-regulation as salinity levels increased, reaching a relative expression of −0.19-fold at 100 mM NaCl. This suggests a potential suppression of cell wall remodeling processes under severe salt stress. The CXE (carboxylesterase) gene showed moderate up-regulation, with expression increasing to 2.36-fold at 100 mM NaCl. However, the PAL (phenylalanine ammonia-lyase) and PX (peroxidase) genes exhibited no significant changes in expression across all salinity treatments, indicating that these pathways may not be directly involved in the salt stress response in this genotype. Overall, these results demonstrate the activation of specific genes related to osmotic adjustment, antioxidative defense, and stress signal transduction under increasing salinity conditions. The up-regulation of SAM, SAMe, HD-Zip, NAC, and MGL highlights their potential role in mitigating salt-induced damage, while the down-regulation of XTH reflects an alteration in cell wall remodeling mechanisms. These findings provide valuable insights into the molecular mechanisms underlying salinity sensitivity in Jatropha curcas and identify key genes that could be targeted in future breeding or biotechnological programs to enhance salinity tolerance in this species (Table 2).

This table shows the variation in relative gene expression of the studied genotype across five salt stress levels (0–100 mM NaCl). Genes such as SAM, SAMe, HD-Zip, NAC, and MGL displayed progressive up-regulation (UR) as NaCl concentration increased, reaching peak expression at 100 mM. Conversely, XTH exhibited significant down-regulation (DR) at higher salinity levels. Genes like PAL and PX showed no significant changes (n.s.) under specific salt treatments.

4. Discussion

4.1. Germination of Jatropha curcas Seed After Imbibition

The study on the germination of J. curcas seeds subjected to different imbibition times reveals essential insights into the effects of hydration on seed viability and vigor. Initial findings demonstrate a reduction in germination percentage as the imbibition time increases. Seeds without imbibition showed the highest germination rate (85%), while seeds imbibed for extended periods (up to 24 h) experienced decreased germination rates and extended MGT from 4.8 to 7.1 days. The negative correlation between imbibition time and germinability suggests that prolonged hydration may induce imbibition damage, possibly due to uncontrolled tissue hydration disrupting cellular integrity and resulting in solute leakage, as similarly observed in studies on corn and rice seeds [46,47]. The uncontrolled water entry, especially in seeds with low initial moisture content, has resulted in reduced vigor, as evidenced by elevated EC values associated with damaged membranes and decreased germination capacity. In this state, the seeds may become vulnerable to environmental stresses, including rapid imbibition and cool temperatures, which can lead to solute leakage and dysfunctional metabolic reactivation [48]. On the other hand, seeds without pre-imbibition demonstrated higher uniformity in germination, attributed to the controlled rehydration process that mitigates cellular damage [49]. Controlled hydration, particularly with a gradual increase, can aid in stabilizing membrane functions and reactivating metabolic processes.

As previously postulated, the fast imbibition of J. curcas confirms they are non-waterproof [6,35]. Bewley et al. [50] highlight the importance of understanding seed imbibition, a critical factor in determining the germination success and desiccation tolerance in seeds. This research emphasizes that while the triphasic model is widely used to explain seed water absorption stages, it does not universally apply to all species, suggesting a need to refine this model for specific types of seeds, especially those used in reforestation and conservation efforts. Our study implies that non-imbibed seeds may be more advantageous for direct sowing.

For J. curcas, employing an electrical conductivity test could serve as a reliable vigor assessment tool, especially for seeds with low initial moisture, allowing for detecting those most viable for cultivation purposes. These findings underscore the importance of managing initial seed moisture and hydration protocols. As commented by Vertucci [48], this research underscores that seed structure and environmental conditions intricately control imbibition kinetics. Optimized hydration rates promote germination, preventing damage from excessive speed or delayed water uptake. Understanding the kinetics of seed hydration makes it possible to better manage germination processes, especially in challenging environments where seed vigor is paramount [48].

