Assessment of the phenotypic and physicochemical traits of nine Iranian endemic fenugreek (Trigonella foenum-graecum L.)

Abstract

Considering the significance of fenugreek as a valuable medicinal and food plant, assessing the genetic diversity of different populations of this species is essential for optimizing performance and adaptability to environmental conditions. This study aims to investigate genetic diversity and identify important phenotypic traits in various Iranian fenugreek accessions (“Mashhad”, “Tehran”, “Yazd”, “Shiraz”, “Birjand”, “Isfahan”, “Kerman”, “Kalat”, “Neyshabur”), an experiment was conducted in a randomized complete block design with three replications and nine treatments (accessions) in Iran. The results showed that the highest seed yield was observed in “Kalat” (120.73 g/m²) and the lowest seed yield was observed in “Neyshabur” (29.28 g/m²) accessions. Also, “Isfahan” had the highest number of branches (10.8/plant), and the highest 1000-seed weight (29.6 g) was recorded in “Shiraz”. It was also found that the number of days to the appearance of the first flower and the arrival of the last pod was the least in the “Yazd” (50 and 85 days) and the highest in the “Kerman” (87.67 and 170 days), respectively. It was also found that the highest content of soluble sugar (0.32%) belonged to the “Mashhad” and the highest amount of total chlorophyll (2.36 mg/gfw) belonged to the “Isfahan” accessions. “Birjand” had the highest antioxidant activity (55.64%). The highest abundance of bluish spots was also seen in the “Kalat” accession. The results of this study showed a considerable diversity between the studied accessions. The most suitable accession for harvesting seeds was “Kalat” and the “Isfahan” accession was the most appropriate for harvesting herbal yield. The results of this study provide a valuable foundation for future research aimed at identifying specific genotypes with superior traits, such as higher yield or stress resistance. Future studies can build upon these findings by focusing on selecting and breeding elite fenugreek lines for use in crop improvement programs.

Introduction

Fenugreek (Trigonella foenum-graecum L.), belonging to the legume family of Fabaceae (Leguminosae), is an annual, self-pollinating, and dicotyledonous plant. This plant is very fragrant and has many medicinal utilization. Also, it is rich in vitamins, protein, minerals, fiber, nutrients, and medicinal properties thanks to sapogenins, alkaloids, and other compounds¹. Fenugreek is native to Asia and Southern Europe. The seeds and leaves of this plant contain anti-diabetic compounds², antimicrobial^3,4, anti-inflammatory^3,5, anti-cancer⁶, immune modulation², and many antioxidant agents³ that are used in medicine. In general, the biomedical activities of fenugreek are attributed to its bioactive compounds such as diosgenin, 4-hydroxyisoleucine (4-HIL), trigonelline, galactomannan (GM), and polyphenols such as quercetin². In addition, fenugreek seeds contain mucilaginous fiber, lysine, LTR proteins (rich in L-tryptophan), coumarin, folic acid, phytic acid, nicotinic acid, sapogenins, scopoletin⁷. The results showed that the fenugreek reduced the TG and LDL and increased HDL levels in diabetic subjects more effectively. Fenugreek supplementation significantly improved lipid profile (LDL, TG, TC, and HDL). It could be considered an effective lipid-lowering medicinal plant⁸. This wide variety of medicinally important compounds has made this plant to be cultivated as an attractive source as well as an opportunity for the global pharmaceutical industry^9,10.

Fenugreek is an old global product and currently has a wide distribution in different parts of the world¹¹. Genetic diversity is one of the most important criteria for producing a crop. The use of genetic diversity is useful and necessary to introduce a crop for a new agro-climatic condition and to screen for a high-yield trait(s), or to identify potential disease or stress resistance germplasm to create a new economically viable and productive variety for commercial release^{7,12,13,14,15,16}. Fenugreek is reported to be rich in different species distributed in various countries across the major continents and has considerable genetic diversity^10,14,17. The morphological and seed yield values showed differences among the fenugreek genotypes and cultivars. It is important to identify plants with high adaptability to changing climatic conditions. The use of genetic diversity to obtain a sustainable yield in dryland conditions with water deficiency is a well-practiced crop development strategy. The use of differences found among the fenugreek genotypes from different origins serves as guidance for breeders who are aiming to develop drought-resistant fenugreek cultivars¹⁸.

McCormick et al. (1998) evaluated 207 accessions of fenugreek collected from 20 countries and compared flowering time and duration, growth habit, and seed yield. Among these populations, flowering commencement varied from 98 to 147 days after planting. Also, the duration of flowering was between 14 and 52 days. Growth habits were observed in these stands from prostrate to erect, and seed yield varied from 0 to 3487 kg/ha. In this research, Iranian accessions were also examined. Their flowering time was between 98 and 123 days after planting, and among the 207 investigated accessions, Iranian accessions were the earliest to flower. Also, these accessions were among the accessions that had the longest flowering period and had the highest yield among other accessions¹⁹.

In Ethiopia, 160 accessions were analyzed to determine the amount and pattern of genetic variation. The results of this research showed highly significant differences for days to 50% flowering, days to 90% maturity, number of pods per plant, number of seeds per pod, number of primary branches, number of secondary branches, thousand seed weight, plant height at flowering, and seed yield kg per hectare, as well as average pod length and seed yield per plant^20,21. For example, the maximum seed yield was 1.47 to 4.36 g/plant. Also, great flexibility was reported for the development of suitable varieties for different agro-ecological regions, and in general, many populations showed shorter days for flowering, and the late types were suitable for low rainfall areas and highland areas with dependable rainfall, respectively²². In another study, seven genotypes of fenugreek were evaluated and the results of this research showed a relatively significant difference between the studied genotypes in terms of the amount of ash, moisture, and some minerals (potassium, phosphorus, calcium, magnesium, sodium, iron, manganese, zinc and copper)²³.

