1. Introduction
Cowpea (Vigna unguiculata (L.) Walp), a legume crop that is grown worldwide, is beneficial for human consumption, animal feed and the environment [1] - [6] . It was estimated that, the total area under production amounts to about 14 million hectares with an annual production of over 5.4 million tones worldwide [7] [8] . Cowpea is widely distributed throughout the tropics, but Central and West Africa amounts to 64 percent of the area with about 8 million hectares followed by about 2.4 million hectares in Central and South America, 1.3 million hectares in Asia and about 0.8 million hectares in East and Southern Africa [9] . Cowpea is also grown as a vegetable (both for the leafy greens and the green peas). It primarily provides protein, starch, soluble sugar, amino acids, fatty acids, minerals and vitamins which benefit human health.
The content of soluble sugar is an important physiological trait which influences cooking quality in seeds and seed production [1] [10] [11] [12] . In fact, cowpea breeding has to feature traits desired by consumers in the market as well as farmers. The “sweet” trait, characterized by a sweeter and milder taste could help broaden cowpea consumption in the market. It not only provides calories as energy to human body, but also improves the cooking quality by producing a desirable taste and mouth feel. Additionally, soluble sugar has been proposed to play an important role in storability and abiotic stress tolerance in seeds, which are highly sensitive to environmental stresses [13] . Increases in levels of sugar in seeds have been correlated with the onset of desiccation tolerance during seed development [14] . Similarly, the loss of these soluble sugars in seeds at the beginning of germination has been found to coincide with the re-acquisition of desiccation sensitivity [15] . Seed longevity and development have also been positively correlated with the level of soluble sugars in seed [16] .
However, this seed soluble sugar content characteristic has not received much attention in the cowpea breeding program, which has been primarily concerned with increasing yield, pest resistance, draught tolerance, protein content and other quality characteristics [3] [4] [17] . A few articles have reported on variable soluble sugar content in different genotypes in cowpea. Baptiste et al. (2011) reported three methods measuring soluble sugar content in cowpea seeds [1] . Nassourou et al. (2017) assessed the amount of total soluble sugar among 15 fully homozygous cowpea cultivars using the phenol sulfuric acid reagent method [3] . The objective of the present work was to evaluate the soluble sugar content in cowpea seeds and investigate elite germplasm with desired soluble sugar contents in order to propose a suitable breeding strategy for improving sweetness of cowpea seeds.
2. Materials and Methods
2.1. Plant Materials
A total of 113 cowpea genotypes (Table 1) were used in this study, including 89 USDA GRIN cowpea accessions and 24 advanced breeding lines from the cowpea
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Table 1. List of cowpea genotypes and their mean soluble sugar content.
breeding program at the University of Arkansas, AR, USA. The field experiment was conducted in three different locations within Arkansas State (Fayetteville, Alma, and Hope) in 2016. During the growing season, no pesticides or herbicides were sprayed to control pests, diseases, and weeds. Cowpea seeds were harvested when the color of pods changed and became mature. Seeds were dried and stored at room temperature until the soluble sugar measurement at laboratory.
Cowpea seed coat color is an important commercial trait to breeders in nursery and consumers in the market because cowpea has a diversity of colorful seed coats (Figure 1). Six seed color patterns were included in this study: black, blackeye, browneye, cream, pinkeye, and red color.
2.2. Sample Preparation
Within each location, seeds from each row were bulk-harvested. Seed soluble sugar content referred to the total sugar content was extracted using the following protocol. An 20 g of cowpea seeds of each sample was ground using a coffee grinder (Hamilton Beach) for 1 min. 2 or 3 g portion of the ground flours was sieved through 100# (nominal wire diameter 0.1 mm, Seedburo Equipment Company). 1.0 g of each sample was weighed and then transferred to a 50 ml microfuge tube. And then 40 ml 80% ethanol was added to each tube, homogenized for 1 min in a vortex and placed in a water bath at 80˚C for 20 min. After this treatment, the tubes were centrifuged at 6000 rpm for 10 min. 2 ml of the supernatant was then transferred into a 100 ml fresh flask to be diluted 50 times.
2.3. Equipment
GENESYSTM 20 Visible Spectrophotometry (Thermo Scientific USA), an instrument which determines the amount of light absorbed while passing through a sample, was used to measure soluble sugar in seeds at laboratories, Department of Food Science, University of Arkansas, Fayetteville, Arkansas, USA.
