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Estimation of Biochemical Parameters in Chickpea (Cicer arietinum L.) Genotypes

Rakesh Kumar Yadav1, Manoj Kumar Tripathi1,2,*, Sushma Tiwari1,2, Ruchi Asati1, Niraj Tripathi3, R.S. Sikarwar1
1Department of Genetics and Plant Breeding, College of Agriculture, Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior-474 002, Madhya Pradesh, India.
2Department of Plant Molecular Biology and Biotechnology, Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior-474 002, Madhya Pradesh, India.
3Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur-482 004, Madhya Pradesh, India.

  • Submitted29-03-2024|

  • Accepted26-06-2024|

  • First Online 18-07-2024|

  • doi 10.18805/LR-5327

ABSTRACT

Background: Cicer arietinum (L.), is a legume being grown world wide as a good source of vegan protein. It is a vital part of human feed. Quantification of biochemical parameters of seed is one of the requirements for breeding programmes to develop cultivars suitable for human consumption and food industry. The objective of the present investigation was to evaluate chickpea genotypes on the basis of biochemical parameters to select superior germplasm for further crop improvement.

Methods: In this investigation, seventy-one chickpea genotypes employed for different biochemical parameters analysis including protein, total free amino acid and sugar content, reducing and non-reducing sugar, phytic acid, total phenol, flavonoid and tannin content along with DPPH radical scavenging activities.

Result: Genotypes showed substantial variation for different biochemical parameters. Maximum seed protein content was found in genotype JG315 (25.1%) and lowest in SAGL-152344 (14.3%), whilst content of amino acid ranged between 2.4 mg/g (SAGL-152318) to 9.51 mg/g (SAGL-152330). Maximum phytic acid content was evident in genotype SAGL22-122 (20.7 mg/g) and lowest in JG315 (4.78 mg/g). Range of total phenol content varied between 0.72mg/g (RVSSG 92) to 1.91 mg/g (ICCV20116).

INTRODUCTION

Cicer arietinum (L.) is the utmost domineering winter season legume also known as garbanzo bean or Bengal gram and Chana in India (Varshney et al., 2013). It is generally classified into two diverse market classes; small seed size, dark in color: the desi type and the larger seed size, light color: kabuli type (Kabuo et al., 2015; Ugandhar et al., 2018; Yadav et al., 2023a). Chickpea occupies about 85% and fifteen percent of the international and national chickpea production areas, correspondingly (Asati et al., 2023; Yadav et al., 2023b).
       
Seeds of chickpea are main and inexpensive source of proteins, carbohydrates, vitamins, fibers, minerals and essential amino acids (Dhankhar et al., 2019; Tiwari et al., 2023 a). It plays an important part in guaranteeing good nutrition and delivers food safety (Tiwari et al., 2023b).
       
At the present, there is a necessity to produce more nutritious and high-quality food crops (Garg et al., 2018; Tiwari et al., 2023c). As chickpea nutritious superiority depends on nutritional and anti-nutritional aspects (Gupta et al., 2021), breeders need to evaluate legumes for these factors to develop superior genotypes and promote their ingesting (Asati et al., 2022). In the current investigation, an effort was made to assess chickpea genotypes for their nutritional and anti-nutritional parameters along with their quality aspects.

MATERIALS AND METHODS

Nutritive composition of seed was analyzed at Biochemical Analysis Laboratory, Department of Plant Molecular Biology and Biotechnology, College of Agriculture, RVSKVV, Gwalior, Madhya Pradesh, India. Seventy-one chickpea genotypes were chosen for analysis and its mature dry seeds harvested in rabi seasons 2022 and 2023 (Table 1). Distant and damaged seeds were removed and the samples were kept for 45 days at room temperature in cotton bags to accomplish an equal level of moisture. The seeds were crushed to fine powder with mortar and pestle and the contents were passed through 80ìm sieve to have even powder, which was stored for extraction and analysis of different biochemical parameters. The analysis was carried out in the normal temperature and humidity range.
 

Table 1: Biochemical parameters in chickpea genotypes.