4.2. Natural and Accelerated Aging on Seed Germination

In general, accelerated aging treatments encompass seed longevity, rapid germination, initial seedling growth, and early stress tolerance, all crucial for the uniform and productive establishment of plants in the field. Similar results were described in this study. The accelerated aging affects the seed germination and vigor between fresh and 1-year-old seeds, showing that seed age plays a crucial role in viability outcomes, as described earlier by Moncaleano-Escandon et al. [11].

The 1-year-old seeds displayed reduced germination rates and higher levels of asynchrony than fresh seeds, which indicates that aging leads to substantial physiological and biochemical modulation. When subjected to accelerated aging (AA), the older seeds showed a partial recovery in germinability, suggesting that this metabolic activation can temporarily enhance performance. However, despite this improvement, the germination rates of the 1-year-old seeds remained lower than fresh seeds, illustrating that accelerated aging cannot fully restore the vigor lost with natural aging.

Accelerated aging and other vigor tests, such as SSAA, proved effective in assessing the viability of J. curcas seeds, although they also highlighted limitations inherent in each method. While beneficial for many crops, the AA test was not as precise for small-seeded species due to rapid water absorption, which can lead to excess stress and skewed results. SSAA emerged as a more reliable alternative, as it maintains controlled humidity levels and slows aging without causing extensive damage to cellular membranes, thus enabling a more gradual assessment of seed vigor [12,32]. This modulation allows SSAA to simulate natural aging more closely, offering repeatable seed viability and vigor measurements.

Controlled deterioration (CD) testing was particularly challenging for J. curcas, as the seeds completely lost their viability when subjected to this method, highlighting the unsuitability of CD for species with high initial moisture content. This extreme outcome in J. curcas seeds indicate that CD may be overly severe for oilseeds or species with high sensitivity to water stress. Increased metabolic activity under high humidity and temperature leads to rapid depletion of reserves and cellular damage [12,51]. These findings caution against the use of CD in similar oil-bearing species and support the need for alternative aging protocols for reliable vigor testing in such crops.

Temperature during storage proved to be a significant factor influencing the longevity and vigor of J. curcas seeds. Seeds stored at 4 °C showed higher germination rates and synchrony than those kept at 25 °C. These reactions increased solute leakage and reduced membrane integrity, indicating compromised seed quality [12,52]. The results align with earlier findings that seed viability declines more rapidly under warmer, tropical conditions, where elevated temperatures hasten deterioration [53,54]. In contrast, storage at lower temperatures better preserves the physiological quality of seeds by slowing these damaging processes, making it a preferred strategy for maintaining the viability of J. curcas and other tropical biodiesel crops [14,55]. Some scholars [53,54,55] emphasize the importance of temperature and moisture management in maintaining seed quality over time. The findings underscore the value of accelerated aging tests and temperature control as effective measures for assessing and preserving seed viability in J. curcas. However, they also call attention to the complexities of seed aging, especially for biodiesel species where high moisture content can significantly impact long-term storability.

With the anticipated impacts of climate change, enhancing seed vigor has become increasingly vital to maintaining crop yields and supporting global food security [56]. By integrating modern genetic tools and breeding strategies, crops can be developed to withstand early-stage stressors, ensuring robust seedling establishment and uniform crop stands. This approach is critical for agricultural productivity, as strong seed vigor contributes to plant health and improves crop yield and profitability by enabling efficient use of resources in challenging environments [56].

4.3. Water Content, Osmotic Potential, and Respiration Rate

The storage of J. curcas seeds at lower temperatures, particularly at 4 °C, combined with the use of desiccants, has shown promise in reducing the metabolic processes that typically lead to seed deterioration over time. Moncaleano-Escandon et al. [11] emphasized that desiccation methods result in lower water content and osmotic potential in stored seeds, significantly decreasing seed respiration rates. This approach is particularly relevant for J. curcas, as its high natural metabolic rate leads to rapid viability loss when stored in warmer, humid conditions [36,57]. In this study, water content in the seeds dropped from 8.8% to 6.2% over 12 months of storage, and the corresponding reduction in osmotic potential, from −16 MPa to −91 MPa, was correlated with a 94% decrease in respiratory rate.

