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Mineral profiling of common bean (Phaseolus vulgaris L.) germplasm

Common bean (Phaseolus vulgaris L.) is the most important edible food legume in the world. It represents 50% of grain legumes consumed worldwide. It is a rich source of protein, complex carbohydrates, poly unsaturated fatty acids (linoleic acid and lenolenic acid), fibre, vitamins and minerals. So far as, minerals are concerned, they are not only important for plant growth and development but for animal growth and development spatially humans as well. In present study, we have evaluated 84 genotypes of common bean for various mineral elements and we have observed wide variation in mineral contents and have also identified the genotypes with high mineral contents. The presence of variability in mineral content among the genotypes could be used as an asset by plant molecular breeders for biofortification of common bean which in turn can be used to address the problem of malnutrion in developing and under developed countries, where common bean is a major source of protein.


INTRODUCTION
Common bean (Phaseolus vulgaris L.) is the most important edible food legume in the world. It represents 50% of grain legumes consumed worldwide Talukder et al. (2010). It is a rich source of protein, complex carbohydrates, poly unsaturated fatty acids (linoleic acid and lenolenic acid), fibre, vitamins and minerals Sathe et al.(1984). It serves 15% of the protein and 30% of the caloric requirement to the world's population Mc Connell et al. (2010). Being a good and cheap source of protein and energy, it is a food of choice in developing countries, as it can combat malnutrition and hunger-related problems Reyes-Mareno et al. (1993). It is not only an energy legume but has anti-diabetic and anticancer activities Camara et al. (2013).
As the domestication of common bean had occurred independently in South America and Central America/ Mexico, so there are two different domesticated gene pools, the Andean and Middle American, respectively De bouck et al. (1993). It has been reported that common bean genotype of Middle American origin has higher mineral content in seeds than the genotypes of Andean origin. Small (16.9g per 100 seeds) navy bean seeds of Middle American origin contain on an average, 90% more calcium than the large kidney bean seeds (47.8g per 100 seeds) belonging to Andean origin (Moraghan and Grafton, 1997). Twenty nine common bean genotypes were assessed by Talukder et al. (2010), for zinc and iron contents, and it was observed that the cultivars of Middle American genotypes had 16.1 and 11.3% more zinc and iron respectively, as compared to that of Andean genotypes. Advances in common bean genomics, such as the availability of complete sequenced genome Schmutz et al.( 2014), have resulted in the development of common bean as a model crop for understanding genetics of some other related and important legumes that includes other members of papilinoid legumes like, cowpea, pigeon pea. Therefore, a better understanding of common bean genome will also facilitate a better comprehension of other important legumes through comparative genomics studies.
Boron is required by plants for development and strengthening of cell wall, cell division, fruit and seed development, sugar transport and hormone development Ahmad et al. (2009). In humans, B is associated with brain function, helps reverse arthritis, strengthen bones, balances hormones, promotes healthy muscle mass, prevents and treats yeast infections, heals skin and prevents infections, fights diabetes (https://draxe.com/boron-uses/). Phosphorus in plants is also critical in root development, crop maturity and seed production (https://www.noble.org/news/publications/ ag-news-and-views/2007/january/back-to-basics-the-rolesof-n-p-k-and-their-sources/-). Consequently, P is the second most abundant essential mineral in the human body after calcium. It not only plays a vital role in various biological processes, including energy metabolism and bone mineralization. But also provides the structural framework for DNA and RNA. As a part of signal transduction, phosphate is used in cyclic AMP (Golub, 2011). So far as is concerned, it is important for the formation of chlorophyll and acts as a key component of various plant enzymes and vitamins (http://www.wengfuaustralia.com/_literature_ 71737/The_Importance_of_Sulphur). It plays an important role in the biosynthesis of essential amino acids Métayer et al. (2008). Sulphur containing compounds are found in all body cells of humans and are quite important for maintenance of life. The important S containing compounds are, methionine, cysteine, homocysteine, cystathione, S-adenosyl methionine (SAMe), taurine, α-keto-γ-CH3-thiobutyrate, methanethiol, thiamin, biotin, alpha-lipoic acid (ALA), coenzyme A, glutathione (GSH), chondroitin sulfate, glucosamine sulfate, fibrinogen, heparin, metallothionein, and inorganic sulphate (Baker,1986). Cu is a key element in reducing anti-oxidative stress (Gaetke and Chow, 2003). It is important in plant reproductive growth stages and has indirect role in chlorophyll production. In humans, it is important for formation of red blood cells, heart and muscles (https://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/ agdex789/$file/531-3.pdf?OpenElement). Mg is important for chlorophyll formation in plants. It also helps in the synthesis of amino acids and some cell proteins. In animals, it is important for bone formation, cell replication, hormone regulation, nerve impulse transmission (stress) and muscle contraction (http://www.oxyfertil.com/royaume-uni/role-camg-plante/animal.html). Mn plays an important role in oxidation and reduction processes in plants, such as the electron transport in photosynthesis. It also plays a role in chlorophyll production, and its presence is essential in Photosystem II. Mn acts as an activating factor that causes the activation of more than 35 different enzymes (Mousavi et al. 2011). In animals, it is required for growth and development and plays a role in immune response, blood sugar homeostasis, adenosine triphosphate (ATP) regulation, reproduction, digestion and bone growth. It is necessary component of numerous metalloenzymes, such as Mn superoxide dismutase, arginase, phosphoenol-pyruvate decarboxylase and glutamine (https://www.sciencedirect.