Chlorophyll content is a crucial indicator of photosynthetic capacity and overall plant health. Variations in chlorophyll levels across accessions can reflect differences in their adaptability to environmental conditions and productivity potential^24,25,26,27. Similarly, total soluble sugar content is a key biochemical trait that indicates carbohydrate metabolism, serving as an energy source and osmotic regulator under stress conditions^28,82. Antioxidant activity, which measures a plant’s ability to neutralize free radicals, is directly linked to its stress tolerance and medicinal properties, making it a vital parameter for evaluating fenugreek accessions^18,29,30. These chemical properties also highlight plants’ metabolic and physiological status and provide essential insights into their potential applications in breeding programs.

Previous research showed that diverse genetic varieties of fenugreek are suitable for commercial cultivation in low-input agricultural systems of underdeveloped countries in Asia, Africa, and Latin America⁷. Many plant breeders worldwide are working on fenugreek breeding¹¹. The breeding goals of these groups are the release of locally adapted cultivars for specific agro-climatic regions with stable herb and seed yield, as well as the enrichment of the chemical compounds of these cultivars for human health and nutrition^31,32. Also, developing high-yielding, disease-resistant, and suitable early-maturing genotypes for specific climatic regions to produce high-quality seeds is another goal of fenugreek breeding programs to create high-quality seeds every year¹⁷.

In this study, the hypothesis is that distinct phenotypic and physiological traits exist among various fenugreek accessions in Iran, influenced by their specific environmental conditions. This hypothesis is grounded in previous research that has established the significance of genotype and environmental interactions in shaping plant characteristics and adaptability. This study aims to assess the genetic variability and morphological, biological, and biochemical traits of different fenugreek (Trigonella foenum-graecum L.) accessions collected from various regions of Iran. The research seeks to identify promising accessions with desirable traits for breeding programs, aiming to improve yield, stress tolerance, and adaptation to diverse agro-ecological conditions. By examining phenotypic diversity, the study provides valuable insights into the genetic potential of fenugreek accessions for future agricultural development and cultivation strategies.

Materials and methods

The experimental site, plant materials, and treatments

This research was conducted in the research field of Ferdowsi University of Mashhad, Iran (latitude 36° 16ʹ N, longitude 59° 36ʹ E, and 985 m altitude) in 2015–2016. The climatic data for Mashhad, relevant to the environmental conditions in which the fenugreek accessions were grown, indicates an average high temperature of 28 °C and a low temperature of 14 °C, resulting in a mean temperature of 21 °C. Additionally, the precipitation level is recorded at 26.4 mm, reflecting the rainfall typical for this region. The soil tested is characterized as clay loam, and the results from its chemical analysis are presented in the table below. The soil analysis revealed that the soil texture was classified as clay loam. The potassium (K) content was 263 mg/kg, while phosphorus (P) measured 11.62 mg/kg, and nitrogen (N) was 553 mg/kg. The organic matter content was 1.3%, indicating moderate fertility. The soil had an electrical conductivity of 0.532 dS/m, which falls within acceptable limits, and the pH level was slightly alkaline at 7.93. These values suggest that the soil was suitable for plant growth without the need for additional fertilizers (Table 1). Based on the soil analysis and to provide natural growth conditions for the plants, no fertilizer treatments were applied. The land preparation, including plowing, disking, and smoothing was done. Then the ground was prepared as a ridge and farrow by 2 m long and 50 centimeters apart. Irrigation was applied until one week before harvest, after which it was stopped.

Table 1 Physical and chemical properties of the soil at the experimental site

Fenugreek seeds (Trigonella foenum-graceum L.) were collected from different regions including “Mashhad”, “Tehran”, “Yazd”, “Shiraz”, “Birjand”, “Isfahan”, “Kerman”, “Kalat” and “Neyshabur” (Fig. 1). The seeds were planted in early May. Before planting, the seeds were disinfected with 3% hypochlorite for 2 min and then rinsed 3 times with distilled water and planted in the ground. The planting area was prepared in furrows and ridges, each measuring 2 m in length and spaced 50 centimeters apart. The total area of each experimental unit was also 2 square meters. During the growing season, agricultural operations including irrigation (once every 2–3 days) and weed control were done manually and equally for all groups. The experiment was conducted as a randomized complete block design with three replications and nine treatments for one replicate randomized within each block. The seeds of all accessions were obtained under national and international guidelines and the seeds were prepared under the supervision and permission of Ferdowsi University of Mashhad and all authors comply with all the local and national guidelines.

Fig. 1

The geographical locations of collected Trigonella foenum-graecum L.

Morphological studies

At the end of October (about six months later) traits were evaluated in two stages, including vegetative and reproductive stages. Three replications were harvested before flowering, and their vegetative characteristics were measured. Additionally, three replications were harvested after flowering and fruit set, and their reproductive characteristics were analyzed. The dry weight (DW) of roots and aerial parts (g) were measured. After measuring the fresh weight of different parts of the plant, the samples were dried in an oven at 72 °C for 48 h and then weighed with a digital scale. Also, 10 plants of each accession were selected, and the number of branches was recorded; the average was calculated for this trait. Before the flowering stage, to calculate the leaf area, the average lateral shoot leaf area was measured using the Leaf area meter of the Delta-T Devices model.

