Introduction

Selecting suitable plants for dry and semi-arid regions is of utmost importance because water scarcity is one of the most significant environmental threats currently facing the global population. Plants use a wide range of physiological strategies to maintain crop productivity in water-limited environments. Therefore, understanding physiological mechanisms is essential for increased drought tolerance and ensuring food security in the face of climate change1,2.

Photosynthesis, the most fundamental and complex physiological process, is vital for all green plants. Drought stress can significantly affect the physiology of plant photosynthesis, leading to leaf yellowing, stomatal closure, and weakened photosynthesis1. The reduction in photosynthetic capacity caused by drought stress not only disrupts plant growth but is also directly or indirectly associated with reduced yield and quality of medicinal plants. Therefore, photosynthesis is a widely used physiological indicator for evaluating the impact of drought stress on medicinal plant quality1. Osmotic regulation, involving the accumulation of organic and inorganic solutes to absorb water and maintain cellular turgor potential, is another critical response to drought stress. These solutes, such as proteins, proline, and sugars, act as osmotic regulators, enhancing the ability of roots to absorb water, maintaining cell turgor, improving stomatal conductance, preventing membrane damage, and scavenging free radicals2.

Plants require an active antioxidant system to counteract oxidative damage under drought stress conditions. The enzymatic antioxidant system, particularly peroxidases and ascorbate peroxidases, is rapidly activated in response to drought stress and helps eliminate excess hydrogen peroxide and prevent lipid peroxidation2. High expression of these antioxidant enzymes, such as isoforms of glutathione peroxidase, can enhance the antioxidant system’s capacity to neutralize reactive oxygen species and improve drought tolerance in plants1,2. Thus, a high level of antioxidant enzyme activity is vital for drought tolerance and ensuring desirable quality.

Recent advancements in drought tolerance in cereal crops, such as maize and wheat, provide valuable insights into improving resilience in crops facing water scarcity. For instance, genomics-assisted breeding and molecular tools have facilitated the development of drought-tolerant cultivars with enhanced yield stability and physiological traits, such as osmotic adjustment and antioxidant enzyme activities1,2. These approaches have demonstrated that improving drought tolerance involves complex traits governed by a combination of genetic and physiological mechanisms, including reactive oxygen species (ROS) scavenging and metabolic adjustments2. Integrating such strategies into breeding programs can inform efforts to develop drought-resilient varieties in medicinal plants like cumin, which are critical for agriculture in arid and semi-arid regions.

Cumin (Cuminum cyminumL.) is the second most popular seed species after black pepper and has a wide range of applications in the beverage, food, alcoholic beverage, pharmaceutical, perfume, and cosmetic industries3. This annual herbaceous plant from the Apiaceae family is widely cultivated in dry and semi-arid regions of the world, such as China, Egypt, Saudi Arabia, the Mediterranean region, India, and Iran, due to its short growth period, low water requirements, fewer pests, and high economic value4.

Cumin is a self-pollinated and self-compatible plant, which raises doubts about hybrid production. Therefore, the primary breeding method, similar to other self-pollinated medicinal plants, involves creating synthetic varieties through the crossbreeding of superior genotypes as parents. This process utilizes heterosis to improve seed yield performance and drought tolerance in cumin5. This method has also been applied to medicinal plants such as Eastern wormwood (Artemisia scoparia), red sage (Salvia miltiorrhiza), motherwort (Leonurus japonicus), caraway (Carum carvi), and French lavender (Lavandula angustifolia)6,7. Additionally, successful results have been achieved in breeding fennel (Foeniculum vulgareMill.), one of the oldest medicinal plants in the Apiaceae family, through the creation of synthetic varieties for drought tolerance. Under drought stress conditions, the synthetic varieties exhibited higher seed yield and essential oil content compared to the parental genotypes8.

Synthetic varieties, due to their wider genetic diversity and superior performance under drought stress conditions, can provide the necessary foundation for developing drought-tolerant commercial varieties9. Drought tolerance is a complex trait involving a combination of morphological and physiological characteristics such as relative leaf water content, relative water loss, proline and abscisic acid accumulation, chlorophyll fluorescence, osmotic regulation, root size, and stomatal conductance9. Physiological traits are crucial for the survival and adaptation of plants to environmental stresses, and the quantitative and qualitative formation of medicinal plants is directly or indirectly influenced by their physiological status.

Loss of genetic diversity can potentially limit the ability of agricultural systems to withstand drought stress. Therefore, understanding the level of diversity and genetic distance between genotypes and synthetic varieties is crucial in plant breeding10. Phenotypic traits (morphological and physiological traits) are commonly used to estimate genetic diversity because they are easy to measure. However, assessing diversity based solely on these traits may not be completely reliable as they are limited and influenced by the environment11. Therefore, complementing phenotypic assessments with molecular markers, which provide a more direct measure of genetic variation, is often recommended to obtain a more comprehensive understanding of genetic diversity. ISSR molecular markers are PCR-based markers that can quickly distinguish genetic differences between individuals and have been proven useful in genome fingerprinting studies, phylogenetic analyses, and gene tagging. This technique uses a single primer designed based on a microsatellite sequence with 2 to 4 additional nucleotides added at the 5’ or 3’ end and does not require prior knowledge of the genome for primer design. ISSR markers are useful for detecting genetic polymorphisms by generating a large number of microsatellite markers distributed throughout the genome12.