com/topics/medicine-and-dentistry/manganese).In case of plants, Ca is an important component of cell wall structure.
In humans bone development is associated with Ca, whose intake in adequate amounts prevents fractures and osteoporosis (Nordin, 1997;Martínez et al., 2002;Bass and Chan, 2006;Rizzoli et al., 2008).

MATERIALS AND METHODS
Plant material: Seeds material of 84 common bean genotypes was used as an experimental material for mineral profiling detailed in Table 1.
Chemical analysis: Dry seeds from each genotype were ground into fine powder and 0.5g of each sample was taken for di-acid digestion. To this 20ml of the digestion solution (a mixture of nitric and per chloric acid at the ratio of 9:4 v/v) was added. The mixture was left at room temperature for overnight. Next day, this mixture was kept on hot plate and heated slowly till we got a clear solution, the brownish smoke has stopped being released, that indicated the complete digestion of the organic matter has occurred. This clear solution was allowed to cool at room temperature. Then, the digested samples were transferred to 50 mL volumetric flasks. The transfer was done by using ash-free quantitative filter paper (Whatman No.1) (modified) Palma et al. (2015). The volume of the solutions was raised to 50 mL using distilled water. The samples were then further diluted. The concentration of these minerals was determined by using of ICP-OES/ AAS (inductively coupled plasma optical emission spectrometry atomic absorption spectrometry).The concentration values were further calculated using the following formula: Conc.(mg/kg) = ppm x final vol. after digestion(ml) x dilution factor Weight of sample (g) Statistical analysis: All the observations were taken in three replicates and values were then averaged. One-way ANOVA was applied to evaluate the variance of mineral contents (B, P, S, Cu, Mn, Mg, Ca) among the genotype. Paired-t test was used to determine variance of minerals within the genotype. The Pearson's correlation coefficient between these elements was determined by Pearson's correlation analysis using SPSS (version 16).