Physiological and biochemical studies

Chlorophyll content

To measure chlorophyll content 50 mg of young and completely developed leaves were weighed. Then, it was placed in 10 mL of 98% methanol )Sigma-Aldrich, St. Louis, MO, USA) to extract pigments, followed by centrifugation for 10 min with a speed of 1600 rpm. The extent of absorption from the resulting extract was read at wavelengths 653 nm, and 666 nm using a spectrophotometer (Cecil Bio Quest, CE 2502, Cambridge, UK). Finally, chlorophyll a (Chl. a) and chlorophyll b (Chl. b) contents were calculated using the following equations³³.

Chl.a (mg/g fw) = [15.65 (A₆₆₆) – 7.340 (A₆₅₃)] V/ W.

Chl.b (mg/g fw) = [27.05 (A₆₅₃) – 11.21 (A₆₆₆)] V/ W.

CHLt = Chl. a + Chl. b.

Which, V is the volume of extract; W is the mass of leaf tissue.

Total soluble sugar content

Total soluble sugar content was estimated using the Anthrone reagent method (Sigma-Aldrich, St. Louis, MO, USA)³⁴. Fresh leaf tissue (500 mg) was homogenized in 10 mL of 95% ethanol (Merck, Germany). The extracts were centrifuged at 3500 rpm for 15 min. The supernatant was collected and 1 ml aliquot was taken for analysis. Standards were prepared using 0.2-1.0 ml of the working standard (glucose). 1 ml of water served as a blank. All were adjusted to1.0 ml with distilled water. 4.0 ml of anthrone reagent was added to each tube, and mixtures were heated in a boiling water bath for 8 min. After cooling, the absorbance was recorded at 630 nm. A standard curve was plotted using 20–100 µg of glucose. The absorbance values of the standard solutions were plotted against their corresponding concentrations to create a standard curve. A linear regression line was fitted to the data points to derive the equation of the line (y = mx + b)​

Assay of antioxidant activity

To measure the destruction capacity of active radicals, the method described in^35,36 was used. Fresh leaf material (100 mg) was completely homogenized in liquid nitrogen and extracted with 96% ethanol. Centrifugation was performed for 5 min at 3500 rpm to separate insoluble solids. An appropriate amount of the upper transparent solution was mixed with 800 µl of DPPH solution (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, USA) in ethanol (0.5 mmol) and the amount of light absorption after the samples were kept in the dark for 30 min was read at a wavelength of 517 nm. The destruction capacity of active radicals was calculated using the following equation:

$$\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\:\text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}=\left(\frac{\left(\text{A}\text{b}\text{s}\text{o}\text{r}\text{p}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:-\text{A}\text{b}\text{s}\text{o}\text{r}\text{p}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\right)}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{p}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:}\right) \times 100$$

Flowering and seed traits

After the fenugreek fruits ripped, the plants were harvested. After harvesting, they were taken to the laboratory. Traits such as the number of days until the last pod ripening, the number of days until the beginning of flowering, and seed yield (g/m²).

Seed traits

Seed traits such as length, width, diameter (mm), and thousand seed weight were measured in two stages (primary and after plantation).

Statistical analysis

Statistical analysis was performed by MSTAT_C and Excel. For Mean comparison, the Least Significant Difference (LSD) was used at p ≤ 0.05 to establish significant differences. The Principal Component Analysis (PCA) and hierarchical clustering dendrogram were generated using the statistical software R, utilizing the prcomp function for PCA and hclust with the “average” method for cluster analysis.

Results

Phenotypical assessment

Plant growth type

The results showed that there is an erect growth type in “Mashhad” and “Neyshabur”, a semi-erect growth type in “Yazd”, “Shiraz”, “Birjand” and, “Kerman” and a prostrate growth type in “Tehran”, “Isfahan” and “Kalat” (Fig. 2). According to McCormick, (1998), growth habits in fenugreek range from very prostrate to very erect. In other study, of 20 fenugreek landraces originating from various regions of Iran, three growth habits were identified: prostrate, semi-erect, and erect growth habits¹⁹.Similarly, Other researchers have reported that Fenugreek seedlings typically grow either erect or semi-erect reaching a height of 30 to 60 cm³⁷.

Fig. 2

Growth type in fenugreek a erect, b semi-prostrate, c prostrate

Leaf area

There was a significant difference between the accessions in terms of leaf area. Since the leaves and stems of this plant are more widely consumed, improving and increasing this trait plays an important role in enhancing the plant’s productivity and efficiency³². The results of this research also showed that “Isfahan” accession had the highest leaf area (18.34 cm²) while the “Shiraz” accession exhibited the smallest leaf area (3.1 cm²), resulting in a notable difference of 17 cm² between these two accessions (Fig. 3). Variation in growth parameters among varieties is an outcome of genomic, environmental, and agronomic interaction^12,38,39. Considering that these accessions were grown under similar agronomic and climatic conditions, therefore, the observed change in the growth of the accessions may be due to the genetic structure. This observation aligns with findings from other studies that emphasize the critical role of genetic diversity in determining morphological traits such as leaf size⁴⁰. In a study conducted in Oman on nine fenugreek accessions, the maximum leaf area was 3.05 cm², while the minimum was 1.8 cm², with an average leaf area of 2.31 cm²⁴¹. In contrast, the average leaf area observed in present study was 6.84 cm², indicating a significant enhancement in the potential leaf area expansion. This suggests that Iranian accessions possess a considerably higher genetic potential for leaf area development. Such differences may arise from variations in breeding history or local adaptations. Additionally, researchers have noted that larger leaf sizes are often associated with superior photosynthetic efficiency and increased biomass production⁴².