Since no study has been conducted on the physiological and genetic aspects of synthetic cumin variety and its potential for enzymatic changes under drought stress, this study aims to compare the resistance potential of the synthetic cumin variety and its parental genotypes to drought stress. It also seeks to evaluate the physiological indicators and enzymatic antioxidant activity under drought stress to assess the synthetic cumin variety as a potential genetic resource for drought tolerance.

Results

The combined analysis of variance revealed significant effects of genotype and irrigation conditions on all measured traits (Table 1). However, interactions between year and irrigation conditions, as well as between genotype and year, were not significant, suggesting consistent trait variation among genotypes across different years. Consequently, the average of three years was used for all statistical and genetic calculations. Seed yield and photosynthetic pigments were more influenced by environmental conditions, showing higher coefficients of variation compared to other traits.

Table 1 Combined analysis of variance for Cumin under normal irrigation and drought stress conditions over three growing seasons. **, Ns: significant at 1% probability level and non-significant at 5% probability level, respectively.
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Seed yield

Under normal irrigation and drought stress conditions, the seed yield for the synthetic variety was 207.30 g/m² and 172.38 g/m², respectively. In contrast, for the parental genotypes, it was 155.55 g/m² and 100.46 g/m², respectively (Table 2). The synthetic variety showed superior seed yield, outperforming parental genotypes by 33.27% under normal irrigation and 71.59% under drought stress conditions. Although drought stress reduced seed yield in both the synthetic variety and parental genotypes, the reduction was significantly less for the synthetic variety (16.85%) compared to the parental genotypes (35.42%).

Table 2 Mean traits studied in the synthetic variety (SYN) and parental genotypes, percentage change of mean traits under drought stress compared to normal conditions, and percentage superiority of the synthetic variety (SYN) over parental genotypes.
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Photosynthetic pigment content

Water deficit caused a significant decrease in chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents. In the mean values of parental genotypes, these decreases were observed from 7.59, 3.52, 11.11, and 13.57 to 5.19, 2.77, 7.96, and 8.74, respectively. While in the synthetic variety, the reductions were from 10.40, 3.65, 14.01, and 16.57 to 7.61, 2.26, 9.87, and 14.37, respectively (Table 2). The impact of drought stress on photosynthetic pigment content exhibited a distinct pattern between the parental genotypes and the synthetic variety. Carotenoids in the parental genotypes showed a 35.62% reduction, while chlorophyll b in the synthetic variety demonstrated a 38.18% decrease under drought stress. Regardless of the irrigation conditions, the synthetic variety consistently exhibited higher photosynthetic pigment content compared to the parental genotypes. Under normal irrigation, the synthetic variety showed increases of 36.95%, 3.70%, 26.04%, and 22.05% in chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, respectively, compared to the parental genotypes. Under drought stress, these increases were 46.53%, −18.43%, 24.03%, and 64.42%, respectively.

Physiological datasets measured in the present study provide important insight into the mechanisms underlying drought tolerance in synthetic varieties of cumin. Photosynthetic pigments, such as chlorophyll a, chlorophyll b, and carotenoids, are a reflection of the plant’s capacity to maintain photosynthesis under stress conditions. The values of the above-mentioned parameters were significantly higher in the synthetic variety both in normal and in drought conditions, which means that it had higher efficiency in photosynthetic processes and maintained higher energy production.

Osmoregulators (proline, protein, and sugar)

Drought stress significantly affected the levels of osmotic regulators, resulting in increased proline and sugar contents, and decreased protein content (Table 2). Notably, the synthetic variety exhibited higher levels of osmotic regulators under both irrigation conditions compared to the parental genotypes. Under water deficit condition, the proline content in the mean of the parental genotypes and the synthetic variety was 3.10 and 4.73, respectively, representing increases of 71.77% and 78.45% compared to normal irrigation condition. While the percentage increase in soluble sugar under drought stress was higher in the mean of the parental genotypes (40.55%) than in the synthetic variety (8.51%), the absolute levels of soluble sugar were higher in the synthetic variety (1.24 under normal and 1.35 under drought conditions) compared to the parental genotypes (0.68 under normal and 0.95 under drought conditions). Soluble protein content ranged from 0.74 to 0.92 under normal irrigation and from 0.46 to 0.48 under drought stress. The reduction in soluble protein content under drought stress was less pronounced in the parental genotypes (37.63%) than in the synthetic variety (48.04%).