Refrigeration with controlled humidity offers advantages for maintaining seed viability despite some limitations. While a controlled, drier atmosphere slows respiration, it does not eliminate it completely, leading to gradual reserve mobilization over time [36]. This mobilization is necessary to support the embryo’s viability; however, overextended storage periods could potentially exhaust reserves, impacting germination negatively [11,57]. The preservation of critical biochemical components, like starch and proteins, is essential for maintaining seed quality, as shown by Moncaleano-Escandon et al. [11], who observed that without desiccant use, J. curcas seeds stored at similar conditions experience more rapid declines in germinability.

Akowuah et al. [57] and Chidananda et al. [36] reported that prolonged seed respiration under low humidity storage settings can progressively consume proteins and carbohydrates essential for germination. Moncaleano-Escandon et al. [11] also observed that increased seed storage duration correlated strongly with a decline in soluble sugars. These data further supports the idea that desiccants help extend viability by slowing these metabolic demands. Consequently, the best outcomes for J. curcas storage in terms of viability and oil preservation appear to rely on an optimized balance of temperature and humidity control [11,36,57]. The germination of 12-month-aged seeds was 31% of that observed in fresh seeds. The strategic storage of J. curcas seeds under conditions of low oxygen, low humidity, and reduced temperature has emerged as a pivotal technique in preserving seed viability and quality. This approach significantly diminishes the respiratory rate of the seeds, a critical factor in prolonging their shelf life and maintaining their germinative potential. Producers can effectively slow down the deterioration process by lowering the metabolic activity through controlled environments—specifically using 1.5 g of silica gel per gram of seed to manage humidity and storing at 4 °C. This technique is particularly valuable for J. curcas seeds, which are poised to become a cornerstone in biodiesel production due to their high oil content and adaptability to poor soils. Looking towards a future where J. curcas might dominate the biodiesel market, it becomes imperative for producers to adopt advanced storage strategies. The research outlined in this study provides a framework for optimizing seed storage to enhance both direct planting and biodiesel production. Producers can ensure the seeds’ longevity and vitality by investing in technologies that minimize oxygen exposure, control humidity, and maintain low storage temperatures. Improving storage conditions is crucial as it allows for the strategic release of seeds in response to market demands, thereby stabilizing prices and increasing profitability. The findings suggest that with proper storage techniques, the degradation of oil content is minimal, ensuring that the seeds retain their commercial value. As global demand for renewable energy sources grows, the ability to store seeds effectively will be a competitive advantage, enabling producers to meet market needs efficiently. This strategic approach not only supports the economic viability of Jatropha cultivation but also contributes to the broader goal of sustainable energy production, making it a key player in the future energy landscape.

The trademark of commodities leads countries to produce strategies to guarantee superiority in the low availability of these commodities. One example is that the Brazilian soybean industry faces significant logistical challenges due to inadequate storage infrastructure [58]. This perspective is complemented by Woerfel [59], who emphasizes the importance of proper storage conditions to maintain soybean quality, suggesting that infrastructure improvements could also address storage deficits. Together, these insights underscore the need for a comprehensive overhaul of Brazil’s logistics to enhance its position in the global market. This debate is mirrored by Zulauf and Kim [60], who analyze the profitability of soybean storage in the U.S., suggesting that individual storage decisions significantly impact market dynamics. They challenge the notion that soybean storage is less profitable than corn, highlighting the importance of reassessing storage strategies. Storage conditions are vital in maintaining soybean quality, advocating for controlled environmental conditions to prevent deterioration. These diverse perspectives highlight the complexity of optimizing soybean logistics and storage, suggesting that a balanced approach considering both immediate and long-term strategies is essential. Both perspectives stress the significance of controlling moisture and temperature to prevent quality loss, which directly impacts the economic value of soybeans. This loss mitigation is particularly relevant in the context of global market competitiveness [58]. The integration of advanced storage technologies and infrastructure improvements could enhance the efficiency and sustainability of soybean logistics [60]. Collectively, these studies underscore the need for ongoing research and investment in infrastructure and storage technologies to support the soybean industry’s growth and sustainability.