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Figure 1. Six seed coat color patterns in cowpea: (A) Black color (ex. PI354832, 32.55 mg/g); (B) Blackeye color (breeding line 09-925, 80.94 mg/g); (C) Browneye color (PI583202, 84.53 mg/g); (D) Cream color (PI663140, 45.98 mg/g); (E) Pinkeye color (cultivar Empire, 86.05 mg/g); and (F) Red color (PI582868, 40.42 mg/g).
2.4. Standard Curve Establishment
The amount of total soluble sugar was measured by the phenol-sulfuric acid reagent method [18] . A total of 50 ml of glucose (AR, Sigam-Alorich) stock solutions were prepared at the following concentrations: 0, 10, 30, 50, 70, and 100 µg/ml standard curve for glucose. Then 0 ml; 0.5 ml; 1.5 ml; 3.5 ml; and 5.0 ml of glucose stock solution were each poured into a 10 ml recipient. 1 ml was taken from each solution and poured into a test tube for measurement of soluble sugar content with colorimetric phenol-sulfuric acid by Spectrophotometry.
After many repetitions, a standard glucose curve with concentration 0, 10%, 30%, 50%, 70% and 100% was set up in this study (Figure 2). Six points look smooth and straight without double to be determined, known as the smooth curve. The best standard curves having the equation Y = 0.0039X + 0.0235 (R2 = 0.99878) was achieved and accomplished consummation, where X is absorbance coefficient at 490 nm wavelength on Spectrophotometer and Y is soluble sugar content (%) in cowpea seed storage. Daily repeats allowed a soluble sugar standard curve to be constructed which was used to determine the soluble sugar concentration in each cowpea sample.
2.5. Quality Assessment
The use of phenol-sulfuric acid with a phenol concentration of 2% provided a relatively simple and reliable colorimetric method to quantify the total soluble-sugar concentration. Spectrophotometer measures the absorbance with wavelength at 490 nm. Absorbance was estimated using Beer’s law:
I0: incident radiation or radiation transmitted.
I: radiation transmitted by the sample.
abc: absorbance of the sample.
a: absorbancy coefficient.
b: path length of the sample.
c: concentration of the sample.
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Figure 2. Glucose standard curves was computer generated based on the five standard concentrations.
Based on each sample’s absorbent value, the concentration of total soluble sugar in each cowpea sample was estimated using the regression equation form the standard curve. Individual extractions were replicated two times for each sample.
2.6. Date Collection and Analysis
Descriptive statistics including mean, minimum, maximum, variance and standard error were computed using the “Tabulate” option of JMP Genomics 7 (SAS Institute, Cary, NC). The distribution graph of the cowpea soluble contents was drawn using the “Distribution”. Analysis of variance (ANOVA) was performed using the general linear model (GLM) of JMP Genomics 7. Student T-test at α = 0.05 was used for multiple comparisons of the least square mean (LSM) among the genotypes and the different seed colors.
3. Results and Discussion
3.1. Soluble Sugar Quantification in Cowpea Seeds
The soluble sugar content of cowpea seeds affects the final flavor of cowpea food and cowpea-derived products. This work was to use a simple, low-cost, spectrophotometric method for soluble sugar quantification in cowpea seeds. Significant variations were observed for soluble sugar contents in 113 diverse cowpea genotypes tested in this study (Table 1). The range in soluble sugar contents among tested samples was from 32.6 mg/g to 86.1 mg/g with an average of 54.5 mg/g, based on the standard cures, indicating seed soluble sugar contents having wide variations among the 113 cowpea genotypes. Soluble sugar content has a bimodal distribution (Table 1 and Figure 3).
The result of soluble sugar content in this experiment was apparently higher than previous report from other studies. Akpapunam et al. (1979) reported that the average of sucrose contents was 22 mg/g from 13 American cowpea (Vigna sinensis) varieties extracted by 70% ethanol [19] . Onigbinde and Akinyele (1983)
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Figure 3. Distribution of soluble sugar content in cowpea seeds among 113 genotypes.
reported that the average content of the sucrose was 8 mg/g in mature dry seeds of 20 varieties of cowpea grown in Nigeria [20] . Nassourou et al. (2017) reported the total soluble sugar content in cowpea seeds using the phenol sulfuric acid reagent method among 15 cowpea cultivars and observed that the highest values were 40.79 mg/g (IT93K-693-2) and 40.64 mg/g (BR1) with a mean of 22.31 mg/g [17] . The content of soluble sugar in cowpea seeds in this study was obviously higher than what Onigbinde et al. (1983) and Nassourou et al. (2017) reported, which may be caused by different cowpea genotypes and extraction method. The result also was shown higher than that Akpapunam et al. (1979) reported due to their different species.