Osmoregulators, such as proline and soluble sugars, play a key role in maintaining cellular turgor and protecting against dehydration. Under drought stress, the synthetic variety exhibited a 78.45% increase in proline content compared to a 71.77% increase in the parental genotypes. This suggests enhanced osmotic adjustment and water uptake efficiency, contributing to the synthetic variety’s ability to withstand water-deficient conditions.

Antioxidant enzymes

Drought stress enhanced the activities of catalase, peroxidase, and ascorbate peroxidase enzymes. In the synthetic variety, their levels increased from 8.70, 7.41, and 8.20 to 16.11, 14.52, and 15.11, respectively. In the mean of the parental genotypes, these enzymes’ levels rose from 2.29, 2.19, and 2.04 to 6.95, 6.54, and 4.14, respectively (Table 2). The synthetic variety’s higher antioxidant enzyme activities resulted in a reduction of the negative impacts of drought stress by 58.12% for catalase, 51.72% for peroxidase, and 18.23% for ascorbate peroxidase, and increased the rate of free radical scavenging compared to the parental genotypes. Under normal irrigation conditions, the activity levels of catalase, peroxidase, and ascorbate peroxidase in the synthetic variety were elevated by 279.85%, 238.46%, and 301.97%, respectively, compared to the parental genotypes. Under drought stress conditions, these increases were 131.81%, 122.05%, and 264.78%, respectively.

Antioxidant enzyme activity, including catalase, peroxidase, and ascorbate peroxidase, provides a defense against oxidative damage caused by drought-induced ROS. The synthetic variety showed a 131.81% increase in catalase activity under drought stress compared to a 203.60% increase in the parental genotypes. This indicates a more efficient ROS scavenging system in the synthetic variety, minimizing cellular damage and enhancing stress resilience.

Essential oil content and composition

The synthetic variety demonstrated the highest essential oil content under both normal irrigation (3.07%) and drought stress (3.27%) conditions. Its superiority over the parental genotypes was 155.39% under normal irrigation and 95.02% under drought stress. Drought stress increased essential oil content by 39.27% in the parental genotypes and by 6.35% in the synthetic variety (Table 2).

Table 3 presents the identification results of volatile compounds in cumin essential oil. Eight chemical compounds were identified, constituting over 85% of the total essential oil composition. Under drought stress, the synthetic variety showed increased levels of cumin aldehyde, cis-beta-Farnesene, (+)-Acoradiene, carotol, and phthalic acid. In contrast, the parental genotypes exhibited increased levels of (+)-Acoradiene, carotol, and phthalic acid under drought stress. Cumin aldehyde was the major component of cumin essential oil, with its levels in the synthetic variety improving by 9.87% under normal conditions and 21.85% under drought stress compared to the mean of the parental genotypes. Under drought stress, the synthetic variety surpassed the parental genotypes in all compounds except p-Cymene and gamma-Terpinene. Beta-Pinene showed the highest percentage increase in the synthetic variety compared to the parental genotypes under both normal irrigation (118%) and drought stress (88.31%) conditions.

Table 3 Essential oil composition of the synthetic variety (SYN) and parental genotypes of Cumin, percentage change of mean traits under drought stress compared to normal conditions, and percentage superiority of the synthetic variety (SYN) over parental genotypes.
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Molecular evaluation using ISSR markers

The genetic diversity of the parental genotypes and the synthetic cumin variety was assessed using 10 ISSR primers (Table 4). The ISSR primers collectively produced 56 bands, of which 11 were monomorphic and the remainder polymorphic. Each primer produced an average of 5.1 bands across the parental genotypes and the synthetic variety. Primers ISSR_03 and ISSR_06 generated the most bands (7 each), whereas primers ISSR_04 and ISSR_08 produced the fewest bands (3 each).

Table 4 Number of amplified loci, number of polymorphic loci, percentage of polymorphism, PIC, MI, and EMR in the primers used. Y=(C, T), V=(A, C,G), D=(A, G,T), B=(C, G,T).
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The average polymorphism percentage among the parental genotypes and the synthetic cumin variety was 87.90%. Specifically, the polymorphism percentages for primers ISSR_01, ISSR_02, ISSR_03, ISSR_04, and ISSR_10 were 66.67%, 80.00%, 85.71%, 66.67%, and 80.00%, respectively, with the other primers showing 100% polymorphism. The average polymorphic information content (PIC) for the primers was 0.28. The highest PIC values were recorded for primers ISSR_06 (0.41) and ISSR_05 (0.38), indicating their superior ability to resolve genetic distances between the parental genotypes and the synthetic variety. Conversely, primer ISSR_04 had the lowest PIC value (0.18), suggesting its limited discriminatory power.

Furthermore, the mean marker index (MI) and effective multiplex ratio (EMR) were 1.37 and 4.51, respectively. Primers ISSR_06 and ISSR_05 exhibited the highest MI and EMR values, while primer ISSR_04 had the lowest values, reflecting its reduced effectiveness in genetic differentiation.