RESULTS AND DISCUSSION
Mineral profiling of common bean genotypes for, P, S, Ca, Mg, B, Cu & Mn: Evaluation of seed material of 84 genotypes of common bean was done to determine the content of P, S, Ca, Mg, B, Cu and Mn. Wide range of variation ranging from 1000.45mg kg -1 (KD-17) to 1989.97 mg kg -1 (B-18) with an average 1690.7 mg kg -1 was found in phosphorus content. Sulphur content in 84 genotypes varies from 1032.77mg kg -1 (WB-956) to1345.95 mg kg -1 (VLR-125) with an average of 1233.53mg kg -1 . Calcium content was found to range between 1055.64 mg kg -1 (K-14) to 2875.44 mg kg -1 (MR-2) with an average of 1765.87 mg kg -1 and Magnesium content was found to range from 1057.52 mg kg -1 (WB-371) to 2654.18 mg kg -1 (K-16) with an average of 1860.14 mg kg -1 . Boron content of 84 genotypes ranges from 1 mg kg -1 (WB-956) to 3.67 mg kg -1 (UD-1) with an average of 1.73mg kg -1 . Wide variation ranging from 4.2 mg kg -1 (R-4) to 62.8 mg kg -1 (SR-1) with an average of 15.75 mg kg -1 was found in copper content. Manganese content varies from 12.16mg kg -1 (R-4) to 96.54 mg kg -1 (UG-10) with an average of 53.53 mg kg -1 . Genotype WB-956 was found to have minimum boron and Sulphur content. Table 1 and represent the mean values of mineral contents observed in seeds of 84 diverse common bean genotypes.
In a previous study, Beebe et al. (2000) have reported that the common bean contain, 3684, 2120, 1466, 10, 9 and 15 mg/kg (mean values) of P, S, Ca, B, Cu, and Mn respectively. According to Moraghan and Grafton, (2001), P, Mg and B contents of common bean ranged between 4500 to 5400, 1700 to 2300, 10.1 to 12.7 mg/kg, respectively. A study carried out by Mesquita et al. (2007), have indicated the P, S, Ca, Mg, Cu and Mn content ranged from 4500 to 7300, 300 to 2800, 1800 to 3400, 2800 to 4700, 11.37 to 17.73, 14.93 to 28.90, mg/kg, respectively. In yet another study, carried out by Mario et al. (2009) P, S, Ca, Mg, B, Cu, and Mn content were reported to range between 4000 to 5600, 2200 to 2800, 1000 to 2600, 1300 to 2300, 9.3 to 15.3, 8.8 to 12.8 and 9.6 to 18.9 mg/kg, respectively. Our results were almost in concurrence with that of the previous data. Except for P and B for which our data presented lower concentrations as compared to the previous reported data. Whereas, Cu and Mn have presented good variability and better concentrations, i.e, SR1 is having high Cu concentration of 62.8mg/kg and UG10 is having high Mn concentration of 96.54mg/kg. This fact propounds that unique alleles might exist in this germplasm not only for Cu and Mn but also for several other important nutrients. Our data presented a good genetic variability in all the above mentioned elements except S which showed less genetic variability. This genetic variability could be exploited by plant breeders to produce the lines which would be not only nutrient rich but would be high yielding as well. So that the farmers will interestingly adopt such seeds for their fields. As such, seeds could assure them health plus wealth. Another, most important aspect which needs to be dealt with is to be sure enough that the extra nutrients contained in the seed should be of sufficient magnitude and should be sufficiently bioavailable to the gut.
In developing countries, people usually take cereal grains and legumes because of being cheap. But their intake does not satisfy the mineral requirements of these people Guzman-Maldonado et al. (2000). In last decade, poor health, lower worker productivity, high rate of mortality and morbidity, increasing rates of chronic diseases (coronary heart disease, cancer, stroke, and diabetes) and permanent impairment of cognitive abilities of infant born from micronutrient deficient mother were being found associated to poor micronutrient nutrition (Welch and Graham, 2000;Graham et al., 2001;Sanghvi et al., 2007). Earlier the micronutrients like, Fe, Zn, folate were discussed because of their concern with the public health Sanghvi et al. (2007). But nowadays, Se and B deficiency is also gaining importance for their inevitable role in human nutrition Graham et al. (2001). Besides, other beneficial effects of B; it improves the brains electrical activity, cognitive performance, and short-term memory for elders. It had demonstrated preventive and therapeutic effects in a number of cancers, such as prostate, cervical, and lung cancers, and multiple and non-Hodgkin's lymphoma; and may help ameliorate the adverse effects of traditional chemotherapeutic agents (Pizzorno, 2015). On the other hand, Ca had also proven promising against osteoporosis, hypertension, and colon cancer Mario et al. (2009). In fact all the mineral elements are beneficial to health in one way or the other. So, the need of the hour is to develop such strategies which can improve the micronutrient quality and quantity of some staple food crops. One such strategy is to use bio fertilizers for instance, in a study carried out by Saikia et al., (2018), an increase in nitrogen and potassium content has been reported in a common bean cultivar after the application of consortium of biofertilizers. But biofertilizers mostly provide primary nutrients to the plants. So this strategy can be used to biofortify limited nutrients and to a certain extent beyond which the increase in bio fertilizers cannot lead to any improvement in mineral content. Therefore, a much potential strategy/ies is/are needed and plant breeding is explicitly one such practicable strategy. A plant breeding strategy, if successful, will not eliminate the need for supplementation, fortiûcation, dietary diversiûcation, and disease reduction programs in the future to combat micronutrient malnutrition. Nevertheless, this strategy does hold great promise for signiûcantly reducing recurrent expenditures required for these higher-cost, shortrun programs by signiûcantly reducing the numbers of people requiring treatment. .05) whereas positive but non-significant correlation was found between B and Mn, P and Mg, Mn and P, S and Ca, S and Mg, Mn and S and Cu and S. However, negative and significant correlation was found between B and Ca (r=-0.300, p<0.01) and negative but non-significant correlation was found between B and S/ Mg/Cu, S and P, Ca and P/Mg, Cu and P/Mn, Mn and Mg (Table 2).

CONCLUSION
We evaluated common bean germplasm for various minerals -B, P, S, Cu, Mn, Mg and Ca and got wide variability in content of each mineral element analysed. The existence of genetic variability showed that selection was possible for the common bean germplasm with a high mineral content for diet enrichment (biofortification). Molecular characterisation followed by identification of genomic loci contributing for accumulation of these minerals through molecular breeding may lead to the production of fortified common bean with high mineral content. Biofortification in a crop like common bean, which is known as the 'Poor Mans' Meat' can really help to address the problem of malnutrition in world, especially the poor countries wherein, common bean is used as a staple food. We also need to focus on the bioavailability of these biofortified minerals in gut. So that, the aim of dealing with the malnutritioned populations (trace element deficient) would be served.