Initial Growth and Stomatal Characteristics of Capsicum Accessions Under PEG-Induced Water Stress In Vitro

1. Introduction

Pepper plants, belonging to the genus Capsicum of the Solanaceae family, are vegetables that stand out for their phenotypic characteristics. The structure of the plants, the morphology of the flowers, and the diversity of shapes and colors of the fruits, in addition to other aesthetic attributes, generate growing interest in this crop, showing great potential for development as ornamental plants [1]. In Brazil, the ornamental plant sector has contributed significantly to the economy. It has gained prominence over the years, generating around R$19.8 billion, creating more than 800,000 indirect jobs and 272,000 direct jobs, involving approximately 8,300 producers in a cultivated area of 15,600 hectares [2]. These data show a growing demand for varieties with high aesthetic value and good adaptability to different cultivation conditions.

However, water stress, often associated with high temperatures, is the environmental variable that most affects crops [3]. This problem is further aggravated by global warming and climate change, which expose the varieties to abiotic and biotic stresses, compromising initial growth and, consequently, productivity [4]. In the work by Molla et al. (2023) [5], a significant reduction in the length of pepper cultivars was observed compared to the control group as a consequence of water stress. In the initial and reproductive stages, pepper plants are more susceptible to stress caused by drought [6]. Bernau et al. (2020) [7], by inducing water stress conditions, observed a slower and, in some cases, incomplete germination process in chili pepper (Capsicum spp.) seeds. To meet the demands of the ornamental sector, the selection of genotypes that are more tolerant to water stress is essential for the development of new cultivars [2].

Polyethylene glycol (PEG) is a compound commonly used to simulate water stress (drought) conditions in mainly controlled environments. Its application allows for the assessment of crop tolerance to water scarcity, which contributes to the selection of better-adapted varieties [8]. Recent studies have shown great variation in the germination and initial growth of pepper plant accessions from various species (Capsicum annuum L., C. frutescens L., and C. chinense Jacq.) when subjected to concentrations of PEG [7,9,10]. These authors examined the influence of genotype and the collection site of the germplasm on drought stress tolerance. However, studies using PEG in the selection of pepper germplasm for ornamental use have not yet been reported.

In a study conducted in Iran, researchers used PEG to induce different levels of water stress during the germination of the ornamental plant Alcea rosea, allowing for the assessment of morphological, physiological, and biochemical changes. [11]. In this study, the authors concluded that the compound is effective for simulating water stress under controlled conditions, aiding in the selection of more drought-tolerant ecotypes even in the initial development phase. This reinforces the importance and efficiency of the technique for selecting tolerant accessions in breeding programs for ornamental plants.

Given the need to select genotypes that are better adapted to adverse conditions, the present study aimed to evaluate germination, initial growth, and stomatal characteristics of ornamental pepper (Capsicum spp.) accessions subjected in vitro to different levels of water stress induced by PEG 6000.

2. Materials and Methods

2.1. Vegetal Material

In this study, seven accessions of pepper plants (Table 1) with ornamental potential were used, previously selected based on earlier studies, as well as five concentrations of Polyethylene Glycol (PEG) 6000: 0% (control), 5%, 10%, 15%, and 20%, which was added to the culture medium to simulate in vitro water stress conditions.

2.2. Seed Preparation and Inoculation

The culture medium used in the study was the Murashige & Skoog (MS) medium [12] (Dinâmica^®) and consists of macronutrients, micronutrients, and vitamins. Sucrose was added to the medium (Dinâmica^®) (30g L^-1) and polyethylene glycol (PEG) (Dinâmica^®), with concentrations according to the treatment. 8 g L^-1 of agar (Mericlone Biotecnologia) were also added, and the medium pH was adjusted to 5.8 using 0.1 N NaOH and 0.1 N HCl solutions. After preparation, the medium was distributed into glass jars containing 40 mL of medium each, totaling four jars per concentration. The jars were organized in plastic bags and were then autoclaved at 120 °C±2 °C and pressure of 1kgf/cm².