Multivariate statistical analyses

To categorize the parental genotypes and the synthetic cumin variety, cluster analysis was employed using the Ward method and the squared Euclidean distance similarity coefficient for physiological data, as well as the UPGMA method with the Jaccard similarity coefficient for molecular data. The cluster analysis of both data sets clearly separated the synthetic variety from the parental genotypes (Fig. 1).

Fig. 1
figure 1

Dendrogram of the synthetic variety and parental genotypes. (a) Physiological data and (b) Molecular data. (The synthetic variety is shown with SYN and the parental genotypes are shown from P1 to P8).

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Principal coordinates analysis (PCoA) was performed to further explore and delineate the relationships between the parental genotypes and the synthetic variety based on physiological and molecular data. The analysis revealed that the first two principal components explained 89.48% of the variance in physiological data and 49.89% of the variance in molecular data between the parental genotypes and the synthetic variety. The three-dimensional scatter plot, derived from the first two principal components, confirmed the cluster analysis groupings (Fig. 2).

Fig. 2
figure 2

Biplot of the synthetic variety population and parental genotypes. (a) Physiological data and (b) Molecular data. (The synthetic variety is shown with SYN and the parental genotypes are shown from P1 to P8).

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Discussion

Drought stress exerts numerous adverse effects on plants, including the inhibition of photosynthesis and growth, as well as impacts on osmotic regulators and enzymatic activity. Additionally, severe drought can result in reduced crop yields and plant1,2. Developing and cultivating drought-tolerant varieties is a strategic approach to mitigate the adverse effects of drought stress on plants. The synthetic cumin variety developed in this study showed improved drought tolerance and seed yield compared to the parental genotypes. These improvements are attributed to increased photosynthetic pigment retention, higher antioxidant enzyme activity, and greater osmoregulatory compound accumulation. These findings suggest that the synthetic variety represents an excellent genetic resource for agricultural production under arid and semi-arid conditions (Fig. 3).

Fig. 3
figure 3

Hypothetical mechanistic model illustrating drought tolerance in cumin. Under drought conditions, reduced CO₂ availability and elevated oxidative stress (via reactive oxygen species, ROS) diminish photosynthetic pigments and impair photosynthesis. In response, cumin increases osmotic regulators (proline, sugars), antioxidant enzymes (catalase, peroxidase, ascorbate peroxidase), and essential oil production—including the main active component, cumin aldehyde—to mitigate stress and enhance drought tolerance. In the figure, (−) indicates reduction while (+) indicates increase of the respective parameters.

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In cumin, seed yield is the most critical and economically relevant trait. It is controlled by numerous physiological and morphological processes, environmental conditions, and the plant’s genetic structure19. Improved seed yield under both normal and drought conditions demonstrates the genetic advantage of the synthetic variety. Compared to parental genotypes, the synthetic variety achieved a yield increase of up to 71.59% under drought stress, supported by higher photosynthetic pigment retention and osmoregulatory efficiency observed in this study. This highlights the role of physiological traits as key factors in drought resilience.

The findings are consistent with earlier studies on medicinal plants and their drought tolerance. Drought stress significantly decreased seed yield by impacting various traits, aligning with the findings of Noori et al.20, and Yadav and Yadav21in cumin. Factors contributing to yield reduction under drought stress include reduced carbon metabolism, stomatal conductance, and water uptake due to impaired root growth30. Under optimal irrigation, increased photosynthesis and assimilate production enhance the seed filling rate, seed weight, and ultimately yield. Drought stress leads to reduced leaf water content, stomatal closure, and decreased photosynthesis, along with impaired enzymatic activities, resulting in lower seed yield. Additionally, drought stress causes flower drop and reduced seed weight, further diminishing seed yield1. The observed increase in antioxidant enzyme activities (e.g., catalase, peroxidase, and ascorbate peroxidase) likely contributed to the synthetic variety’s ability to detoxify reactive oxygen species under drought conditions, protecting cellular structures and maintaining physiological processes. Additionally, higher proline and soluble sugar levels in the synthetic variety supported osmotic adjustment, improving water uptake and turgor maintenance.

One of the primary breeding goals for cumin is to increase seed yield, which can be achieved through the development of synthetic varieties. The synthetic cumin variety exhibited the highest seed yield under both stress and non-stress conditions (Table 2). Furthermore, this variety was less negatively affected by drought stress compared to the parental genotypes. The enhanced seed yield of the synthetic variety over the parental genotypes can be attributed to increased genetic diversity and recombination22. The combination of genes from the parental genotypes provides a broader genetic base and potential for new gene combinations in the synthetic variety, increasing yield potential, especially under adverse environmental conditions23. This enhances the suitability of the synthetic variety for cultivation in arid and semi-arid regions. Significant seed yield increases through the development of synthetic varieties have also been reported by other researchers23.

