Cicer arietinum L. (chickpea) is an annual, predominantly self-pollinating pulse crop belonging to the family Fabaceae (Vélez et al., 2025). It is widely accepted that chickpea originated and underwent primary domestication in the region encompassing present-day southeastern Turkiye and northern Syria. Subsequent diversification is believed to have occurred across the Middle East, Central Asia, the Indian subcontinent and the Mediterranean basin (Igolkina et al., 2023; Zhang et al., 2024). Currently cultivated in more than 50 countries worldwide, chickpea represents one of the most important grain legumes and is ranked as the third most widely produced food legume globally, following dry beans and dry peas (Zhang et al., 2024).
       
The high content of proteins, amino acids, vitamins, minerals and bioactive compounds in chickpea seeds has made chickpea an important plant-based food source in human nutrition (Kaur and Prasad, 2021; Begum et al., 2023; Jha et al., 2024; Yadav et al., 2025).
       
Cicer arietinum has attracted considerable attention not only because of its high nutritional value but also because of its functional and potential health-promoting properties. Bioactive compounds present in chickpea have been associated with antioxidant and health-promoting properties (Begum et al., 2023; Jha et al., 2024). Furthermore, experimental evidence suggests potential effects on glycaemic regulation, lipid metabolism and gut health (Faridy et al., 2020; Begum et al., 2023).
       
The seed proteins of Cicer arietinum L. consist predominantly of globulins and albumins, which account for approximately 55-60% and 8-14% of total seed protein, respectively. Globulins represent the principal storage protein fraction, whereas albumins comprise mainly water-soluble proteins with enzymatic and metabolic functions. In addition, albumin fractions may contain antinutritional compounds, including trypsin inhibitors (Zha et al., 2021; Grasso et al., 2022). Chickpea proteins are considered valuable sources of essential amino acids (Ipekesen et al., 2022; Begum et al., 2023). Tryptophan is generally present at relatively low concentrations and may not be detected in some analyses because of methodological limitations or detection thresholds. However, other studies have identified chickpea genotypes characterised by elevated tryptophan contents (Salmanova et al., 2026). Tryptophan plays an important role in neuronal function, immune regulation and gut homeostasis (Luo et al., 2026).
       
Chickpea is a nutrient-dense and affordable source of plant-based protein with potential to support food security in developing regions. Genotype-dependent variation in biochemical composition affects its nutritional and functional properties and may contribute to stress adaptation, defence responses and metabolic regulation (Thudi et al., 2017; Rajput et al., 2023). Due to its low production requirements, adaptive capacity, extensive root system and ability to fix atmospheric nitrogen through symbiosis, chickpea is also an important component of sustainable agricultural systems, improving soil fertility and reducing reliance on mineral fertilizers (Mohsenzadeh, 2024). Therefore, this study aimed to characterize technological traits, biochemical composition and globulin storage-protein profiles of local and introduced chickpea genotypes and to identify promising accessions for breeding programmes targeting improved nutritional quality.

Plant material
 
The field and laboratory experiments were conducted from 2023 to 2025 at the Absheron Experimental Base Station of the Institute of Genetic Resources, Ministry of Science and Education of the Republic of Azerbaijan. The research site is located on the Caspian Sea coast, southeast of Baku, at 40.413010^oN and 49.833421^oE.
       
The Absheron Peninsula is characterized by a dry subtropical climate. Mean annual air temperature ranges from 12 to 15^oC; minimum temperatures during winter generally range from -2 to 4^oC, whereas maximum temperatures in summer reach 28-35^oC. Mean daily air temperature remains above 0^oC. Annual precipitation is approximately 200-250 mm. These climatic conditions are considered favourable for the cultivation of legume crops.

The investigated chickpea accessions were sown under open-field conditions during the autumn season at the experimental site of the Genetic Resources Institute. Seeds from 51 mature chickpea accessions were used as the experimental material. The local cultivar Cemile and Cemile 1 originated from Azerbaijan and was maintained in the chickpea germplasm collection of the Institute of Genetic Resources. The remaining 49 chickpea accessions were introduced into Azerbaijan from the International Center for Agricultural Research in the Dry Areas (ICARDA) and subsequently maintained at the Institute’s Absheron Experimental Base Station (Supplementary Table S1).