4.4. Metabolism and Metabolic Pathway Under Storage

The biochemical changes in J. curcas seeds stored for 12 months and significant shifts observed in storage compounds affected seed viability and germination potential. The oil content initially remained stable but declined after 12 months of storage, aligning with earlier findings by Akowuah et al. [57], who noted that storage time could influence oil content and lead to increases in free fatty acids. This degradation is associated with ongoing respiratory activity in the seeds, albeit at a reduced rate, leading to a gradual breakdown of essential energy sources.

Starch content, another critical reserve, showed an 11% reduction over the storage period, similar to trends observed by Moncaleano-Escandon et al. [11], who describe significant biochemical changes in stored J. curcas seeds. The decline in starch points to its role as a primary energy source under the low-humidity storage conditions as applied in this study. In parallel, amino acids, total sugars, and glucose increased substantially by 37%, 29%, and 90% during storage. This increase suggests that the vast majority of seed reserves were consumed during the storage period, contributing to carbon skeletons and enabling seed metabolism to persist under prolonged storage. This metabolic shift reflects the findings of Chidananda et al. [36], who showed that proteins might be mobilized for respiration or as osmoprotectants. Carbohydrate as sucrose decreased 47% by 12 months, while glucose levels increased 3-fold in the first 6 months and then declined twice compared to non-stored seeds. Similar trends have been reported by Alencar et al. [17] in studies on seed stress responses, where glucose and other soluble sugars play critical roles in maintaining energy flow under reduced respiration conditions.

The metabolic pathway was defined by all metabolic data of J. curcas seeds stored from 0 to 12 months. Over time, compounds from the TCA cycle, such as citrate, isocitrate, α-ketoglutarate, and succinate, initially increase sharply, suggesting active metabolic adjustments likely aimed at maintaining respiration and basal cellular functions. After 12 months, citrate and isocitrate concentrations remain substantially elevated, nearly double the levels observed in fresh seeds. Conversely, α-ketoglutarate and succinate display a marked reduction at approximately 32% and 38% of initial levels, respectively. This pattern reflects a complex metabolic regulation, where the seeds maintain certain TCA intermediates, possibly to ensure a minimum energy threshold for eventual germination while gradually depleting others, highlighting an adaptive prioritization in energy utilization pathways, the observed decrease in oil content by 26% aligns with the activation of the glyoxylate cycle and β-oxidation processes, consistent with the seeds’ shift towards lipid mobilization as a primary energy reserve.

This lipid breakdown supports the TCA cycle via acetyl-CoA production and illustrates how storage duration imposes a trade-off between reserve preservation and energy demand. Proteins metabolized into amino acids, such as glutamate, further feed into this cycle, supporting the reduced glycolysis activity implied by the data. These results are like those presented by seeds in the germination phase [61,62,63], where endosperm reserves were metabolized to provide carbon skeletons for mitochondrial respiration and ATP production by the TCA cycle and cellular respiration. These metabolic adaptations demonstrate the seeds’ capacity to conserve key intermediates necessary for survival during prolonged storage, albeit at the expense of immediate germination readiness. This carefully regulated metabolic downshift allows for extended viability, albeit with delayed germination times once activated post-storage.

Thus, the results reported here suggest that, even though the respiratory rate reduced by 94%, the seeds still maintained a basal level of respiration, a fact that demonstrates that J. curcas seeds can significantly reduce their metabolism, at the cost of lower consumption of reserves. However, regarding biofuels, reserves, especially oil, must be maintained at an elevated level so that the stored seeds can serve as an alternative source of biodiesel. Nevertheless, even considering the seeds stored for 12 months, it was found that the oil concentration in the seeds was 24%, which still characterizes J. curcas as a promising source of oil for biodiesel when considering those of soybean, canola, and corn. According to Raja et al. [64], seeds of J. curcas are the primary source of oil because they contain 18% protein, 38% fat, and 17% carbohydrates; data followed our results.