Fig. 3

Leaf area of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Dry weight of aerial organs and roots

There was a significant difference in the amount of dry weight in the aerial organs. The highest biomass was observed in the “Isfahan” accession, while the lowest was found in the “Neyshabur” accession (Fig. 4). This difference underscores the potential of selecting superior accessions to improve biomass yield in fenugreek cultivation. A previous study reported that the dry weight of fenugreek cultivars varies from 0.77% in the Shahryar cultivar to 0.144% in the Borujerd cultivar⁴³. This suggests that genetic diversity significantly influences biomass production across different environments and accessions. Regarding root dry weightsignificant differences were observed among the fenugreek accessions. The maximum root dry weight was observed in the “Tehran” accession, while the minimum was found in the “Neyshabur” accession(Fig. 5). These findings suggest that genetic factors may play a crucial role in determining root biomass, which is vital for nutrients and water uptake

The photo synthetically active leaf area is crucial for absorbing and utilizingradiant energy, ultimately leads to maximum dry matter accumulation³⁹. In agreement with this theory, the highest leaf area and dry weight of shoots and roots were observed in the “Isfahan” and “Tehran” accessions, This correlation can be explained by the capacity of these accessions to harvest maximum sunlight, facilitating photosynthesis and, ultimately leading to increased dry weight The increase in leaf area likely contributed to the enhanced ability to capture sunlight and optimize photosynthesis, which in turn resulted in higher biomass accumulation. These findings align with previous studies on fenugreek, reinforcing that both leaf area and genetic factors significantly influence biomass production in this species^44,45. Further investigation into the specific genetic traits responsible for these variations could provide valuable insights for improving fenugreek cultivation practices. Also, the integration of genetic, physiological, and agronomic factors will provide a comprehensive strategy for improving fenugreek’s productivity under diverse environmental conditions.

Fig. 4

Dry weight of aerial organs of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Fig. 5

Dry weight of roots of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Number of branches

There was a significant difference in the number of branches between the fenugreek accessions. The highest number (10.8) was observed in the “Isfahan” accession, while the lowest (2.6) was found in the “Tehran” and “Yazd” accessions (Fig. 6). In another study, researchers found a significant difference in the number of branches between fenugreek accessions at the 1% probability level⁴¹. The present findings were partially in agreement with those of others, who reported that the number of branches varied between 2.3 and 7.5 in 245 fenugreek genotypes⁴⁶. Also, in another study, the number of branches in fenugreek was reported to range from 2.40 to 4.90⁴⁷, and from 1.00 to 4.33⁴⁸, further highlighting the impact of genetic and environmental factors on this trait. The branch number values of the fenugreek genotypes and cultivars were reported to vary in different conditions with values ranging from 2 to 4.93¹⁸. These results highlight the genetic diversity among accessions and suggest that branching is influenced by both inherent genetic potential and environmental conditions. Increased branching in the “Isfahan” accession could be attributed to its superior genetic potential for vegetative growth, as branches often originate from nodes that develop due to high photosynthetic activity. On the other hand, the lower number of branches observed in the “Tehran” and “Yazd” accessions might be due to limitations in either genetic expression or less efficient resource allocation for vegetative growth. Environmental factors like nutrient availability, light exposure, and planting density could also influence the observed variation in branching⁴⁹. In another study, a significant variation in the number of branches was observed among the fenugreek genotypes, ranging from 1.86 to 5.96. This variation is crucial for breeding programs, as the number of branches can directly influence the overall biomass and seed yield⁵⁰. Genotypes with a higher number of branches, which also demonstrated superior seed yield, are particularly valuable for selection in future breeding efforts. The diversity in branching patterns provides breeders with opportunities to select genotypes that are better suited for different cultivation environments and yield optimization. Additionally, branching can serve as an indicator of adaptive capacity in response to varying environmental conditions, such as water stress or nutrient limitation.

Fig. 6

Number of branches of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Number of days to flowering

There was a significant difference between the accessions in terms of the number of days to flowering. The “Yazd” accessions began to flower only after 50 days, while the days required for flowering in the “Kerman” accessions was 89 days (Fig. 7). In McCormick et al. (1998) research, which tested about 207 fenugreek genotypes, it was found that the flowering time varies between 98 and 147 days after planting¹⁹. Other researchers divided the native fenugreek accessions into three groups according to the flowering time. The early flowering accessions entered the reproductive stage between 39 and 45 days after planting. Borazjan, Kashan, “Isfahan”, Borujerd, “Kermanshah” and “Kerman” accessions were in this group. The mid-flower accessions entered the reproductive stage between 39 and 65 days after planting and included the accessions of Araghi, Ardestan, “Neyshabur”, “Yazd”, Khash, Sisakht, “Shiraz” and Kashan. Late flowering groups entered the reproductive phase between 65 and 75 days after planting and included Khorramabad, Qaenat, Semnan, Ahvaz, and Shahrerei accessions⁵¹. Another study reported on 36 geographically diverse Ethiopian fenugreek accessions and found 42.5 to 52.5 days to flowering⁵². Also, there was significant variation in the days to 50% flowering among the 160 fenugreek accessions, ranging from 41.14 to 57.04 days, with a mean of 51.34 days⁵³. The wide range of days to flowering observed among the fenugreek accessions in this study highlights the potential for developing varieties tailored to specific agro-ecological zones. Accessions with early flowering, such as “Yazd,” may have evolved mechanisms to optimize resource use in short growing seasons or under drought-prone conditions, where early reproduction can ensure survival and seed production. Conversely, late-flowering accessions like “Kerman” might favor longer vegetative growth, enhancing biomass and seed yields in favorable environments. The differences observed in our study suggest that flowering time is controlled by complex interactions between genotype and environment. These findings can guide breeding programs, prioritizing early-flowering genotypes for regions with limited growing seasons and selecting late-flowering accessions for areas with extended cultivation periods.