 
Biochemical analyses
 
Determination of protein content
 
Total protein content in chickpea grains was determined using the Kjeldahl method. 100 mg of finely ground chickpea flour was transferred to a 100 mL Kjeldahl digestion flask, followed by the addition of 7 mL of concentrated sulfuric acid (H₂SO₄). A selenium-copper mixture was used as the digestion catalyst.
       
The samples were digested in a fume hood until a clear solution was obtained. After cooling, the inner walls of the flask were rinsed with distilled water and the samples were heated for an additional 10 min to ensure complete digestion. Subsequently, the digested samples were subjected to distillation using a Kjeldahl apparatus.
       
For ammonia collection, 25 mL of 0.02 N H₂SO₄ and 1-2 drops of indicator were added to each 200 mL receiving flask. The digestion flasks were connected to the Kjeldahl apparatus and the digested samples were alkalized with a 40% alkaline solution. Distillation was continued for 20-30 min. The solution collected in the receiving flask was then back-titrated with 0.02 N NaOH until the endpoint, indicated by a distinct color change, was reached.
       
The total protein content was calculated according to the following equation:
 

 
Where
V =      Volume of 0.02 N NaOH used for back-titration (mL).
25 =    Initial volume of 0.02 N H₂SO₄ added to the receiving flask (mL).
0.28 = Nitrogen-equivalent coefficient for 0.02 N H₂SO₄.
m =     Mass of the analyzed sample; 100/89 is the dry-matter correction factor.
6.25 = Nitrogen-to-protein conversion factor (Vinklárková, 2015).

Determination of lysine content
 
Lysine content was determined in duplicate using the ninhydrin-based colorimetric method described by Museyko and Sysoev (1970), with minor modifications. Briefly, 30 mg of finely ground chickpea flour was transferred to a 100 mL flask, followed by the addition of 1 mL of 2% Na₂CO₃ solution and 250 mg of finely powdered glass. The mixture was thoroughly homogenized with a glass rod and incubated in a water bath at 80^oC for 10 min.
       
After the initial incubation, 2 mL of ninhydrin reagent was added to each sample. Calibration standards were prepared by mixing 0.5 mL of each L-lysine standard solution (0-100 µg/mL L-lysine) with 0.5 mL of 4% Na₂CO₃  solution and 2 mL of ninhydrin reagent. A reagent blank containing all reagents but no chickpea sample or L-lysine standard was prepared in parallel. The samples, standards and reagent blank were incubated in a water bath for 30 min under identical conditions.
       
Following colour development, the mixtures were cooled to room temperature, supplemented with 5 mL of 95% ethanol, thoroughly mixed and centrifuged at for 5 min. The absorbance of the clear supernatant was measured against the reagent blank using a photoelectric colorimeter (Ultrospec™ 2100 pro UV/Vis spectrophotometer (Amersham Biosciences, UK). Lysine concentrations were determined from the linear calibration curve constructed using the L-lysine standards. The calibration equation was expressed as:

A=aC+b
 

Where
A = Blank-corrected absorbance.
C = L-lysine concentration.
a = Slope.
b = Intercept.
       
Calibration performance was evaluated using the coefficient of determination.
       
Lysine content was calculated according to the following equation:
 

Lysine (mg/100 g DW)= mC × V × DF × 100

 
Where
C= Lysine concentration obtained from the calibration curve (mg/mL).
V = Final extract volume (mL).
DF = Dilution factor.
m = Dry mass of the analysed sample (g).
         
The results were expressed as mg/100 g dry weight (DW).
 
Determination of tryptophan content
 
Tryptophan content in synthetic chickpea grains was determined according to the colorimetric method of Ermakov and Yarosh (1969), with minor modifications. Briefly, 200 mg of finely ground chickpea flour was transferred into a 100 mL volumetric flask and 2 mL of distilled water was added. Subsequently, 1 mL of a 4% gelatin solution prepared in 25% KOH was added. The flasks were tightly sealed with rubber stoppers and the samples were incubated at 40^oC for 18-20 h (Ermakov and Yarosh, 1969).
       