 
Estimation of different biochemical parameters
 
Protein and total amino acid content
 
The procedure described by Lowry et al., (1951) was used to extract the total protein content. Whereas, the procedure described by Moore and Stein (1948) was employed to obtain the total free amino acid content. 
 
Sugar estimation
 
A 0.1 g sample of seed was mixed with 10 ml of 50% alcohol and the mixture was centrifuged for 15-20 minutes at 5000 rpm. The supernatant was collected and allowed to evaporate in a water bath until two to three milliliters were left. After thoroughly mixing and letting it rest at room temperature for ten minutes, 10 ml of CCl4 was added. Two layers developed with the addition of CCl4. The top layer was separated and 10 ml of distilled water was added to it, while the lower layer was thrown away. Total and reducing sugars were estimated from this extract.
 
Reducing sugar
 
Reducing sugar content was assessed following the Miller (1959) method.
 
Total sugar
 
Dubois et al., (1956) method was used to calculate total sugar using a phenol reagent.
 
Non-reducing sugar
 
The non-reducing sugar content was obtained by following formula:
 
Non-reducing sugar = Total sugar - Reducing sugar
 
Extraction of tannin and phytic acid content
 
Tannic acid equivalents were used to estimate the tannins after the extraction process was carried out as per by method described by Schandrel et al., (1970). Following Wilcox et al., (2000) method, the phytic acid was determined from the dry seeds.
 
Extraction of total phenol content (TPC)
 
According to Swain and Hillis (1959) method, the total phenolic contents were extracted and calculated.
 
Estimation of total flavonoid content
 
Total flavonoid content was determined by the method given by Khoo et al., (2013).
 
DPPH radical scavenging assay
 
Radical scavenging activity was measured using the Bersuder et al., (1998) method. The following equation was used to compute DPPH radical scavenging activity:
 
  
 
Where,
Ablank= Absorbance of the control reaction.
Asample= Absorbance of sample.

RESULTS AND DISCUSSION

Characterization of biochemical parameters
 
Chickpea seeds contain essential amino acids, these amino acids are important for the human body for several cell metabolic and biological activities (Rajput et al., 2023). In the current research, protein content of JG315 (25.1%) followed by JAKI 9218 (24.8%) genotypes were higher amongst 71 genotypes, whilst lowest protein content was evident in genotypes SAGL-22122 (16.8%). Highest free amino acid content was found in genotypes SAGL-152330 (9.51 mg/g) followed by ICCV 201206 (8.87mg/g), whereas, lowest was evident in SAGL-152318 (2.4 mg/g) (Table 1). Our findings agree with those of Bhagyawant et al., (2018). Reducing sugar content differed in chickpea seeds from 0.86% (SAGL-152250) to 2.37% (JAKI 9218) and total sugar content varied between SAGL-152222 (2.15%) to JAKI 9218 (5.67%), whereas range of non-reducing sugar content arrayed between 1.2% (SAGL-152336) to 3.3% (JAKI9218). Rajput et al., (2023) and Tiwari et al., (2023c) observed similar results.
       
Two anti-nutritional factors viz., tannins and phytic acid were estimated in 71 chickpea genotypes. Maximum phytic acid content was seen in genotype SAGL22-122 (20.7 mg/g) and minimum in JG315 (4.78 mg/g). The highest tannin content was investigated in genotype viz., SAGL-153226 (9.45 mg/g) and lowest in SAGL152258 (4.121 mg/g). Kaur et al., (2013) found similar consequences.
       
The phenolic chemicals in grains are primarily responsible for their antioxidant activity. Phenols are essential for deactivating metal-ions. Additionally, the polyphenols help to prevent cardiovascular disorders by scavenging hydroxyl and peroxyl radicals (Luo et al., 2002). In the present study, range of total phenol content for chickpea genotypes varied between 0.72 mg/g (RVSSG92) to 1.91 mg/g (ICCV20116). Whilst, total flavonoid content arrayed between 0.28 mg/g (SAGL-152278) to 1.59 mg/g (SAGL 22-124). These findings are in accordance with results of Kaur et al., (2013) and Jameel et al., (2021).
       