Photosynthetic pigment content is a crucial and straightforward physiological indicator for assessing drought stress impact and determining drought tolerance. Chlorophyll, located within the thylakoid membrane, is essential for photosynthesis, and drought stress triggers the rapid translocation of nitrogen and carbohydrates from vegetative organs to seeds, accelerating their maturation2. According to Table 2, drought stress resulted in a significant reduction in photosynthetic pigment content. Similar reductions in cumin under drought conditions have been documented by Noori et al.20, Timachi et al.24, and Pishva et al.25. Thus, photosynthetic pigment content can be effectively used alongside seed yield to screen for drought tolerance. In this study, the synthetic variety achieved the highest seed yield under both normal and drought stress conditions (Table 2). Additionally, the synthetic variety showed the least reduction in chlorophyll a and carotenoid content under drought stress, maintaining higher levels of photosynthetic pigments and carotenoids than the parental genotypes. This indicates that the synthetic variety sustains higher net photosynthesis and water content, showcasing greater drought tolerance compared to the parental genotypes. The reduced decline in photosynthetic pigment levels in the synthetic variety under drought stress optimizes the expression of stress-related genes, which plays a critical role in enhancing the plant’s resilience to drought. This presents a promising approach for improving agricultural sustainability in water-scarce environments.

The initial physiological response of plants to drought stress involves osmotic regulation and stomatal closure, which are crucial for maintaining cellular moisture and enhancing water uptake from the environment. This process helps preserve normal physiological and biochemical functions within cells20. Osmotic regulation, by generating a negative osmotic potential within cells, enhances the plant’s ability to absorb water from the soil, thereby improving its drought tolerance. In our study, the levels of three osmotic regulators proline, sugar, and protein exhibited differential changes under drought stress (Table 2). Both proline and sugar levels increased, with the highest accumulation observed in the synthetic cumin variety, suggesting its superior drought tolerance.

Proline functions as an antioxidant, maintaining cellular redox balance, reducing lipid peroxidation, and inducing the expression of stress-responsive genes while activating antioxidant enzymes1. Soluble sugars are vital for cellular biosynthesis and metabolism, serving as essential energy sources during developmental processes. Under drought conditions, soluble sugars aid in osmotic regulation, reduce water potential, and protect cells from oxidative damage by stabilizing cellular membranes and proteins1.

Drought stress led to a reduction in soluble protein concentration in the leaves. Nevertheless, the synthetic variety exhibited higher protein levels under both normal and drought conditions. This reduction in protein concentration can be attributed to decreased protein synthesis or increased proteolysis due to heightened protease activity under drought conditions26. Drought stress enhances protease activity, resulting in protein degradation and an increase in free amino acids, including proline. The activation of protease-encoding genes promotes protein degradation and the synthesis of compatible solutes26.

Previous studies on cumin have similarly reported an accumulation of proline and sugar, along with a decrease in protein content under drought stress, aligning with our findings24,25. Therefore, it can be concluded that the higher accumulation of proline and soluble sugars and the reduction in protein content in the synthetic variety, compared to the parental genotypes, significantly contribute to the increased drought tolerance of cumin. This adaptation facilitates improved physiological activity and reduced membrane lipid peroxidation, offering a promising strategy for enhancing agricultural sustainability in water-limited environments.

Drought stress led to an increase in the essential oil content of cumin (Table 2). This observation aligns with the findings of previous studies, which reported that drought stress enhances the essential oil percentage in cumin19. Medicinal plants tend to produce more essential oils under drought conditions as a defensive mechanism to prevent cellular oxidation19. Our study showed that the synthetic variety had the highest essential oil content under both normal and drought stress conditions, suggesting improved drought tolerance.

The chemical composition of essential oils is influenced by the plant’s genetic makeup and environmental conditions27. Analysis of the chemical constituents in cumin essential oil indicated that the synthetic variety had higher levels of the primary active ingredient compared to the parental genotypes under both normal and drought stress conditions (Table 3). Although the changes in essential oil components due to drought stress did not follow a consistent trend, the combination of superior parental genotypes in the synthetic variety resulted in increased levels of most compounds, particularly under drought stress, compared to the parental genotypes.

This enhanced chemical profile under stress conditions indicates that the synthetic variety is more adept at producing valuable metabolites, which may contribute to its superior drought tolerance. This finding underscores the potential of using synthetic varieties to improve the resilience and productivity of crops in water-limited environments.

The antioxidant system in plants is composed of enzymatic and non-enzymatic antioxidants28. Enzymatic antioxidants are pivotal in neutralizing free radicals and maintaining the balance of ROS within plant cells2. In this study, drought stress enhanced the activities of antioxidant enzymes, including ascorbate peroxidase, peroxidase, and catalase (Table 2). The synthetic variety demonstrated the highest antioxidant activity under drought condition. Elevated antioxidant enzyme activity strengthens the plant’s physiological and biochemical defense mechanisms against drought, aiding in the maintenance of cellular water content and reducing drought-induced damage.