The PCA explains all datasets to confirm the previous idea. The analysis demonstrated that group 1, incorporating the non-storage seeds, was supported by a high respiration rate and germination percentage. Also, this group comprises a high concentration of starch, oil, and sucrose and a 4-fold higher fumarate compared to seeds stored for 12 months. Those features represent at least 27% of all factors that explain the PCA. Similar results were described by Lozano-Isla et al. [13]. Öpik [65] describes that during the germination process, the oxygen uptake of the whole cotyledon peaks between the third and fifth days. However, mitochondrial activity starts to decline after about 36 h. The continued increase in respiration rate beyond this point could potentially be explained by the rise in soluble oxidative activity, which progresses as mitochondrial function decreases. Group 2, formed by seeds stored for 3 months, is promoted by osmotic potential, WRC, proteins, and pyruvate. These feature groups are due to the low osmotic potential promoted by silica gel in the containers. However, the WRC was only 9.8% lower than non-stored seeds, encouraging the respiration activity showed by an increase in 42% of pyruvate, maybe the metabolism of proteins and starch. Seeds stored for 6 months significantly increase starch metabolism, with consequent glycolysis and TCA increase. The increase in 1.8-, 1.9, 2.0-, 3.6-, 4.8-, and 10.6-fold in Fructose, Maltose, Fructose-6-P, Succinate, α-ketoglutarate, and Glucose-6-P are strong evidence that the reduction in 15.5% in WRC is not able to reduce the seed respiration to low rates to stop the metabolism of reserves. It is worth noting that seeds stored for 6 months present an osmotic potential 267% lower than non-stored seeds. Roberts and Ellis [66] describe that the lower limit for the relation in various species coincides with a seed moisture content between 2 and 6%. Below this level, there is little or no improvement in longevity with reduced moisture content. In this storage point, the J. curcas seeds showed a WRC of 7.4%. According to these authors, above 6%, and providing oxygen is available to sustain respiration, seed longevity increases with an increase in water potential except that, unless the seeds are dormant, the fact that may have occurred in consonance with the decrease in osmotic potential inside the containers. The TCA activity was drastically reduced in seeds stored for 9 months, as shown by citrate, isocitrate, glucose, and soluble sugars. These data were in agreement with other species, like maize [67], Arabidopsis [68], and pea [69]. Seeds stored for 12 months presented very high MGT and a higher increment of amino acids, which promote osmotic adjustment [70]. In this process, the proline is the primary amino acid involved in osmotic adjustment in accordance Braccini et al. [71].

4.5. Effect of NaCl on Seed Germination After Storage Under Air-Humidity Control Container

The germination of J. curcas seeds stored for three months, particularly under increased salinity conditions, shows significant shifts in germinability and MGT, as illustrated in Figure 10A,B. After a storage period of three months, seeds displayed robust germinability in non-saline environments, with germination initiating within 2–4 days. However, as NaCl concentrations rose, germination percentage and MGT diminished, aligning with previous findings in J. curcas [13,18] and other species [72,73], where salinity induces osmotic stress, inhibiting water uptake and delaying germination. Moderate salinity levels (50–75 mM L⁻¹ NaCl) led to minor delays in MGT, consistent with the moderate tolerance exhibited by J. curcas, but at higher concentrations (100 mM L⁻¹ NaCl), MGT extended considerably, with intervals reaching 17–18 days. This delay likely reflects both osmotic and ionic toxicity, corroborating the findings by Alencar et al. [17] and Lozano-Isla et al. [13], who noted delayed germination and reduced seedling vigor in J. curcas exposed to similar salt stress levels.

Extended storage under refrigeration, while effective in maintaining overall seed viability, appears to impose subtle metabolic constraints that manifest during germination, especially under stressful conditions such as salinity. The gradual increase in MGT following storage suggests a decline in the seeds’ metabolic readiness, further compounded by salt-induced stress during germination [11,13,36]. Thus, while cold storage preserves the fundamental germinative capacity of J. curcas, its efficacy in saline environments diminishes over time.