Fig. 7

Number of days until the beginning of flowering of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Number of days to the last pod ripening

There was a significant difference in the number of days to the last pod ripening between the accessions (Fig. 8). The difference between the days from planting the fenugreek accessions to the last pod ripening varied between 85 and 170 days. The lowest value was observed in the “Yazd” accessions, and the highest was observed in the “Kerman” accessions (Fig. 8). In another experiment on other accessions of this plant, the days to maturity ranged from 111.00 to 133.00 days⁵⁴. Also, the minimum and maximum days to maturity were reported as 124 and 133 days respectively⁵⁵. In another study, there was a wide range of days to 90% maturity differences among 160 fenugreek accessions and ranged from 97.67 to 138.21 days, with an overall mean of 119.32 days²⁰. The observed differences in the time it takes for accessions to flower and reach maturity present significant opportunities for breeders to create varieties that are well-suited to diverse agroecological conditions. Accessions that mature early may be more advantageous in regions with limited rainfall and shorter growing seasons, while those that mature later could thrive better in highland areas where rainfall is more reliable²⁰. Additionally, it has been suggested that early maturing genotypes may help reduce the risks associated with climatic variability. At the same time, late-maturing varieties could have more time to accumulate biomass and increase yield potential. Early-maturing accesions, such as those observed in the “Yazd” accession, could be advantageous in regions prone to drought or with limited growing seasons, where early maturation can ensure seed production before harsh environmental conditions prevail. In contrast, late-maturing accesions like “Kerman” may be better suited to regions with reliable rainfall and longer growing periods, allowing them to maximize biomass production and improve seed yields.

Fig. 8

Number of days to the last pod ripening of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Seed yield

A significant difference was observed between the yield of accessions. The highest yield (120.73 g/m²) was observed in the “Kalat” accession and the lowest yield (29.28 g/m²) was observed in “Neyshabur” accessions, so the difference between the highest and the lowest yield was around 90 g/m² (Fig. 9). In similar research in Canada on fenugreek cultivars, a significant difference was observed between different cultivars in terms of yield. It was found that in the irrigation conditions of the first year, the PI 211,636 cultivar had the highest yield and the L3675 cultivar had the lowest yield⁵⁶. Also in another study, seed yield showed differences in the cultivation years among the fenugreek genotypes and cultivars grown under irrigated and dryland conditions and variable between 31.14 and 142.02 (kg/ha)¹⁸. Seed yield in fenugreek genotypes has shown significant variability, critical for selecting promising accessions in breeding programs. In the study that was conducted on 111 fenugreek genotypes, seed yield ranged widely between 0.71 and 8.60 g per plant, highlighting the genetic diversity across the 111 fenugreek genotypes It has been stated that morphological properties such as plant height and branch number are closely related to seed yield in fenugreek⁵⁷. This study, also observed that the accessions with the lowest number of branches (“Tehran”, “Yazd” and “Neyshabur”) had the lowest seed yield. This broad variation is supported by other studies where seed yield per plant varied significantly based on genotype and environmental conditions. For example, one study on fenugreek genotypes reported seed yield variations from 0.05 to 8.61 g per plant, with the highest yield observed in Turkish and Pakistani genotypes¹⁸. Based on this, extensive research has been carried out, which has led to the modification and introduction of new and high-yielding cultivars. Both genotype and environmental factors, along with their interactions, were observed to affect forage and seed yield in fenugreek grown in western Canada⁵⁸. Therefore, the difference in seed yield can be due to the difference in accessions and environment and genotypic variation may be utilized in developing new cultivars⁵⁹. The higher yield observed in accessions like “Kalat” could be attributed to a combination of favorable genetic traits and its ability to adapt to local environmental conditions, while the lower yields of “Neyshabur” may reflect constraints related to both genetic limitations and less optimal environmental factors. Understanding these interactions can help optimize breeding strategies, particularly in regions where water availability and climate conditions fluctuate. Moreover, incorporating genetic traits associated with higher yields, such as increased branch number and plant height, could potentially enhance productivity and facilitate the development of more resilient fenugreek cultivars in response to changing environmental challenges.

Fig. 9

Seed yield of nine Iranian endemic fenugreek. The values with the same letters are not significant according to the least significant difference test at the 5% level.

Seed size

The results showed that there is a significant difference between the accessions in terms of the seed length. The highest seed length recorded in “Tehran” (3.65 mm) and “Isfahan” (3.66 mm) accessions, while after seed cultivation and seed collection from grown plants, the measurements determined that the “Mashhad” accession (4.17 mm) had the longest length among other accessions. The seed width was the highest in the “Isfahan” accessions (2.61 mm) and the lowest in the “Kalat” accessions (1.60 mm), but after cultivation, the lowest seed width (1.72 mm) was observed in the “Isfahan” accessions (Table 2).

