Cowpea (Vigna unguiculata [L.] Walp.) is annual herbaceous plant, cultivated for its leaves, green pods, grains and adaptability to diverse agro-climatic conditions. Because of the high protein content and superior biological value on a dry weight basis of the green leaves, cowpeas are sometimes referred to as "vegetable meat" or "poor man's meat." They also supply fibre, vitamins, and minerals. Additionally, it is grown as a green manure, fodder, cover, or catch crop. Cowpea grains have higher percentage of vital minerals than meat, fish, or eggs, including calcium (826 mg/kg) and iron (53.2 mg/kg), both of which are excellent for lowering blood cholesterol (Rangel et al., 2003; Achuba, 2006; Boukar et al., 2019). In India, the mean grain yields of cowpea are between 249 to 980kg/ha which is far less than the potential yield 3t per hectare elsewhere (Molosiwa et al., 2016). A major constraint to achieve this production of cowpea grains in the tropics and sub tropics is lack of high yielding cultivars and poor cultivation practices. Therefore, development of best performing, locally adaptable potential cultivars offer a simple and cost-effective method to produce higher yield. Seed quality traits are critical determinants of yield potential and crop performance, influencing germination, seedling vigour, and overall productivity. In cowpeas seed size is considered as an important trait as it directly influences productivity along with seed colours, which determine grain quality for marketing (Wirianto et al., 2024). Characterizing germplasm based on seed morphological and physiological traits provides essential insights for breeding programs. Seed colour, shape, and eye colour are key morphological descriptors that vary significantly across genotypes, reflecting genetic diversity. Physiological traits, including seed germination, vigour indices, protein content, and dehydrogenase activity, further highlight genotype-specific performance under different conditions. 

Although morphological and physiological traits are recognized as important, data on their variation across cowpea genotypes under stress conditions remains limited. This lack of information hinders the identification of genotypes with superior adaptive traits, which are crucial for improving crop performance. While previous studies have emphasized the importance of evaluating seed quality attributes, there is insufficient research on the response of cowpea genotypes to controlled deterioration-a key factor in simulating stress conditions (Sathya et al., 2023). To address this gap, this study aims to characterize cowpea germplasm lines based on seed traits and quality parameters, identify superior genotypes, and evaluate their performance under controlled deterioration. The findings will offer valuable insights for breeding programs focused on enhancing seed quality and yield potential, particularly under stress conditions.

Seed material. The experiment was conduct in the laboratory of the department of seed science and technology, G.K.V.K. at University of Agricultural Sciences Bangalore. 118 diverse germplasms along with two check varieties (C-152 and KBC-9) were evaluated for seed quality such as germination, seed moisture content, total dehydrogenase activity and protein content. Further, they were subjected to controlled deterioration test to study the effects of the ageing on the physiological statuses of seeds.

Seed morphological characterization. Seed traits of each genotype were observed at physiological maturity. Eye colour was recorded as tan brown, red, or black; seed colour as white, brown, red, or black; and seed shape as kidney, elliptical, or rhomboid. 

Hundred seed weight. Cleaning was done to remove the cracked, broken and abnormal seeds, other seeds, foreign matters, etc. by hand picking. Hundred seeds were counted from the harvested plants of each plot and were weighed and expressed in grams.

Standard germination test. The germination test was conducted as per ISTA guidelines in the laboratory by using between paper method (Anon., 2021). One hundred seeds are randomly selected from each genotype in three replications and placed equidistantly on the paper towel, they are further rolled and kept in a germination chamber with a temperature of 25 ± 1°C and Relative humidity of 90 per cent. The first count and the final count of the germinated seedlings was taken on the 5th and 8th day respectively and the percentage of germination was expressed based on the number of normal seedlings present.

Seed germination = [No. of normal seedlings/No. of seed put for germination] × 100

Shoot length, root length and mean seedling length. From the standard germination test, shoot length (collar region to shoot tip), root length (collar region to primary root tip), and seedling length (primary root tip to apical shoot tip) were measured on ten randomly selected normal seedlings per genotype replication. Measurements were taken on the final count day and expressed as mean lengths in centimetres.

Seedling dry weight. Ten normal seedlings used for shoot and root length measurements were dried in a hot air oven at 80°C for 17 ± 1 hour, cooled in desiccators for 45 minutes, and weighed. The mean seedling dry weight was expressed in milligrams (mg).

Seedling vigour indices. Seedling vigour indices were calculated following (Abdul-Baki and Anderson 1973). Vigour index-I and Vigour index-II were computed as

Vigour index-I = Germination (%) × [Root + Shoot length (cm)]

Vigour index-II = Germination (%) × Mean seedling dry weight (mg)

Seed moisture content. Seed moisture content (%) was determined using the oven-dry method (Anon., 1985). Five grams of seeds were dried in aluminium cups at 103°C for 17 hours, and then cooled in desiccators for 30 minutes before weighing. The moisture content was calculated using the formula:

Seed moisture content (%) = [M2 - M3] / [M2 - M1]× 100

Where, M1 = The weight of the container with its lid; M2 = The weight of the container with its lid and seeds before drying; M3 = The weight of the container with lid and seeds after drying.