Following incubation, the samples were cooled to room temperature. Subsequently, 0.5 mL of p-dimethyl aminobenzaldehyde reagent prepared in 10% HCl, 0.5 mL of 1% sodium nitrate (NaNO₃) solution and 28 mL of concentrated HCl were sequentially added to each flask. The reaction mixtures were thoroughly mixed, resealed and incubated in a thermostatically controlled chamber at 25°C for 90 min.
       
After color development, the volume of each sample was adjusted to 100 mL with distilled water. The resulting solutions were filtered and their absorbance was measured at 590 nm against a reagent blank using a spectrophotometer (Amersham Biosciences, UK). Tryptophan concentration was determined using an external calibration curve prepared from analytical-grade L-tryptophan standards subjected to the same color-development procedure.
       
Tryptophan content was calculated according to the following equation:

 
Where
C =    Tryptophan concentration obtained from the calibration curve (mg mL^-1).
V =    Final volume of the sample solution (mL).
DF = Dilution factor.
m =   Mass of the analyzed grain sample (g).
       
The results were expressed as milligrams of tryptophan per 100 g of grain dry matter.
 
Electrophoretic analysis (A-PAGE)
 
Globulin storage proteins from 51 chickpea genotypes were extracted and analysed by acid polyacrylamide gel electrophoresis (A-PAGE) using a modified Poperelya (1989) protocol. Since the original method was developed for wheat gliadin analysis, adaptations for chickpea storage proteins included two preliminary extractions with 70% ethanol, two washes with 0.03% acetic acid-acetone solution, incubation for 15 min and centrifugation at 3500 rpm for 5 min after each step, followed by solubilization of the pellet in 500 µL of 9 M urea-acetic acid solution. Protein extracts were subsequently separated in a vertical A-PAGE system using glycine–acetate buffer (pH 3.5).
       
These modifications were introduced to improve extraction and electrophoretic resolution of chickpea globulin storage proteins while maintaining the original principles of the Poperelya method.
       
For consistent band scoring, each electrophoretic profile was divided into four operational mobility regions (Regions I–IV), arranged from the upper to the lower part of the gel. These regions were defined solely according to relative electrophoretic mobility and were used to facilitate consistent comparison of banding patterns among chickpea accessions; they were not interpreted as biochemically homologous to the α-, β-, γ-, or ω-gliadin fractions of wheat (Bushuk and Zillman, 1978). For each operational mobility region, an electrophoretic pattern class was defined as a unique combination of reproducible presence/absence bands. Accessions with identical band combinations were assigned to the same pattern class, while any reproducible difference in at least one band defined a different class. Pattern diversity was calculated as:
                                              

H = 1 - ∑Pi²

Where
H =  Storage-protein pattern diversity index.
Pi = Frequency of the i^th electrophoretic pattern within each zone.
       
Because the input data represent electrophoretic protein-band phenotypes rather than directly genotyped loci, H was interpreted as an index of storage-protein pattern diversity and not as a genome-wide estimate of storage-protein pattern diversity.
 
Cluster analysis of electrophoretic profiles
 
Only clear and reproducible globulin bands were used for cluster analysis. Pairwise dissimilarities among accessions were calculated using the Jaccard coefficient was constructed using DARwin software version 6.0. As no bootstrap analysis was performed, the clustering was considered an exploratory representation of variation in globulin storage-protein profiles rather than statistically supported genetic relationships.
 
Statistical analysis
 
All technological and biochemical traits were evaluated in three replicates for each of the 51 chickpea accessions and results were expressed as mean ± standard deviation (SD). Differences among accessions were analyzed using one-way ANOVA, with accession as the fixed factor. Variance homogeneity was assessed using Levene’s test and significant differences were further evaluated by Tukey’s HSD post-hoc test at P<0.05. In tables, means sharing the same lowercase letter indicate no significant difference according to Tukey’s HSD test.
       
Pearson’s correlation analysis was performed using accession mean values (n = 51) to assess relationships among water absorption capacity, cooking time, moisture content, 100-seed weight, protein, lysine and tryptophan contents. Correlation significance was tested using two-tailed tests at P<0.05, P<0.01 and P<0.001.
       