Under drought stress, the synthetic variety exhibited increases of 131.81%, 122.05%, and 264.78% in catalase, peroxidase, and ascorbate peroxidase activities, respectively, compared to the average of the parental genotypes (Table 2). Catalase, an iron-containing enzyme, is crucial for the detoxification of hydrogen peroxide29. Enhanced catalase activity in response to drought stress has also been observed in plants by other researchers1,2.

Peroxidase aids in eliminating ROS to prevent excessive plasma membrane damage, with malondialdehyde serving as an indicator of lipid peroxidation due to ROS damage. Peroxidase helps remove hydrogen peroxide and malondialdehyde, preserving cellular membrane integrity. In this study, drought stress resulted in a twofold increase in peroxidase activity. Similar increases in peroxidase activity due to drought stress have been observed in various other plants1,2. Ascorbate peroxidase reduces oxidative stress damage by scavenging free radicals, particularly hydrogen peroxide. The primary detoxification system for hydrogen peroxide in plant chloroplasts involves the ascorbate-glutathione cycle, where ascorbate peroxidase is a key enzyme. It catalyzes the conversion of hydrogen peroxide to water using ascorbate as an electron donor28. Overexpression of ascorbate peroxidase in various plant species underscores its critical role in defending plants against abiotic stress1,2. Thus, the increased antioxidant enzyme levels in the synthetic cumin variety suggest enhanced tolerance to oxidative stress and better adaptability to environmental challenges.

Recent studies on drought-tolerant cereal crops have emphasized the critical role of enzymatic antioxidant systems in improving drought resilience. For instance, Reza et al.1 reported that improved catalase and peroxidase activities in wheat (Triticum boeoticum) significantly reduced ROS damage under drought conditions, leading to enhanced yield stability. Similarly, Bhardwaj et al.2 demonstrated that ion homeostasis and mesophyll regulation in barley play vital roles in maintaining cellular integrity and photosynthetic activity during water stress. These findings align with our results, where catalase, peroxidase, and ascorbate peroxidase activities increased significantly in the synthetic cumin variety under drought conditions. The universal relevance of these mechanisms across plant species underscores their importance in developing drought-tolerant varieties.

A comprehensive understanding of genetic diversity is crucial for breeding programs aimed at exploiting heterosis and increasing crop resilience. ISSR primers revealed substantial genetic diversity between the synthetic cumin variety and the parental genotypes, confirming the findings of Ebrahimiyan et al.30 on the high genetic diversity of Iranian cumin genotypes. Primers ISSR_06 and ISSR_05, which demonstrated high diversity according to PIC, EMR, and MI indices, are particularly useful for analyzing the subsequent generations of the synthetic variety (Table 4).

Cluster analysis of physiological and molecular data revealed that the parental genotypes and the synthetic variety were grouped into two distinct clusters (Fig. 1). The cluster analysis results were consistent with the PCoA diagram, which illustrated the distribution of the parental genotypes and the synthetic variety (Fig. 2). The greater genetic distance observed during the random crossing of parental genotypes in the polycross test for producing the synthetic variety increased the number of heterozygous loci and the potential for heterosis. Moreover, genetic distance might be associated with desirable traits31. Therefore, it can be concluded that the parental genotypes were aptly selected, facilitating the transfer of superior genes to the synthetic variety.

The findings of this study demonstrate that the synthetic cumin variety exhibits significantly enhanced seed yield and drought tolerance compared to parental genotypes. This improvement is attributed to a combination of physiological adaptations, such as increased antioxidant enzyme activity and osmoregulatory compound accumulation, as well as the broader genetic base achieved through synthetic variety breeding. These traits are not only pivotal for the resilience of cumin but can also serve as a model for improving other medicinal and aromatic crops cultivated in arid and semi-arid regions.

The breeding strategy employed here could be applied to other cross-pollinated crops where hybridization is less feasible, providing a promising approach for improving seed yield and stress tolerance. Additionally, the integration of physiological, biochemical, and molecular markers demonstrated in this study can be adopted in breeding programs targeting climate-resilient crops.

Beyond cumin, these findings hold relevance for addressing global food security challenges, particularly in regions experiencing increasing water scarcity due to climate change. Developing drought-tolerant varieties of crops with low water requirements will contribute to sustainable agricultural practices and ensure the productivity of economically valuable plants under adverse conditions.

While this study provides a comprehensive evaluation of the synthetic cumin variety under drought stress, further investigations are warranted to fully exploit its potential. Future research should focus on unraveling the molecular pathways and gene networks that confer drought tolerance, which could lead to the identification of novel genetic markers for marker-assisted selection. Additionally, multi-location and multi-year trials are essential to confirm the stability and adaptability of the synthetic variety across diverse agro-ecological zones. Moreover, investigating the effects of other abiotic stresses, such as salinity or extreme temperatures, on the synthetic variety could expand its utility and relevance in stress-prone regions, ensuring broader applications in sustainable agriculture.