In terms of seed diameter, although no significant difference was observed between the accessions, after cultivation, the smallest (1.08 mm) and largest (1.37 mm) diameters were related to “Mashhad” and Neyshabour accessions, respectively. The differences observed in seed size among the accessions indicate that there is considerable genetic variation, which is essential for breeding programs aiming to improve fenugreek’s productivity. Larger seeds, particularly those found in the “Mashhad” accession, are typically associated with higher thousand-seed weight and better seed yield. This can directly enhance the productivity and commercial value of fenugreek as a crop. Additionally, seed size is a valuable indicator of overall plant vigor and may be linked to other important agronomic traits, such as germination rate and early seedling growth. In the study of other researchers on fenugreek accessions, the different accessions of fenugreek vary in seed size, it was found that the largest seed size is related to Sarab accession and the smallest one is related to Qom accession, and it was found that the effect of the environment plays an important role in the quality and quantity of the seeds⁶⁰. Another study reported that the seeds have lengths ranging from 3.02 mm to 4.09 mm and the seeds have widths ranging from 2.02 mm to 2.09 mm. The average length and width of fenugreek seeds were 3.461 mm and 2.061 mm, respectively⁶¹. Our results were somewhat different from these results, which can be attributed to the type of accessions or growth conditions. Given that environmental conditions significantly influence seed size, breeding programs should focus on selecting accessions that perform well under specific regional conditions to maximize seed quality and yield. Furthermore, selecting for larger seeds, while considering environmental factors, could contribute to the development of high-yielding fenugreek varieties that are well-suited to different agro-climatic conditions. Moreover, seed size is often correlated with other important traits, such as thousand-seed weight and seed yield, making it a critical selection criterion in fenugreek breeding programs. Larger seeds contribute to higher thousand-seed weight, which, in turn, can enhance the crop’s overall productivity and commercial value^20,53.

1000-Seed weight

The weight of 1000 seeds showed a significant difference among different accessions of fenugreek. The comparison of the average weight of 1000 seeds between fenugreek populations also showed that the highest and lowest weight of 1000 seeds before cultivation belonged to “Shiraz” (14 g) and “Kalat” (8 g), respectively; However, after planting, the highest weight per 1000- seeds belonged to “Shiraz” and “Isfahan” accessions with an average of 29 g while the lowest weight observed in the “Birjand”, “Kerman” and “Neyshabur” accessions with an average of10 g.

The trait of thousand-seed weight is a key parameter in fenugreek breeding programs because it strongly correlates with overall seed yield and quality. Studies have shown significant variability in 1000-seed weight among different fenugreek genotypes and accessions. For example, in one study, 1000- seed weight values ranged between 6.18 g and 23.22 g, indicating a broad genetic diversity that can be exploited for breeding purposes⁶². In the other study, the weight of 1000 seeds of fenugreek varied from 5.56 g in “Semnan” accessions and 19.44 g in “Kermanshah” accessions⁵¹. Also, the 1000 seed mass of Indian fenugreek seeds ranged from 14.05 to 14.85 g⁶¹. The average weight of a thousand seeds was 14.45 g. similar results have been reported⁶³. The thousand-seed weight is a critical trait in fenugreek breeding programs as it directly impacts the yield potential and seed quality. In the study, a wide variation in 1000-seed weight was observed among the different origin 111 fenugreek genotypes, ranging from 6.18 to 23.22 g. This variation highlights the genetic diversity present in the fenugreek accessions, making it possible to select genotypes with higher 1000-seed weights for breeding purposes⁵⁰. Therefore, accessions with higher 1000-seed weight, which also exhibited superior seed yield, are promising candidates for improving fenugreek’s commercial value. This trait is especially important for markets where larger seed size is preferred for its potential health and nutritional benefits. The observed diversity in 1000-seed weight offers breeders flexibility in selecting accessions best suited for different environmental conditions and market demands. Additionally, larger seeds generally contribute to higher thousand-seed weight, which is often associated with improved plant vigor, enhanced germination rates, and ultimately higher yields. By focusing on breeding fenugreek varieties with optimized 1,000-seed weight, breeders can develop cultivars that not only perform better in terms of yield but also meet market preferences for seed size, adding value to the crop.

Percentage of bluish spot

There was a significant difference between the accessions in terms of the percentage of pigment abundance (Fig. 10). So that the abundance of pigments was higher in the “Kalat” accessions, but this percentage was 0 in the “Mashhad”, “Tehran”, “Birjand”, and “Yazd” accessions; This showed the diversity among the accessions. The presence of pigment in the seed coat of different fenugreek cultivars has been seen, and these cultivars are classified as “colorata type”. For example, Kenyan (RH 2698) belongs to the colorata type. The advantages of this cultivar are the very high diosgenin content of seeds, the high seed-yielding nature, the earliness in maturity, the absence of sprouting in a pod, the resistance to damp weather and winds because of the strong stems, and the secondary shoots that arise from the base. Also, Ethiopian (RH 2699) is another cultivar of colorata type that has a high percentage of crude protein and fixed oils are advantages of this cultivar (Petropoulos 2002). In the current research, “Kalat” had the highest pigment abundance and the highest performance, which is consistent with the results of the research by¹⁴, but in terms of the earliness in maturity, “Kalat” needed more days to reach the last pod than other accessions.The “Kalat” accession stands out not only for its high pigment abundance but also for its overall performance in terms of seed yield, although it requires more time to mature compared to other accessions. The diversity in pigment presence and its relation to other agronomic traits such as diosgenin content and seed yield makes this trait an important consideration in fenugreek breeding programs, particularly for regions with specific climatic challenges.