The seeds of each accession were disinfected in a laminar flow chamber using a 1:1 solution (v/v) of distilled water and sodium hypochlorite for 10 minutes. After disinfestation, three seeds from each accession were inoculated into each pot containing culture medium. The material was kept in a growth room at 25 °C ± 2 °C with a 12:12h photoperiod. The experiment was conducted in a completely randomized design in a 5 x 7 factorial scheme (PEG concentrations x accessions) with four replications, each containing three seeds in each.

2.3. In Vitro Germination

Germination was evaluated daily for 20 days, always in the afternoon, completing 24 hours after inoculation. A seed was considered germinated when there was a protrusion of the radicle, according to the guidelines established by the reference “Regras para Análise de Sementes” (2009) [16].

At the end, the germination rate was calculated based on the number of inoculated seeds over the total and the germination speed index (GSI), following the formula proposed by Maguire (1962) [13]:

$$GSI=\sum {\displaystyle \frac{G_i}{n_i}}$$

where:

$G_i$ = number of germinated seeds on i-th day

$n_i$ = i-th day at which counting is being made

2.4. Seedling Morphology Assessment

The growth of pepper seedlings was observed 35 days after the seeds were inoculated in a medium with different concentrations of PEG. For this, two seedlings, randomly selected from each replicate, were evaluated. The length of the shoot and the root (mm) was measured using a digital caliper (Pantec^®). The fresh and dry weight (g) of the aerial part and the root were determined using a precision balance (Bel engineering^®, model s203). Soon after, the aerial part and the root were placed in properly labeled paper bags and set to dry in a drying oven (Tecnal^®, model TE-394/2) at 60 °C until constant weight was achieved, the dry weight of the shoot and the root was then determined. The dry matter content in the aerial part and in the root were calculated based on the following expression:

$$Drymattercontent(\%)={\displaystyle \frac{DryWeight}{FreshWeight}}x100$$

2.5. Stomatal Assessment

The stomatal evaluations were carried out with the aid of a trinocular microscope (Leica^® DM2500) with 400x magnification equipped with a digital color camera system (Leica^®, DFC700T). For this procedure, the adaxial side of four leaves/treatment was glued onto the slide for 5 minutes with glue (tekbond^®) until the impression was made. The measurements were taken with the aid of the Leica LAS Interactive Measurements module, using the ruler tool. Two stomata per leaf were randomly measured for the length (SL) and width (SW) of the stomata (µm), drawing a straight line along the longest axis of the stoma, from the end of one guard cell to the other, and the diameter by drawing a straight line across the opening (ostiole), measuring the width of the central region of the stoma, then it was calculated the ratio between length/width of the stomata (SL/SW). Stomata midpoint length (SML) (µm) and stomata midpoint width (SMW) (µm), were measured in the internal region of the stomata, by drawing a straight line in the longitudinal and transverse directions of the ostiole, respectively. The number of stomata in an area of 0.29mm² for the calculation of stomatal density as number of stomata per area unit (number of stomata per mm²).

The evaluation of stomata was carried out for the accessions CPCE 005, CPCE 007, CPCE 010, and CPCE 011, at two PEG concentrations (0% and 5%). A completely randomized design was used, in a 2 x 4 factorial scheme (concentration x accessions).

2.6. Statistical Analysis

All data processing, analyses, and classifications were performed in R v4.0.3 (The R Foundation for Statistical Computing, Vienna, Austria). The data were subjected to analysis of variance (ANOVA) using the package easyanova [14]. The homogeneity of variances was verified by the Bartlett test and the normality of the residuals by the Shapiro-Wilk test. When the assumptions were met, the means of the factors—accessions, PEG concentrations, and interaction—were compared using Tukey’s test (p < 0.05). For the variables whose assumptions of normality and homoscedasticity were not met, the analysis was performed using Generalized Linear Models (GLM), selected based on the Akaike Information Criterion (AIC) [15]. The model with binomial distribution was used for the germination variable; the model with Inverse Gaussian distribution for the percentage of shoot dry matter; and the model with Gamma distribution for the percentage of root dry matter. For the stomatal variables, the Gamma model was used for density and Inverse Gaussian for SL/SW. Significant differences between factors and interaction for the GLM models were compared by Tukey’s tests (p < 0.05) with confidence intervals adjusted by the Šidák method using the ‘emmeans’ function in the multcomp.