Electrical conductivity (μS/cm/gm). Twenty-five seeds from each genotype replication were soaked in 25 ml distilled water for 24 hours at 25 ± 1°C. The electrical conductivity (EC) of the seed leachate was measured using a digital conductivity meter (Model: Systronic 306), adjusted by subtracting the EC of distilled water, and expressed in μS/cm/gm (Anon., 2021).

Total Dehydrogenase (TDH) activity (A480). The seed coats of 10 seeds of each genotype in three replications were removed, and the embryonic axes were soaked in 0.5% tetrazolium chloride solution, incubated at 25 ± 1°C in the dark for 4 hours. After thorough washing, the red formazan from stained embryos was eluted in 5 ml of 2-methoxy ethanol for 24 hours in an airtight container. The extract was decanted, and color intensity was measured at A480 using a spectrophotometer. Dehydrogenase activity was expressed as optical density at A480 (Perl et al., 1978).

Seed protein content. The total soluble protein content (%) was estimated as per the method prescribed by Lowry et al. (1951). Reagent solutions included sodium carbonate (Solution A), sodium potassium tartarate (1.35%), copper sulphate (5.5%), and Folin Ciocalteu reagent (FCR, 1:1 dilution). Solution C (A + B) was prepared fresh, and BSA served as the standard. 100 mg of dried sample was extracted with 0.1M sodium phosphate buffer (pH 7.0), centrifuged, and the supernatant was reacted with reagents, incubated, and absorbance was recorded at 660 nm. Protein content was calculated using a BSA standard curve and expressed as a percentage.

Adjustment of Seed Moisture Content for CD. After determining the initial seed moisture content (SMC). The moisture content of the seeds was adjusted to the desired value based on the ISTA (Anon., 2017 by using the formula.

W2 = [100 – A] × [W1/100 – B]

Where, A = initial seed moisture content (%), B = desired seed moisture content (%), W1 = initial weight of the seeds (g), W2 = final weight of the seeds with desired moisture content (g)

Seeds of each replication imbibed on a moist germination /filter paper, placed in a suitable container.

Controlled Deterioration test. Once seeds have reached the required weight, each replication was placed in sealed aluminium foil packed and equilibrated at 4°C overnight to ensure an even distribution of moisture. Seeds package was then placed in a plastic envelope, allowing no ingression of water, and kept in a water bath at ±40°C for up to 24hr ± 15min.  After which routine germination test is followed, only number of normal seedlings were and expressed as the percentage of germination. 

Statistical Analysis. The statistical analysis and interpretation of the experimental data for controlled deterioration test was done by using Fisher’s method of Analysis of Variance technique as outlined by Gomez and Gomez (1984). The level of significance used in ‘F’ and ‘t’ tests was at five per cent. Critical difference values were calculated wherever F test was significant.

Seed morphological characterization. Cowpea germplasm lines were characterized based on seed colour, shape, and eye colour. Variations included 19 lines with white seeds, 24 with red, 4 with black, and 73 with brown (Fig. 1). Tan brown seed eye colour was observed in 92 lines, red in 15, and black in 13. Seed shapes were elliptical (48 lines), rhomboid (41), and kidney-shaped (31) (Table 2). Kabas et al. (2007) reported diverse seed coat colours, including white, cream, green, buff, red, brown, and black in cowpea. Henshaw (2008) noted various seed coat textures and shapes like kidney, rhomboid, ovoid, globose, and crowder.

Hundred seed weight. Cowpea genotypes and checks showed significant differences in 100-seed weight. Genotype EC-075180 recorded the highest weight (18.70 g), while among checks, KBC 9 had a higher seed index (10.67 g) (Table 3). This variation is attributed to genetic variability, efficient photosynthesis, and nutrient distribution during seed filling. Similar findings were reported by Peksen et al. (2004).

Seed germination. The primary objective of seed production is to achieve high germination rates. According to minimum seed certification standards, cowpea seeds should have at least 75 per cent germination. In this study, all cowpea genotypes exceeded this threshold. Seed germination varied significantly among different genotypes and checks. Genotype V- 578 recorded the highest seed germination (96.00 %) (Table 3). The high germination percentages may be attributed to genetic traits, water use efficiency, and nutrient uptake, which contribute to increased storage of food reserves that are utilized during germination and plant growth (Ranjitha et al., 2016). A similar effect of variety on seed germination and its components was observed by Olasoji et al. (2013).

Seed moisture content. Seed moisture content of different genotypes and checks resulted in non-significant difference for seed moisture content. Minimum moisture content was recorded in genotype NBC 51 (7.54 %), while maximum moisture content was recorded in genotype GW HOPE (11.29 %). Whereas, among checks C 152 recorded minimum moisture content (8.41 %) (Table 3). Similar results were reported by Ranasingh et al. (2021).