Range of DPPH for chickpea genotypes varied from 28.84% to 47.53%. It was seen that the genotypes varied significantly in respect of DPPH %. Highest DPPH % was evident in genotype SAGL-152222 (47.53%) followed by SAGL-161025 (47.12%), while, lowest in SAGL 22-121 (28.84%). These findings are in accordance with outcomes of Bhagyawant et al., (2015).
 
Correlation analysis among biochemical parameters
 
Protein had significant and positive association with total free amino acid (r=0.5475) and significantly and negatively correlated with phytic acid content (r= -0.7982). Total free amino acid was significantly and positively associated with total sugar content (r=2517) and reducing sugar content (r=0.2401) whereas significantly and negatively correlated with phytic acid content (r=-0.4966). Reducing sugar content was significantly and positively associated with total sugar content (r=0.8356), whilst total sugar content had positive and significant correlation with non-reducing sugar (r=0.8711). Total flavonoid had positive and significant correlation with DPPH (r=0.2667) (Table 2). These associations between different parameters may help for the selection of elite genotype (s).
 

Table 2: Correlation analysis between different biochemical parameters.


 
Phylogenetic cluster analysis and expression analysis among biochemical parameters
 
Based on heat map dendrogram (Fig 1) we can classify 71 genotypes in two major groups A and B having 16 and 55 genotypes respectively. Group A is further divided into A1 and A2 having 6 genotypes and 10 genotypes correspondingly, whereas group B is divided into B1 and B2 which comprised of 13 and 42 genotypes respectively and further division goes on. The heat map is ranged between -2 to 2 and it represents the level of expression of different biochemical parameters (Fig 1). The heat map revealed two major clusters, the first comprising the DPPH and protein, while second major cluster comprising the remaining biochemical parameters. Similar kind of studies were also performed by Sharma et al., (2021) and Sistu et al., (2023) as they have also estimated different biochemical parameters and represented heat map for showing the level of expression.
 

Fig 1: Heat map of chickpea genotypes based on different biochemical parameters.


 
Principal component analysis of biochemical parameters
 
A principal component analysis (PCA) was conducted employing all investigated traits. A biplot was built by plotting the PC1 grooves (x-axis) against PC2 notches (y-axis) for each parameter and all genotypes (Fig 2). Scree plot (Fig 3) displayed that, among the 10 principal components, PC1, PC2, PC3, PC4 had mined Eigen values >1. The rest principal components had Eigen values <1 so have not been deliberated further. Cumulatively, these four principal components donated 73.25% of the total variability in the genotypes. Out of a 100% collective variation, the PC1 exhibited maximum variability (27.26 %) followed by PC2 (22.81%) (Table 3).
 

Fig 2: PCA biplot analysis for different biochemical parameters of chickpea genotypes.


 

Fig 3: Scree plot between eigen value and principal components of biochemical parameters.


 

Table 3: Principal Component analysis (PCA) for different biochemical parameters.


       
On the basis of angle between the vectors, protein, total free amino acid, total flavonoid, reducing sugar content, total sugar content and non-reducing sugar content were strongly and positively correlated traits among studied biochemical parameters (Fig 4), while phytic acid content was negatively correlated and DPPH, total phenol content and tannin content not correlated with each other. These findings are in accordance with results of Bhagyawant et al., (2015) and Jameel et al., (2021).
 

Fig 4: Scatter plot with correlation value for biochemical parameters of 71 chickpea genotypes.

CONCLUSION

Chickpea is the most consumable pulse crop and efficient source of protein, vitamin and minerals. Programmes for improving the chickpea crop are now being actively pursued around the world. Selecting germplasm with improved nutrients is essential. In the present investigation, chickpea seed of various genotypes contain considerable amount of protein and other studied biochemical parameters. Maximum protein, amino acid, sugar (reducing sugar, total sugar, non-reducing sugar), phytic acid, tannins, phenol, total flavonoid and DPPH was investigated in genotypes viz., JG315, SAGL-152330, JAKI 9218, SAGL 22-122, SAGL-153226, ICCV 20116, SAGL 22-124 and SAGL-152222 respectively. This information therefore, may be proved useful for selecting the donor parent (s).

CONFLICT OF INTEREST

Authors declare there is no conflicts of interests.

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