3. Results

3.1. Influence of Water Restriction on Seedling Germination and Morphology

The high concentrations of PEG tested completely inhibited germination in ornamental pepper accessions. Germinated seeds and developed seedlings were observed only in the control treatment (0% PEG) and at the 5% concentration. Therefore, in the analyses carried out in this study, only two PEG concentrations were considered.

Germination, germination speed index (GSI), length of the aerial part (AL), root length (RL), percentage of dry matter in the aerial part (ADMP) the percentage of dry matter in the root (RDMP) are parameters that explain the significant effects (p<0.05) observed between the accessions and the PEG concentrations (Table 2). The results indicate that both the accessions and the concentrations of PEG significantly influenced the evaluated parameters.

Germination was strongly influenced by the genotypic factor and the PEG concentration (5%), with the CPCE011 accession showing superior performance, reaching 100% germination, followed by CPCE018 (91.62%), CPCE005 (91.5%), and CPCE010 (83.25%), which did not differ from each other (Figure 1A). On the other hand, the CPCE007 and CPCE019 accessions showed the lowest germination rates, with only 41.25% and 33%, respectively.

The water restriction simulated by PEG reduced the germination percentage regardless of the accessions (Table 2 and Figure 1B). The average germination in the control treatment of 80.75% dropped to 67.67% at the 5% PEG concentration, a reduction of approximately 13.1%. It can be observed that water restriction influenced germination; however, it did not alter the germination speed (GSI), as no differences were observed between the PEG concentrations (Table 2). However, the effect of genotype was significant (Table 2). Accessions CPCE 018 (0.29±0.03) and CPCE 020 (0.26±0.04) showed higher GSI values compared to the other accessions, regardless of PEG concentration.

Regarding the length of the aerial part (AL), there was a significant difference between the accessions evaluated independently, with CPCE 020, CPCE 010, CPCE 011, and CPCE 018 showing, respectively, more developed seedlings, which may indicate good tolerance (Table 2). A significant interaction effect was also observed (Table 2 and Table 3). Considering the PEG concentration of 0%, once again the accessions CPCE 10, CPCE 11, CPCE 18, and CPCE 20 showed seedlings with greater length of the aerial part. Under water restriction conditions (5% PEG), the CPCE 010 and CPCE 020 accessions stood out. It is important to emphasize, however, that the CPCE 010 accession was strongly influenced by the PEG dose, with a reduction of approximately 10 mm in shoot length at the 5% concentration (Table 3 and Figure 1C). No significant reductions were observed between the two concentrations for the other accessions.

When observing root length (RL) development, the accession CPCE 005 stood out with the greatest average length (124.16±28.7), followed by accessions CPCE 007 and CPCE 010 (Table 2). On average, the water restriction imposed by PEG did not influence the root length of the seedlings.

Regarding dry matter percentage, differences were observed among the accessions (Table 2). Accessions CPCE 007, CPCE 010, CPCE 011, and CPCE 019 showed the highest average percentages. It is important to highlight accession CPCE 020, which showed seedlings with greater shoot length but low dry matter percentages. The accessions did not influence the dry matter percentage of the root. There was an effect of PEG concentrations on the shoot dry matter percentage (ADMP) and root dry matter percentage (RDMP), with higher percentages observed at the 5% PEG concentration, suggesting possible physiological adaptation mechanisms of the seedlings induced to water stress (Table 2).

3.2. Stomatal Characteristics

Significant changes were observed in the stomatal characteristics of the seedlings in response to the different accessions and PEG concentrations, and an interaction effect was observed for stomatal density, stomatal length, midpoint length, and midpoint width (Table 4).

Stomatal density varied significantly among the accessions (p < 0.001) and in interaction with PEG (p = 0.01), with the CPCE007 accession showing the highest number of stomata per area (99.9±49.32) (Table 2 and Figure 2A,B).