Materials and methods

Plant materials

Breeding for drought tolerance in cumin relies on two major factors: the presence and accessibility of genetic diversity in terms of tolerance, and the utilization of this genetic diversity through the screening and selection of superior genotypes in stress environments32. Previous studies have reported the presence of these factors for drought tolerance in cumin genotypes16,19. Consequently, a genetic pool was established by randomly crossing superior genotypes in a polycross scheme. Genotype selection was based on performance traits, late-season drought tolerance, and general combining ability. The resulting seeds from eight cumin genotypes (YAR1, YSA2, GJA3, KBA4, KRA5, SKD6, SSH7, and NKM8) involved in the polycross treasury were mixed equally to create the initial synthetic variety. The synthetic variety and parental genotypes were evaluated over three growing seasons under normal irrigation conditions and late-season drought stress. The parental cumin seeds used in this study were sourced from the gene bank of the College of Aburaihan, University of Tehran. All experimental procedures involving plants complied with relevant institutional, national, and international guidelines and legislation.

Field experiments and data collection

The field trials were conducted using a randomized complete block design (RCBD) with three replications. The experiment was carried out over three cropping seasons (December to May) at the research field of the College of Aburaihan, University of Tehran, Tehran, Iran (35°29’N, 51°40’E, elevation 1027 m). The treatments included two irrigation conditions (normal irrigation and drought stress) and nine genotypes (eight parental genotypes and one synthetic variety). Each plot consisted of four rows, 1 m in length, with 25 cm spacing between rows, resulting in approximately 120 plants per square meter. Randomization of plots was achieved using standard randomization procedures to minimize environmental effects.

Severe drought stress was applied at the flowering stage by withholding irrigation until 70% of the available soil moisture was depleted (30% of field capacity), while normal irrigation was maintained at field capacity19. The soil texture in both normal irrigation and drought stress sites was loamy clay and to determine soil moisture, we used the field capacity (FC) and wilting point (WP) method.

Soil moisture was measured using standard gravimetric methods at cumin root zone depth (0–20 centimeters) using a soil auger33. Irrigation depth was calculated as follows:

$${\rm I = [(FC-\theta)/100]\:\times D \times B}$$

Soil moisture content (SMC) was measured during the experiment using the FC and WP method. To determine FC, we collect a soil sample, weigh it (wet weight), then dry it in an oven at 105 °C until it reaches constant weight (dry weight). The FC was calculated using the formula:

$${\rm FC = (Wet\: Weight-Dry\: Weight)/Dry\: Weight}$$

The available water capacity is the difference between FC and WP. Water was delivered using a drip irrigation system, and the volume was measured.

Extraction and measurement of photosynthetic pigments

Fresh leaves (100 mg) were ground in 80% (v/v) acetone (Sigma, St. Louis, Missouri, USA) at 4 °C. The homogenate was centrifuged at 10,750 g for 15 min at 4 °C to remove cellular debris. The supernatant’s absorbance was recorded at 665, 647, and 461 nm using a UV-Visible spectrophotometer (SpectramaxPlus 384, Molecular Devices, USA). Chlorophyll a, chlorophyll b, total chlorophyll (chlorophyll a + chlorophyll b), and carotenoids were measured34.

Extraction and quantification of proline

Proline content in leaf samples was extracted using 3% (w/v) sulfosalicylic acid (Sigma, St. Louis, Missouri, USA) and determined with ninhydrin reagent (Sigma, St. Louis, Missouri, USA)34. Leaf tissue was homogenized in 2 mL of sulfosalicylic acid and centrifuged at 10,750 g for 15 min at 4 °C. The supernatant (1 mL) was mixed with 1 mL of ninhydrin and acetic acid, incubated at 98 °C for 1 h, cooled on ice, and absorbance at 520 nm was recorded using a spectrophotometer (SpectramaxPlus 384, Molecular Devices, USA). Total proline content was estimated using a standard curve.

Total soluble sugars

Total soluble sugars were extracted from homogenized aerial organ samples in 80% (v/v) ethanol and determined using anthrone reagent (Sigma, St. Louis, Missouri, USA) (0.2% w/v in concentrated H₂SO₄) and a glucose standard curve (0–1000mg/L)34. The ethanol extract (0.25 mL) was mixed with 3 mL of anthrone reagent, incubated at 100 °C for 10 min, cooled to room temperature, and absorbance at 630 nm was recorded using a spectrophotometer (SpectramaxPlus 384, Molecular Devices, USA) to estimate the sugar content.

Extraction of enzymes and measurement of enzyme activity

Leaf samples (control and drought-treated) were homogenized in liquid nitrogen and mixed with extraction buffer containing 50 mM phosphate buffer (pH 7.0) and 0.5 mM EDTA (Sigma, St. Louis, Missouri, USA). The homogenate was centrifuged at 10,750 g for 15 min at 4 °C, and the supernatant was collected for enzyme activity assays of peroxidase, ascorbate peroxidase, catalase, and protein.