The top five genotypes with highest soluble sugar content in cowpea seeds were determined to be “Empire” (86.05 mg/g), PI583202 (84.53 mg/g), 09-655 (82.05 mg/g), PI601085 (81.57 mg/g), and 09-529 (80.94 mg/g). In contrast, the USDA germplasm accession PI354832 (32.55 mg/g) and PI354865 (36.26 mg/g) had the lowest soluble sugar content among the 113 cowpea genotypes tested. Overall, the soluble sugar content in Arkansas cultivars and advanced breeding lines was higher than the USDA germplasm accessions. However, higher soluble sugar content was also observed in some USDA accessions such as PI583202 (84.50 mg/g) and PI601085 (81.60 mg/g). These results indicated that high level of soluble sugar content not only can be discovered from released cowpea cultivars, but also from cowpea germplasm. These cowpea genotypes with higher soluble sugar content will be beneficial to the future breeding program and genetic improvement for meeting the needs of the market and consumers.
3.2. Compare Seed Soluble Sugar Content in Genetic Background
A highly significant difference (P < 0.001) was observed between Arkansas cowpea cultivars/advanced breeding lines and USDA cowpea germplasm accessions (Table 2). Most of the highest soluble sugar contents are found in Arkansas cowpea cultivars and advanced breeding lines. The average soluble sugar content of Arkansas cowpea cultivars/advanced breeding lines was 71.13 mg/g, significantly higher than the average soluble sugar content of cowpea germplasm accessions (50.04 mg/g). Cowpea breeding program in University of Arkansas have had more than 60 years of history since the first variety “Monarch” was released in 1956. After selection and enhancement from generation to generation, the Arkansas cowpea cultivars/breeding lines have developed more closely to
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Table 2. Descriptive statistic for soluble sugar (mg/g) by genetic background.
consumer’s needs, including quality improvement and increasing breeder’s expectation in seeds. Regardless of intentional or unintentional selection, the seed soluble sugar content of cowpea cultivars and advanced breeding lines has been also improved and increased after breeding and genetic improvement in Arkansas State and the Southern region of US (Table 2). These cultivars and advanced breeding lines exhibited more soluble sugar content and better adaptation to growing conditions in Arkansas State and the Southern region than USDA germplasm accessions.
3.3. Soluble Sugar Content Evaluation in Different Seed Color
Cowpea seed coat color is an important morphological character and commercial trait to costumers because of diversity of cowpea seed coat (Figure 4), which provides various products with colorful seed coat for consumers to choose in supermarket or grocery stores. In order to represent detailed cowpea seed coat color description in markets, ix major types of cowpea seed colors were used for this study: black, blackeye, browneye, cream, pinkeye, and red color. The variance of soluble sugar content in color coat seed was measured using GENESYS™ 20 Visible Spectrophotometer in this study (Table 1 and Table 3). The results showed that the pinkeye color cowpea seeds had the highest soluble sugar content averaging 72.42 mg/g, and the black color cowpea contained the
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Figure 4. Average soluble sugar content (mg/g) per seed coat color.
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Table 3. Descriptive statistics for soluble sugar (mg/g) by seed coat color.
lowest soluble sugar, averaging 43.58 mg/g in cowpea seeds. The browneye color (60.80 mg/g) contained almost similar amount of soluble sugar compared with that of blackeye color (59.38 mg/g). Overall, the soluble sugar content in pinkeye color (72.42 mg/g) > browneye (60.80 mg/g) ≥ blackeye (59.39 mg/g) > cream (49.53 mg/g) ≥ red (49.08 mg/g) ≥ black (43.58 mg/g). This study will inform consumers to buy which type of the seed color cowpea that fits their needs in terms of cooking quality and sweetness.
4. Conclusion
A total of 113 cowpea genotypes were measured for their soluble sugar contents in seeds using Spectrophotometer by phenol-sulphuric acid method in this study. A wide range of solublesugar content were observed from 86.05 mg/g to 32.55 mg/g with an average of 54.52 mg/g. Commercial cultivars and advanced breeding lines from Arkansas region, USA contained higher soluble sugar content than USDA germplasm did. The soluble sugar content changed with the seed color coat: pinkeye (72.42 mg/g) > browneye (60.80 mg/g) ≥ blackeye (59.38 mg/g) > cream (49.53 mg/g) ≥ red (49.08 mg/g) ≥ black (43.58 mg/g).