Considering the interaction, under the control condition (0% PEG), there was no difference in stomatal density among accessions CPCE 007, CPCE 010, and CPCE 011 (Table 5). Under water restriction (5% PEG), accession CPCE 007 does not differ from accession CPCE 011. Although the means observed are much higher for accession CPCE 007, greater data variability was observed, as reflected by the standard deviation (Figure 2A). In both conditions, accession CPCE 005 showed lower stomatal density. When comparing the effect of water restriction simulated by PEG, a reduction in stomatal density was observed for Accession CPCE 010 (Figure 2C). The stomatal density of 55.2±11.94 observed at the 0% PEG concentration decreased to 19.5±13.9 in the 5% PEG treatment. The other accessions maintained stability under both conditions.

The stomatal length (SL) and stomatal width (SW) did not show significant differences between the accessions or between PEG concentrations. However, a significant interaction between the two factors was observed for length (Table 4). The accession CPCE 007 showed a reduction in SL (25.08 ± 1.17) with increasing PEG concentration, while the accession CPCE 010 demonstrated an increase in stomatal length (30.83 ± 6.29) under stress conditions (Table 5).

The mid-point length (SML) and mid-point width (SMW) proved to be sensitive to stress, with significant effects from the interaction and access (Table 4). A reduction in mid-point length (17.90±2.59) was observed for accession CPCE 007 when subjected to water stress, while CPCE 005 and CPCE 010 stood out, with an increase in this variable under the same conditions (Table 5). Accession CPCE 011 showed a significant reduction in mid-point width under the 5% PEG concentration (Table 5), indicating a possible physiological mechanism of the seedling to minimize water loss through transpiration. For the other accessions, no significant differences were observed between the two conditions. The SL/SW ratio, which is related to the diameter/shape of the stomata, did not show significant differences in any of the factors, nor in the interaction between factors.

4. Discussion

4.1. Effect of Water Stress on Seed Germination Characteristics and Seedling Vigor

In the genus Capsicum spp., seed germination is usually evaluated after 14 days [16]. In this study, the evaluations were conducted over a period of twenty days in order to better describe the influence of PEG concentrations on the germination process. PEG concentrations above 5% completely inhibited seed germination. The stress simulated by PEG reduces the seed’s ability to absorb water due to an osmotic effect [17,18]. Keeping seeds in a non-germinated state during periods of drought can allow plants to escape dehydration, one of the strategies used by species for drought resistance [19,20]. Demir & Mavi (2008) [17] also did not observe germination of pepper seeds, at different stages of development, at the highest PEG concentrations.

The results indicated that germination, subjected to a 5% PEG concentration, was reduced to 67.67±36.92% compared to the control (80.75±26.53) (Table 2), confirming the inhibitory effect of simulated water stress on pepper seed germination (Capsicum spp.). Similar results were obtained by Gangotri et al. (2022) [6], who observed a significant reduction in the germination percentage of pepper genotypes with the increase in PEG 6000 concentrations. In the same way, Wickramasinghe & Seran (2019) [21] they evaluated drought stress tolerance in tomato seedlings, which belong to the same family as pepper plants (Solanaceae), and observed significant losses in the germination rate. However, in the present study, some pepper plant accessions maintained high germination rates when compared to the tomato seedlings (Solanum lycopersicum L.) in which germination was below 60% [21].

In this sense, accessions that maintained a high germination rate regardless of the conditions tested are important in breeding programs, as they exhibit tolerance to water restriction. The accessions CPCE 011, CPCE 005, and CPCE 018 show germination rates above 90%. An expected trait of high-quality seeds is the ability to germinate under adverse growth conditions. Rapid germination and emergence are essential for the successful establishment of crops [22].

The significant interaction for the shoot length of the seedlings reveals the differential response of the accessions (genotype effect) to environmental variations, in this study imposed by the concentrations of PEG. Only the accession CPCE 010 (Table 3) had its seedling length reduced by water restriction, demonstrating tolerance among the accessions tested. The reduction of external water potential triggers several important events in plant tissues. For example, at the macroscopic level, osmotic stress inhibits cell growth due to the decrease in turgor pressure [23], what can explain the variability between the accessions, as indicated by the standard deviations associated with the averages of the shoot length.