Table 2 Seed traits of nine Iranian endemic fenugreek

Fig. 10

Bluish spot status in fenugreek seeds

Biochemical evolution

Chlorophyll content

No significant difference was observed in the amount of chlorophyll a, b, and total in different accessions (Table 3). However, in general, the highest content of chlorophyll a (1.98 mg/gfw) was in the “Isfahan” accessions and the lowest (1.51 mg/gfw) in the “Birjand” accession. The content of chlorophyll b was the highest in “Kerman” accession (0.405 mg/gfw) and the lowest in “Mashhad” accession (0.253 mg/gfw), and finally, the maximum total chlorophyll content (2.36 and 2.35 mg/gfw) was in “Isfahan” and “Kerman” accessions, respectively and the lowest amount of total chlorophyll (1.69 mg/gfw) was also in “Birjand” accession (Table 4). In another study, the researchers stated that the chlorophyll content of fenugreek varies from 0.13 (g/kg of fresh weight) in the Ardestan genotypes to 0.23 g/kg fw in the “Kerman” genotypes⁴³. Also, the examination of 30 genotypes of fenugreek (Trigonella foenum-graecum L.) showed great variation (45.47–59.47 SPAD Unit) in chlorophyll content⁶⁴.

Photosynthesis plays a major role in the growth and performance of plants. Chlorophyll is the main factor contributing to the rate of photosynthesis⁶⁵, and it mainly acts as a transfer center in the harvesting of light energy⁶⁶. The chlorophyll content is closely related to crop yield due to its role in the photosynthesis process, and the degradation of chlorophyll limits yield potential⁶⁷. Therefore, increasing the content of chlorophyll in the plant will increase photosynthesis. Following the increase in photosynthesis, plant performance improves. Breeding plant genotypes with long periods of active photosynthesis is a practical approach to increase yield, which requires robust green phenotypes with high chlorophyll content⁶⁸. Therefore, chlorophyll content is one of the traits that can be considered for plant breeding. It is concluded that the accessions of fenugreek that have more chlorophyll content, such as “Isfahan”, “Kerman” and “Yazd” are suitable for breeding programs. Also, it has been proven that drought-tolerant genotypes retain many photosynthetic pigments under drought stress. As a result, genotypes with high chlorophyll content may be more drought-stress tolerant⁶⁹. Genotypes such as “Isfahan,” “Kerman,” and “Yazd,” with higher chlorophyll content, are thus not only valuable for breeding programs focused on yield but also for developing drought-tolerant varieties. As photosynthesis is essential for biomass production, these accessions are particularly suited for breeding programs aiming to improve plant productivity and resilience to environmental stressors.

Total soluble sugar contents

There was a significant difference in the soluble sugar content across the fenugreek accessions (Table 3). The highest content was observed in the “Mashhad” accession (0.32 mg/gfw), followed by the accessions from “Yazd,” “Kerman,” “Isfahan,” and “Neyshabur.” The lowest sugar content (0.15 mg/gfw) was recorded in the “Shiraz” accession (Table 4).

The observed variability in soluble sugar content among fenugreek accessions may be attributed to both genetic factors and environmental conditions. Studies have shown that specific genotypes of fenugreek exhibit significant genetic variability in soluble sugar accumulation, indicating that genetic factors play a crucial role in determining the levels of soluble sugars in these plants. For instance, researchers found that high heritability estimates for traits such as yield and related characters suggest that selection based on these traits could lead to improved sugar content in certain genotypes of fenugreek⁷⁰. Moreover, when compared to previous studies, such as those reporting the highest soluble sugar content in the Guj.methi-2 cultivar (11.01%) and the lowest in the JFG.226 cultivar (4.14%)⁷¹, the current findings reflect lower values overall, possibly due to differences in measurement techniques, environmental stress factors, or even the maturation stage of the plants at the time of analysis. The carbohydrate content of Indian fenugreek seeds (55.49%)⁶¹. Also differs significantly from our results, which may be attributed to variations in genetic background and local cultivation practices. These discrepancies underline the need for further investigation into how both genetic and environmental factors interact to influence sugar accumulation in fenugreek. The environmental conditions in Mashhad, such as temperature, rainfall, and soil characteristics, likely created favorable conditions for sugar production⁷². Research shows that environmental stressors, like drought or high temperature, can lead to enhanced sugar accumulation as a protective mechanism in plants⁷³. In conclusion, understanding these variations can be crucial for breeding programs aimed at enhancing specific biochemical traits such as soluble sugar content. Future studies should explore the genetic markers associated with sugar metabolism in fenugreek and investigate the impact of environmental conditions on sugar synthesis, particularly in different phenological stages and growing environments.

Antioxidant activities

The results showed that fenugreek accessions were significantly different from each other in terms of antioxidant activities (Table 3). So, the highest amount of these activities was observed in “Birjand” accessions (55.64%) and the lowest in “Kerman” (42.86%) (Table 4). The DPPH values obtained in this study were found to be lower than the antioxidant value (80.53%) reported by⁷⁴ and partiallly similar to^{75] and [76}, who reported 43.6–67.30% and 51.60%, respectively. Also¹⁸, reported that the DPPH values changed between 33.58 and 60.59% under irrigated and between 25.59 and 42.64% under dryland conditions. According to previous findings, extraction methods or processes affect antioxidant values in fenugreek and cause changes in antioxidant values⁷⁷. In addition, it has been stated that antioxidant properties are affected by quantitative and qualitative changes in phenolic compounds during the germination period⁷⁸. Therefore, the different results of antioxidant values in the present study with previous studies can be explained by extraction methods and processes, as well as differences in genotype, cultivation time, and ecological conditions, and ecological conditions. Others also concluded that the antioxidant activity of crops significantly differs with genetic diversity, seed color, climatic and soil conditions, and crop time⁷⁹. Also, extraction methods significantly affect the antioxidant values in fenugreek, leading to different results across studies¹⁸.