Principal component analysis (PCA) was conducted using standardized accession means (z-score transformation) based on the correlation matrix. Principal components were interpreted according to eigenvalues, explained variance and trait loadings. Cluster analysis of technological traits was visualized using a heat map based on Euclidean distance and Ward’s hierarchical clustering method.

Chickpea is regarded as an important food source among grain legumes because of its high nutritional value. To evaluate variation in nutritional quality traits among the studied chickpea genotypes, a range of technological and biochemical analyses was performed on their seeds. The results of these analyses are presented in Fig 1.  There is a strong negative relationship betweenwater absorption capacityand cooking time and a strong positive relationship between absorbability and moisture content. The results showed that, among the 51 chickpea accessions evaluated, 12 exhibited superior technological characteristics (Fig 1).


       
Based on the results of the study, the highest 100-seed weight values were recorded in five chickpea accessions: Filip 07/H127 (40.5 g), Filip 06-114 (41.5 g), Filip 07-183 (41.8 g), Filip 07-115 (42.9 g) and Filip 88-85 (43.9 g). These values were 4.5-8.4 g higher than that of the standard cultivar Cemile, corresponding to an increase of 12.5-23.3% in 100-seed weight. The considerable variation observed in 100-seed weight among the evaluated accessions agrees with previous studies reporting significant genotypic variability for this trait in chickpea. Moreover, high heritability coupled with high genetic advance has been reported for 100-seed weight, indicating that this trait may respond effectively to selection (Dehal et al., 2016; Yücel, 2020). Accessions exhibiting comparatively favourable technological characteristics generally showed water absorption capacities of 14-15 mL and cooking times of 29-30 min. However, because no predefined composite selection index was applied, the accessions were evaluated separately for each technological trait rather than classified into a single overall “superior” group.
       
The biochemical composition of the accessions was evaluated based on protein, tryptophan and lysine contents (Fig 2).


       
For descriptive screening, comparatively high protein content was operationally defined as ≥25.6% DW. These values represented relative increases of approximately 6.7-13.3% compared with Cemile (24.0%), equivalent to differences of 1.6-3.2 percentage points. Although these accessions showed higher mean values, Tukey’s HSD indicated no significant differences from Cemile. The overall effect of accession on protein content was significant (P<0.001), Tukey’s HSD test showed that none of these nine accessions differed significantly from Cemile (P>0.05). Thus, their higher mean protein contents should be interpreted as descriptive differences and potential candidates for further evaluation rather than statistically confirmed superiority over Cemile.
       
Tryptophan content in the evaluated chickpea accessions was lower than that of the standard cultivar Cemile. However, among the 51 accessions analyzed, 11 exhibited tryptophan contents ranging from 210 to 250 mg/100 g dry weight. The highest tryptophan content (250 mg/100 g dry weight) was recorded in the Filip 08-27, Filip 08-65 and Filip 08-58 accessions. In a comparable study, the tryptophan content of chickpea seeds was reported as 0.44 ±0.03 mg g^-1 on a dry-weight basis. Among several legume crops investigated, including soybean, common bean, lentil and chickpea, chickpea was found to have the lowest tryptophan content (Köse et al., 2024).
       
High lysine contents were identified in 12 chickpea accessions. These values were approximately twofold higher than those recorded in the standard cultivar, Cemile. The highest lysine content was observed in Filip 08-65, Filip 09-240, Filip 09-285 and Filip 06-114. Previous studies have also reported that lysine is among the most abundant essential amino acids in chickpea seeds relative to other amino acids (Ipekesen et al., 2022).
       
The relationships among protein, lysine and tryptophan contents were further evaluated using Pearson correlation analysis and principal component analysis (PCA). The correlation matrix revealed a significant positive association between protein and lysine contents, whereas tryptophan showed a weaker relationship with the other biochemical traits. The PCA analysis demonstrated the distribution of chickpea accessions according to their biochemical characteristics (Fig 3).