The peroxidase activity was measured using a reaction mixture of phosphate buffer, H₂O₂, guaiacol, and enzyme extract, with absorbance recorded at 470 nm after incubation at 34 °C for 3 min35. Ascorbate peroxidase activity was measured in a mixture of phosphate buffer, EDTA, ascorbate, H₂O₂, and enzyme extract, with absorbance at 290 nm36. Catalase activity was measured using phosphate buffer, H₂O₂, and enzyme extract, with absorbance at 240 nm37. The content of photosynthetic pigments was measured at different wavelengths using a spectrophotometer (SpectramaxPlus 384, Molecular Devices, USA).

Soluble protein

Total protein content was measured using the Bradford method with Coomassie Brilliant Blue G-250 dye (Sigma, St. Louis, Missouri, USA) in 95% ethanol and orthophosphoric acid (Sigma, St. Louis, Missouri, USA) using a spectrophotometer (SpectramaxPlus 384, Molecular Devices, USA)38. Bovine serum albumin (BSA) (Sigma, St. Louis, Missouri, USA) was used to construct the standard curve.

Essential oil content and composition

Essential oil was extracted by water distillation using a Clevenger apparatus from seed samples for 3.5 h, resulting in pale yellow oil39. The composition was analyzed by gas chromatography (Agilent 7890 A) with a flame ionization detector and mass spectrometry (Agilent 5975 C) using an HP Innowax Capillary column. The injection volume and temperature were set at 1 µL and 250 °C, respectively. Helium was the carrier gas at a constant flow of 0.8 mL/min. The oven temperature program was: 60 °C for 10 min, then 4 °C/min to 250 °C, held for 10 min. The mass spectrum was monitored from 35 to 450 amu using electron impact ionization at 70 eV40.

Molecular evaluation using ISSR markers

DNA extraction from seed samples was performed using a modified CTAB method41. DNA quality and quantity were assessed using a nanodrop and 1% agarose gel, then diluted to 30 ng/µL for PCR. The ISSR primers used in this study (Table 4) were synthesized by Metabion company (Munich, Germany). PCR amplification involved 1 µL DNA (25 ng/µL), 1.25 µL primer (1 µl), and 12.5 µL of 2x master mix (1.5 mM MgCl₂, 4.0 mM dNTPs, 2.0 units/µL Taq DNA polymerase) in a 25 µL reaction volume. PCR conditions were: initial denaturation at 94 °C for 5 min, followed by 40 cycles of 94 °C for 1 min, primer-specific annealing for 2 min, extension at 72 °C for 2 min, and final extension at 72 °C for 5 min. PCR products were separated on 2% agarose gel in TAE buffer for 3 h at 100 volts, visualized, and scored after ethidium bromide staining.

Statistical and molecular analysis

Prior to ANOVA, the Kolmogorov-Smirnov test assessed error normality, and Bartlett’s test checked residual variance homogeneity among environments. Combined ANOVA using SPSS version 26 examined differences among breeding populations, years, Irrigation conditions, and their interactions. The PIC was calculated as:

$${\rm PIC}= 1-\sum {\rm PI^2}$$

where P is the sum of band numbers for all genotypes42. MI was calculated as:

$${\rm MI = PIC \times N \times \beta }$$

where N is the total number of bands, and β is the polymorphism ratio for each primer42. EMR was calculated as:

$${\rm EMR = NPB \times \beta}$$

where NPB is the number of polymorphic bands and β is the polymorphism percentage42. Cluster analysis, principal coordinate analysis, and similarity matrix analysis were performed using SPSS version 26 and PAST software.

Conclusion

Under drought stress conditions, ROS have the potential to react with many cellular components, causing damage to membranes and other essential macromolecules such as photosynthetic pigments, proteins, nucleic acids, and lipids. Therefore, their levels must be controlled within the cell. Plants employ complex defense systems to mitigate the adverse effects of ROS during drought stress. Drought-tolerant varieties can counteract drought stress by inducing antioxidant defense systems and enhancing physiological traits. Thus, there is a relationship between physiological traits and stress tolerance.

The results demonstrated that the synthetic drought-tolerant cumin variety improved physiological traits, such as the accumulation of osmotic regulators and essential oils, and increased antioxidant enzyme activity, which contributed to its higher drought tolerance and superior seed yield compared to the parental genotypes. The genetic diversity present in the synthetic variety makes it a valuable resource for breeding programs aimed at developing drought-tolerant cumin varieties. Utilizing the genetic base and adaptability of the synthetic variety, breeders can develop commercial varieties that are better equipped to withstand drought stress, ultimately contributing to sustainable crop production in arid environments.

In conclusion, the synthetic cumin variety offers a promising avenue for improving seed yield and drought tolerance in arid and semi-arid regions. Its development showcases the effectiveness of combining physiological, biochemical, and molecular approaches in plant breeding. Future research should aim to validate these findings in diverse environments and explore their applicability to other crops, paving the way for resilient agricultural systems in the face of global climate challenges.