According to Meloni et al. [74], elevated salinity levels result in reduced uptake of K⁺, Ca²⁺, NO₃⁻, and P_i, while simultaneously leading to an increase in an uptake in Na⁺ and Cl⁻. In this way, the ratios of Na⁺/Ca²⁺, Na⁺/K⁺, and Ca²⁺/Mg²⁺ often increase. This relationship was perceived in this study as already described for J. curcas in another study with young plants [18]. In addition, Marschner [75] suggests that diminished potassium levels might result from the limited movement of this cation within the xylem or potentially from inhibited potassium uptake. This issue is verified by a substantial reduction in K⁺/Na⁺ from 6.5 in NaCl-free plantlets to 0.14 in 100 mM L⁻¹ NaCl. According to Silva et al. [76], the K-leaf concentration in young J. curcas plants decreased by approximately 87% when grown in 100 mM L⁻¹ NaCl compared to NaCl-free plants. This phenomenon was described by Cunha et al. [77], in which the concentration of K⁺ in the root xylem decreased from 38.1 g kg⁻¹ DW to 25.5 g kg⁻¹ DW (a decrease of 33.1%). In this study, any solutes were measured in the xylem; however, the concentration of K⁺ in the leaflet decreased from 33.5 g kg⁻¹ DW to 7.5 g kg⁻¹ DW, a fall of 78%.

A recent publication [76] revealed that Na⁺ and Cl⁻ concentrations increase significantly under salt stress while K⁺ content decreases. This ion imbalance is crucial for understanding the osmotic adjustment mechanisms in J. curcas [16]. In accordance with these authors, the seedlings exhibit a high uptake of Na⁺ and Cl⁻, suggesting a salt-included characteristic, which is essential for osmotic regulation under saline conditions. Additionally, the selectivity of ions is critical to highlight because Na⁺ affects the K⁺ uptake. In addition, these authors suggest different salt tolerance mechanisms, such as ion exclusion and compartmentalization in vacuoles commonly observed in glycophytes. These mechanisms help maintain low cytosolic Na⁺ levels while preserving K⁺ concentrations for normal metabolic functions. However, the findings in J. curcas emphasize its unique strategy of utilizing high Na⁺ and Cl⁻ accumulation for osmotic adjustment, highlighting the species’ ability to maintain osmotic balance through strategic ion and solute management [76,78,79,80], differing from the exclusion mechanisms seen in other species [76]. Regarding K⁺ uptake, Matos et al. [80] indicated that while salinity did not exhibit toxic effects, it caused nutritional imbalances, particularly affecting Ca²⁺ and K⁺ absorption. However, Matos et al. [80] described that Ca²⁺ and K⁺ absorption rise with NaCl, while in the present study, both ions decreased due to NaCl. Those authors underscore the importance of a balanced nutrient supply for optimal plant development and stress tolerance, suggesting that while J. curcas can endure certain nutrient deficiencies and its growth is optimized with a complete nutrient solution [76,80]. These studies provide valuable insights into the physiological responses of J. curcas to nutritional and saline stress. In accordance with Pinheiro et al. [81], some species, like J. curcas [76,78,79,80], exhibit mechanisms like ion exclusion and compartmentalization to mitigate salt damage, whereas castor bean seedlings appear to adjust by altering physiological processes [82]. This distinction highlights the variability in plant responses to salinity and the need for species-specific strategies to enhance salt tolerance [81]. This distinction emphasizes the variability in plant responses to salt stress and the potential for sugar metabolism to play a central role in adaptation [83].