       
Based on the biochemical analysis, Filip 08-65 was the only accession combining the highest observed lysine content (1294 mg/100 g DW) with the highest tryptophan content (250 mg/100 g DW), although its mean protein content was comparatively moderate (25.6% DW). When expressed relative to protein content, these values corresponded to 50.5 mg lysine/g protein and 9.8 mg tryptophan/g protein, calculated by dividing the amino acid content expressed per 100 g DW by the protein content expressed as g/100 g DW. Filip 03-140 and Filip 09-240 were tied for the highest mean protein content (27.2% DW). Filip 09-240 also contained 1294 mg lysine/100 g DW, corresponding to 47.6 mg lysine/g protein. The second-highest tryptophan value was observed in Filip 07-115 (240 mg/100 g DW), corresponding to 9.2 mg/g protein. Although these results indicate comparatively favourable lysine and tryptophan concentrations in selected accessions, the overall adequacy of their protein relative to the FAO/WHO/UNU reference pattern cannot be established because the complete essential amino-acid profile and protein digestibility were not evaluated. Electrophoretic analysis of globulin storage proteins was performed in the seeds of 51 chickpea genotypes selected for the study using acid polyacrylamide gel electrophoresis (A-PAGE). Four electrophoretic zones (I-IV) were identified and analyzed in the electrophoregrams (Fig 4).


    
Nei’s diversity indices for electrophoretic storage-protein patterns were calculated for individual polymorphic zones of globulin storage protein electrophoregrams were determined in the chickpea accessions (Hıv = 0.464, Hııı = 0.664, Hıı = 0.752 and Hı = 0.562). The highest storage-protein pattern diversity was observed in the β region (Hβ = 0.752). Compared with the other zones, the β zone exhibited greater variation in the number, intensity and migration positions of protein bands, indicating its important role in detecting genetic variation. In another study, genetic diversity was investigated in 76 chickpea accessions. Electrophoretic analysis showed that the γ and β regions had the greatest diagnostic value, enabling reliable differentiation among the accessions. These findings confirm that electrophoretic profiles of globulin proteins can serve as a reliable biochemical tool for assessing genetic diversity, identifying cultivars and selecting promising chickpea forms for future breeding programs (Salmanova, 2026).
       
Based on the dendrogram derived from globulin storage-protein electrophoretic profiles, the chickpea accessions were separated into four clusters (Fig 5). Accessions 14, 15 and 36 formed individual branches, indicating that their storage-protein profiles were distinct from those of the remaining accessions. Most accessions were grouped within one major cluster, reflecting comparatively similar electrophoretic banding patterns. These results represent biochemical differentiation based on storage-protein profiles and should not be interpreted as evidence of genome-wide genetic distinctness. Confirmation of genetic relationships requires DNA-based marker analysis, such as SSR or ISSR approaches (Bouri et al., 2020; Janghel et al., 2021).

Based on descriptive screening thresholds, nine accessions showed comparatively high protein contents (≥25.6% DW), 12 showed high lysine contents (≥1035 mg/100 g DW) and 11 exhibited high tryptophan contents (≥210 mg/100 g DW). Several accessions displayed favourable technological traits, although no multi-trait ranking was applied. Electrophoretic analysis of globulin storage proteins revealed considerable polymorphism among the 51 chickpea accessions, with the β region showing the highest storage-protein pattern diversity (Hβ = 0.752). These protein-profile differences represent biochemical variation and require validation through DNA-based molecular-marker analyses to determine their relationship with broader storage-protein pattern diversity.
       
The identified accessions with favourable nutritional characteristics and distinct storage-protein profiles may serve as preliminary candidates for chickpea breeding programmes aimed at improving seed quality. Further evaluation across environments and using molecular approaches is required to confirm their breeding potential.

This research was supported by the Institute of Genetic Resources of the Ministry of Science and Education of the Republic of Azerbaijan through its institutional research facilities.
 
Disclaimers
 
The views and conclusions presented in this article are those of the authors and may not necessarily reflect the opinions or positions of their affiliated institutions. The authors have taken care to ensure that the information provided is accurate and complete; however, they cannot be held responsible for any direct or indirect losses or consequences that may arise from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish,or preparation of the manuscript.