Assessing the morphological variation and characterising the proteins of bambara groundnut (Vigna Subterranea L. Verdc)

Abstract

Bambara groundnut (Vigna subterranea L. Verdc) is an underutilized crop in the African continent. It is a drought tolerant crop and fixes atmospheric nitrogen. Bambara groundnut is primarily grown for the protein content of its seeds and is mainly produced by small scale farmers at the subsistence level. However, despite its importance as a subsistence crop in many African countries, only local landraces of bambara groundnut are still cultivated. Mass selection of a few local varieties for the main agronomic characteristics has been carried out. All the bambara groundnut germplasm in South Africa has not been morphologically characterized. Although the protein of bambara groundnut is of good quality and is rich in lysine, there is no information on the characterisation of these proteins. The presence of antinutritional factors in the crop has also received little attention. This study focused on three major objectives including: (I) to assess the extent of morphological variations among thirty selected landraces of bambara groundnut, (II) to characterize the major seed proteins in these accessions using one dimensional gel electrophoresis, and (III) to determine the presence of any anti-nutritional factors in the seeds of the selected bambara groundnut landraces. 30 accessions of bambara groundnut were evaluated for their variability in agronomic and morphological traits. The field experiment was conducted at ARC-VOPI in Roodeplaat research farm during the 2014/2015 summer cropping season. The field trial was arranged as a complete randomized block design with 3 replications. 18 quantitative traits were recorded to estimate the level of genetic variability among accessions. 4 different methods were employed to extract seed proteins from 30 bambara groundnut accessions in order to ascertain the best method for protein extraction. These methods included: 10%-80% isopropanol, 10% trichloroacetic acid (TCA) in acetone solution, sonication and 2x Lammeli buffer extraction methods. The quick start Qubit® fluorometer protein kit was used to determine the protein concentration in each sample. The samples were then subjected to one dimensional gel electrophoresis. For antinutritional analysis, 5 factors (condensed tannins, free and phytic acid phosphate, polyphenol and trypsin contents) were used to determine the amount of antinutrient in 30 bambara seeds that were ground to a fine powdery flour. 3 replicates of all the samples were ground for each assay evaluated. The flour was then immediately extracted and used for the different assays. The analysis of variance revealed significant differences only in 10 of the 18 phenotypic traits that were evaluated. The UPGMA cluster analysis based on the quantitative traits produced vii four distinct groups of genotypes and a singleton. Genotypes SB11-1A, SB19-1A, SB12-3B and Bambara-12 were found to possess good vegetative characters and are recommended for use as suitable parents when breeding cultivars for fodder production. Desirable yield and yield-related traits were identified in B7-1, SB4-4C, SB19-1A, Bambara-12 and SB16-5A and are recommended as suitable parental lines for bambara groundnut grain production improvement. The quantitative characters therefore provided a useful measure of genetic variability among bambara genotypes and will enable the identification of potential parental materials for future breeding programmes in South Africa. Out of the 4 different seed protein extraction methods exploited for this study, the 2x Laemmli buffer extraction method produced the best result with clear protein bands. A unique feature from all extraction methods was the presence of a common protein band at ̴ 75 kDa. All extraction methods except 10 % TCA-Acetone resolved common banding patterns in all the bambara groundnut samples. This data suggests that there is very little or no intraspecific genetic diversity among the seed proteins of bambara groundnut accessions studied. There was wide variation in the content of the five antinutritional compounds among the thirty bambara groundnut accessions. The mean values for condensed tannin content ranged between 0.20 - 6.20 mg/g. Free phosphate recorded an overall mean of 1.71 mg/g while a range of 1.35 - 4.93 mg/g was observed by phytic acid phosphate (PAP). The polyphenol content had an overall mean of 0.39 mg/g and trypsin inhibitor (TIA) was quite variable among the bambara groundnut accessions ranging from 5.30 - 73.40 TIA/mg. Generally, higher levels of antinutrients were observed in this study compared to the other studies. The results obtained in this study led to a conclusion that although variations exits among the accessions studied, further research is required to verify the extent of morphological variations, the efficiency of protein extractions methods evaluated and the effects of these antinutrients in human and animal feeds.