Root growth was not influenced by PEG concentrations. In the face of osmotic stress, plants maintain root growth and reduce shoot growth, especially in the early stages of development [24]. The optimized architecture of the root system can strengthen the properties of plants facing drought stress, including root length and number, to increase the absorption of deeper water sources, ultimately enhancing drought tolerance [25]. In this way, accessions with more developed roots may show greater tolerance under water-restricted conditions, as seen in accessions CPCE 005, CPCE 007, and CPCE 010.

In the variables of percentage of shoot dry matter (ADMP) and percentage of root dry matter (RDMP), the treatment with PEG promoted a significant increase. Demir & Mavi (2008) [17] they also observed that, in contrast to the decrease in fresh weight, the seedling’s dry weight increases as stress concentrations rise. This result suggests that although stress inhibits germination and initial growth, it may induce adaptation mechanisms at the metabolic level, such as the partitioning of biomass to the shoot and root of the seedling. Sané et al. (2021) [26] used as one of the parameters for evaluating drought stress tolerance in tomatoes (Solanum lycoperiscum L.) the accumulation of Proline by the seedlings, using the colorimetry method proposed by Monneveux & Nemmar (1986) [27]. The accumulation of this compound can be considered a plant response to water stress. Therefore, in future research, it is recommended to quantify proline in order to verify possible relationships between this biomass accumulation and the significant results observed for the percentage of dry matter.

4.2. Stomatal Characteristics in Response to Water Stress

Stomata are essential structural features of most plants; they play a crucial role in gas exchange, photosynthesis, and transpiration in plants in general [28]. Studies explore stomatal behavior in response to water stress and environmental changes [29]. Our study investigated stomatal density and other stomatal characteristics in response to water stress induced by PEG 6000 in four accessions of ornamental pepper plants. The results revealed distinct response patterns, reinforcing the variability among genotypes and the interaction with PEG concentrations simulating water stress.

Stomatal density varied significantly among the accessions (p < 0.001), with accession CPCE007 standing out compared to the others due to its higher number of stomata/area, highlighting the variability among the accessions (Figure 2). A higher stomatal density may be associated with a greater ability of the plants to capture CO₂ from the atmosphere and, consequently, increase photosynthetic efficiency [30].

Different from the results found in this work, Syafriani et al. (2024) [10] they did not observe differences in the number of stomata, but they did observe variation in length and width, emphasizing the genotypic effect as an adaptation mechanism. There was no significant effect of PEG 6000 on stomatal length (SL) or width (SW), nor on mid-point length (SML) (p > 0.05), however, an effect of the accession was observed, corroborating the results for pepper plants reported by Syafriani et al. (2024) [10]. The midpoint width (SMW) was significantly reduced by the application of 5% PEG (p = 0.04). This response is consistent with a plant defense mechanism under water deficit stress conditions, through the closing or fine adjustment of stomatal opening [28]. The reduction in SMW can decrease water loss through transpiration, representing an important stress tolerance strategy.

The significant interaction between accession and PEG concentration for SL (p = 0.03), SML (p < 0.001), and SMW (p = 0.04) reinforces that the response of stomatal morphology to water stress depends on the genotype. This genetic variability is valuable for breeding programs, as genotypes capable of maintaining functional stomata under stress may exhibit better physiological performance under drought conditions. The SL/SW ratio, which is associated with stomatal shape, was not significantly affected by any of the factors studied (p > 0.05), suggesting that stomatal shape is a stable structural characteristic in pepper plants, less susceptible to changes resulting from water stress. Higher values of this ratio indicate greater stomatal functionality due to their ellipsoid shape [30].

5. Conclusions

In this study, water stress induced by PEG significantly impacted germination, the percentage of shoot and root dry matter, and the width of the stomata mid-point. The accessions CPCE 020, CPCE 018, and CPCE 011 exhibited more characteristics of tolerance to water stress, demonstrating great potential for selection in breeding programs. CPCE 019 was the most susceptible to water stress. The results of this work can guide breeding programs for the selection and improvement of tolerant genotypes, aiming at the development of ornamental pepper varieties. It is suggested that further studies be conducted to evaluate the performance of these accessions under greenhouse conditions.