Table 3 Analysis of variance of measured indices in fenugreek accessions

Table 4 Biochemical traits of nine Iranian endemic fenugreek

PCA and cluster analysis

To better understand the relationships among traits and to group the different populations, a principal component analysis (PCA) was performed. According to Fig. 11, the PCA results indicate that the first component accounts for approximately 95.94% of the total variance, while the second component accounts for 4.05%, covering 99.99% of the total data variability. These two components can thus be considered the primary components for analysis. In the first component, the trait bluish spot showed a high correlation, while in the second component, the trait 1000-seed weight had a strong correlation. Based on this chart, populations can be selected or grouped according to specific objectives. For example, the “Isfahan” and “Neyshabur” populations, located in the upper section of the chart, exhibit higher 1000-seed weights.

Fig. 11

Principal component analysis (PCA) of all the traits measured

The cluster dendrogram in Fig. 12 provides insights into the genetic relationships among different Iranian fenugreek accessions based on their phenotypic traits. This hierarchical clustering analysis highlights the similarities and distinctions between the accessions, allowing for an interpretation of their genetic diversity. The representation in the dendrogram reveals that “Mashhad” is categorized into its own cluster, which is notably distanced from the clusters of other samples. This separation underscores the unique phenotypic attributes of “Mashhad” in relation to the other entities analyzed. The comparative analysis reveals that “Mashhad” fenugreek stands out in its morphological and certain phytochemical aspects when juxtaposed with other entities. This superiority may indicate potential benefits for its utilization in diverse fields, warranting further investigation into its applications and benefits. A separate study investigating the variability among various Iranian fenugreek ecotypes found that the leaves of the Karaj ecotype and the seeds of the Mashhad ecotype present promising options for potential commercial use within the food industry⁸⁰. Consequently, the findings underscore the importance of exploring and selecting specific ecotypes for their commercial viability, which could lead to enhanced applications in the food sector and contribute to the diversification of fenugreek utilization.

The spatial closeness between the “Kerman” and “Kalat” classifications suggests that they exhibit a notable degree of similarity in their defining characteristics. Such a relationship may provide insights into their respective traits and could be indicative of shared origins or influences. This clustering of “Isfahan” and “Shiraz” not only highlights their spatial connection but also points to the possibility of underlying shared traits or genetic features that warrant further exploration. In their study, Haliloğlu and colleagues assessed 34 genotypes of fenugreek sourced from 18 different countries, employing 24 inter-primer binding site markers for their analysis. Their findings indicated that the genetic diversity within the “Isfahan” population was marginally greater than that observed in the Indian genotypes, as evidenced by 11 distinct genetic indices. The researchers further categorized the populations into a distinct subcluster, highlighting the unique genetic characteristics of the “Isfahan” group in comparison to the Indian varieties. This classification underscores the importance of geographical and genetic factors in the diversity of fenugreek genotypes⁸¹. These groupings suggest that accessions in close proximity within the dendrogram may perform similarly under similar environmental or agronomic conditions.

The analysis also implies that the diversity observed among these clusters can be advantageous for selective breeding programs. For example, distinct accessions such as “Mashhad” may possess unique traits beneficial for specific purposes, while similar accessions like “Kerman” and “Kalat” could be optimized for enhanced uniformity in yield or other agronomic traits. The exploration of genetic variation among fenugreek accessions serves as a critical basis for strategic breeding programs. By leveraging this information, researchers and breeders can aim to create superior cultivars that not only yield more but also demonstrate enhanced adaptability to various environmental stressors and possess increased medicinal benefits. In summary, this cluster analysis serves as a valuable tool in understanding and utilizing the genetic diversity within Iranian fenugreek accessions, aiding in the identification of suitable candidates for breeding and improvement programs.

Fig. 12

Cluster dendrogram of Iranian fenugreek accessions based on phenotypic traits.

Conclusion

Evaluating the genetic diversity of plants both within and among various geographic areas is essential for the effective management and preservation of fenugreek genotypes. Numerous elements, such as the processes of natural selection, agricultural breeding methods, mechanisms of seed dispersal, and the lifestyle of organisms, can significantly affect the genetic diversity within a species. This study underscores the remarkable genetic diversity present among various fenugreek accessions in Iran, highlighting their potential for breeding and agricultural development. The Isfahan accession exhibited the highest dry weight and leaf area, while Kalat showed the highest overall yield. On the other hand, “Neyshabur” and “Yazd” accessions had the lowest yields, demonstrating the wide variability in growth performance. Biochemical traits further confirmed this diversity, with the “Mashhad” accession having the highest sugar content, while “Isfahan” and “Yazd” were notable for their high total chlorophyll levels. The presence of such variability aligns with findings in previous studies, which have also highlighted the medicinal and nutritional potential of fenugreek across different environments and genetic lines. The diversity identified in traits such as seed weight, leaf area, and biochemical attributes suggests that these accessions can be utilized for targeted breeding programs. This genetic richness is crucial not only for improving yield and growth characteristics but also for enhancing medicinal properties, such as the production of bioactive compounds like diosgenin and trigonelline, known for their therapeutic effects. Given the growing interest in fenugreek for its medicinal uses, particularly in managing diabetes, cardiovascular diseases, and inflammation, it is essential that future studies further investigate the genetic and biochemical diversity of these accessions. Moreover, exploring how environmental factors influence these traits could help optimize fenugreek cultivation for both agricultural productivity and medicinal quality. In summary, the findings from this research emphasize the untapped potential of Iranian fenugreek accessions, which could be crucial for the advancement of breeding programs aimed at improving both the yield and medicinal properties of this valuable plant.