The transcriptional response of J. curcas to salt stress is characterized by significant regulation of key genes involved in osmotic adjustment, antioxidative defense, and stress signal transduction. Several genes showed progressive up-regulation and down-regulation as NaCl concentration increased, indicating an adaptive activation to salinity. The SAMe and SAM genes showed significant up-regulation, reaching maximum fold increases of 2.36 and 10.31, respectively, under the 100 mM NaCl treatment. This trend indicates a significant activation of methylation pathways, which are essential for modulating metabolic processes and antioxidative defense under salt stress. These results align with previous studies suggesting that regulation of SAMe and SAM is linked to the synthesis of osmoprotective compounds and the modulation of enzymatic activity under salinity conditions [84,85]. Similarly, the transcription factors HD-Zip and NAC showed significant up-regulation (5.10 and 5.42 times, respectively) as salinity levels increased, highlighting their role in regulating stress-associated genes and stabilizing photosystem functions, as demonstrated in previous research [86]. These factors are key in stress signal transduction and gene expression regulation under adverse conditions, facilitating adaptation to salt stress. The MGL gene, involved in the synthesis of osmoprotective compounds such as methionine, also showed a progressive increase in expression, reaching a maximum of 7.36 times at 100 mM NaCl. This behavior reinforces the importance of osmotic adjustment strategies in salt tolerance, where the accumulation of osmoprotectants helps protect cells from osmotic damage and maintain cellular integrity [87]. In contrast, the XTH gene exhibited continuous down-regulation as salinity increased, with a relative expression of −0.19-fold at 100 mM NaCl. These data suggests that salt stress may suppress cell wall remodeling, which could impair cell expansion and plant growth, as this process is crucial for establishing new plant structures under stress conditions [85]. On the other hand, the CXE, PAL, and PX genes showed varied responses. The CXE gene exhibited moderate up-regulation (2.36-fold), indicating its involvement in protecting lipids and other bioactive compounds under salt stress. However, PAL and PX did not show significant changes in expression across the salt treatments, suggesting that, in this specific genotype, these genes may not play a critical role in the salt stress response.

5. Conclusions and Future Perspectives

This study on Jatropha curcas presents details of seed viability, metabolic activity, and physiological response under varying storage durations and salinity stress conditions. The storage of J. curcas seeds in containers with controlled relative humidity at low temperatures (4 °C) over extended periods proved beneficial for maintaining both seed viability and oil content. These conditions slow the seeds’ metabolic rate, effectively preserving essential reserves and extending storage life without significant quality degradation. The results demonstrate that seeds stored for up to 12 months at 4 °C exhibited minimal loss in germination potential and oil integrity, making it an economically viable approach for producers. The controlled humidity, achieved with desiccants, further mitigated deterioration by reducing moisture content, which limited respiratory activity and, thus, resource depletion. Such findings suggest that low-temperature, controlled-humidity storage offers an optimal balance, minimizing energy costs associated with storage while preserving germination capacity and oil yield, making this method especially advantageous in biodiesel production settings.

Considering the oil content stability observed over a 3–6-month storage period, J. curcas seeds could be stored during surplus periods if market conditions favor this approach. However, their susceptibility to storage indicates that such storage is beneficial only if it is conducted in an air-humidity container and under cooler temperatures because paper bags and higher temperatures provoke a substantial increase in respiration rates and a reduction in germination vigor and seed reserves. Thus, producers should assess storage costs and requirements to preserve seed lots when deciding on storage for economic purposes.

Seeds stored for three months under controlled conditions retained high germinability and exhibited a mean germination time (MGT) that increased with salinity stress, indicative of osmotic inhibition. Data reveal that while seed germination in a non-saline environment maintained a high germination rate, salinity levels as low as 40 mM L⁻¹ NaCl significantly reduced this rate to about 37%, with further reductions seen at 100 mM L⁻¹ NaCl (24%). The results underscore the critical susceptibility of J. curcas seeds to salinity, with NaCl concentrations above 40 mM L⁻¹ causing delayed germination times, attributed to osmotic stress that limits water uptake and delays early metabolic activation necessary for seedling establishment. Conversely, seeds germinated under non-saline conditions within approximately 4–6 days; exposure to 100 mM L⁻¹ NaCl extended the MGT to around 17–18 days, reflecting a threefold increase.

Also, this study highlights the need for further research into the genetic and environmental factors influencing Jatropha curcas stress tolerance. The findings suggest that while the plant holds potential as a biofuel source, its cultivation in saline and drought-prone areas requires careful management and possibly genetic enhancement. Future studies could focus on breeding programs or biotechnological approaches to improve stress tolerance, ensuring the plant success in diverse environmental conditions.