Shoot length, root length and mean seedling length. The shoot length varied significantly, with IC 97767(10) recording the highest (18.00 cm), followed by NBC 24 (15.50 cm), and IC 45061 with the lowest (6.34 cm). Root length also varied significantly, with SUPER 30 having the longest (22.72 cm), followed by C 33 (22.34 cm), and PMCP1131 the shortest (6.82 cm). Mean seedling length showed no significant variation; however, NBC 016 had the highest (33.07 cm), Overall, IC 97767(10) excelled in shoot length, SUPER 30 in root length, and NBC 016 in total seedling length, indicating its potential for breeding programs due to its balanced growth.

Seedling dry weight. Significant variation was observed, with EC 390287 recording the highest (43.27 mg), followed by CP 98 (41.97 mg), and GW HOPE the lowest (26.52 mg). Among checks, KBC 9 had the highest mean dry weight (36.20 mg). 

Seedling vigour indices. Seedling vigour index-I showed no significant differences, with NBC 016 recording the highest value (2983), driven by high germination percentage and mean seedling length. Seedling vigour index-II varied significantly, with EC 390287 achieving the highest value (3844), followed by CP-98 (3747), and GW HOPE the lowest (2364). KBC 9 also excelled among checks (3222), attributed to high germination percentage and mean seedling dry weight (Table 3).

Electrical conductivity (μS/cm/gm). The electrical conductivity  (mScm^-¹g^-¹) of cowpea genotypes, varied significantly. DC-15 recorded the lowest conductivity (2.67), followed by IC 402159 (3.34), while PCP 1124-1 had the highest (8.99). Among checks, C 152 exhibited the lowest value (4.13) (Table 3). Electrical conductivity, a sensitive indicator of seed quality, negatively correlates with other quality traits, as noted by Hibbard and Miller (1928); Natarajaratnam et al. (1987).

Total dehydrogenase activity (A480nm). Genotype EC- 075180 showed the highest TDH activity (3.98) whereas among the checks, KBC 9 exhibited the highest dehydrogenase activity (2.24) (Table 3). The increased total dehydrogenase activity could be attributed to the high vigour of seeds, which contain more active, living cells, supporting better germination and growth. Similar results were reported by Basu and Parida (2023).

Protein content (%). The significant variation in total seed protein content among genotypes and checks can be attributed to genetic differences, environmental factors, and the interaction between them. Genotype EC- 492292 exhibited the highest protein concentration of 29.93 per cent, indicating it possess genetic traits that enhance protein synthesis and accumulation during seed development (Table 3). Variations in physiological processes, such as nitrogen metabolism and storage protein synthesis, may also contribute to these differences across genotypes and checks. Similar findings were reported by Guo et al. (2022).

Controlled Deterioration (CD) Test. Under controlled deterioration, the percentage of normal seedlings and germination rate significantly declined at the (5%) level, decreasing from 90.88 per cent in NBC 14 to 75.00 per cent after 24 hours, (48%) after 48 hours, and (24%) after 72 hours. Similarly, NBC 12 showed an initial germination rate of (91.83%), dropping to (60%) in 24 hours, 41 per cent in 48 hours, and 25 per cent in 72 hours (Table 4) (Fig. 2). As the ageing conditions intensified, with seed moisture content increasing to (20%) and the ageing period extending from 0 to 3 days, the decline in the percentage of normal seedlings followed a corresponding pattern. The decline was also prominent in all other 15 cowpea genotypes, though their magnitude were overall less in NBC 14 this can be due to higher 100 seed weight (13.64 gm), high germination (90.88%) and genetic factor. Similar findings were reported by Khan et al. (2015).

Table 1: Analysis of variance (ANOVA) for seed quality parameters in cowpea (Vigna unguiculata (L.) Walp) genotypes.

ns P > 0.05; * P <= 0.05

Fig. 1. Variation in seed colour of distint cowpe genotypes.

Table 2: Characterization of cowpea germplasm for seed quality traits.

Table 4: Seed germination of better performing cowpea (Vgina unguiculata (L.) Walp) genotype after controlled deterioration test.

Fig. 2. Germination percentage of better performing cowpea genotypes after controlled deterioration test.

The results give scope for improvement of cowpea breeding and seed quality. The morphological diversity, high germination with V-578, highest seed weight with EC- 075180 and maximum protein content with EC- 492292, present the prospects for breeding for high yielding, nutrient rich verities. Genotype superior in seedling vigour with (IC-97767(10), SUPER 30) can impart better early growth. Indicators of seed quality such as electrical conductivity and dehydrogenase activity support efficient screening, but controlled deterioration tests provide information toward the development of genotypes with better storage potential. Genetic and physiological studies coupled with advanced tools like marker-assisted selection and genomic studies can accelerate the development of robust, high-quality varieties of cowpea specifically tailored to diverse agro-climatic conditions towards sustainable agriculture and food security.