Application of Zinc, Iron and Boron Enhances Productivity and Grain Biofortification of Mungbean

1  Introduction

Mungbean, being recognized as poor man’s meat, contains a sufficient amount of quality proteins [1]. Its nutritional profile revealed that its seed contains 367 mg phosphorus and 132 mg calcium per 100 g of seed; whereas, carbohydrates, protein, ash, fibre and fats are 50%, 26%, 4%–5%, 3%–4.5% and 3%, respectively [2]. It is usually grown in semi-arid and arid regions of the world [3]. It is considered the main pulse crop of Pakistan and was cultivated on an area of 0.231 million hectares with a total production of 0.204 million tonnes [4]. Its average yield (0.88 tonnes ha−1) is however far less than the developing countries. Improving the yield of this vital crop both quantitatively as well as qualitatively can further make it more profitable economically and beneficial from a health’s point of view.

Micronutrients, though required in smaller amounts, are necessary for human health, normal plant growth and the development of all crops [5,6]. A little quantity of micronutrients is needed for better growth and production of all crop plants [7]. Shenkin [8] suggested the application of Zn, B, Se, and Fe to be beneficial in improving the immune system of plants besides increasing their growth and development. Micronutrient malnutrition is threatening the world’s population, especially in developing countries [9]. Approximately 2 billion people across the globe are Zn deficient [10] and its deficiency ranks 5th in causing deaths in developing parts of the world while 11th in the overall world [11]. About 33% of children and 40% of mothers in Pakistan are suffering from Zn deficiency, particularly in the rural areas [12]. Fe deficiency is also a global issue that is affecting about 2 billion of the world’s population especially children and women in Latin America, South Asia, and Africa [13]. About 40% of women in their reproductive age are anemic in South Asia which constitutes 37.5% of global anemic cases [14]. While in South America, the Caribbean and Central America about 46.2%, 42.9%, and 33.9% population respectively is anemic with more prevalence in children under 11 months of age [15]. Bioavailable Zn is deficient in about 50% of the world’s cultivated land [16]. In Pakistan, the situation is even worse with the prevalence of Zn deficiency in 70% of the soils [17]. The prolonged scarcity of Zn leads to poor vegetation, sexual development and low Zn contents in grain [18], reduction in leaf size and internodal length [19]. Zn deficiency in humans is directly linked with the soil’s Zn deficiency [20]. Fe is not deficient in our soils but is less bio-available due to very little accessibility to plants in readily available form (ferric form, Fe3+) and high pH of the soil [21]. In calcareous soils, low Fe bioavailability and chemical solubility are serious problems for crop plants [22]. Pakistani soils (approximately 82%) are also deficient in B. Its availability is affected by high soil pH, calcareousness and low organic matter [23]. Zn is necessary for plants as it plays an important role in enzyme activation and nucleic acid, protein, carbohydrates and lipids metabolism [24]. It improves plant photosynthesis, growth, nitrogen fixation and ultimately the yield of crops [25]. After nitrogen and phosphorus, Zn deficiency ranks third in Pakistan in limiting plant performance [26]. Zn foliar application at flowering and grain filling enhanced pod’s number, pod’s weight, seeds per pod, biological yield, 100-seed weight, and seed yield as compared to control [27]. Negative effects of Zn deficiency in humans are pronounced on immunity, bones, skin, brain and reproductive system [28] causing several disorders including cancer, diarrhea, and pneumonia [29]. Humans consuming Zn deficient food products may suffer from Zn deficiency-related health issues such as poor birth outcome, susceptibility to diseases, stunted growth, enhanced mortality, poor immune system and brain functioning [30,31]. Rehman et al. [32] suggested the economical, feasible and sustainable approach for overcoming Zn deficiency in human food which is the biofortification of Zn in grains to increase the concentration and bioavailability of Zn in food.

Iron is an essential part of hemoglobin and myoglobin proteins which provide oxygen from lungs to tissues and muscles respectively [33,34]. Fe is an essential component for the growth, development, and synthesis of connective tissues and hormones for normal cellular functioning [34]. About half the population of women and preschool children in Asia and Africa is suffering from Fe deficiency anemia (IDA) [35]. IDA reduces immunity and physical as well as cognitive development. It enhances the risk of perinatal and maternal mortality and also affects the work performance of all age groups individuals [36]. In plants, B is essential for carbohydrate and RNA metabolism, sugar transport, membrane transport, bioaccumulation of essential elements and respiration [37]. The cell wall of all the plants is strengthened by minute amounts of B compounds [38]. Some biological functions, i.e., calcium and insulin metabolism, bone growth and maintenance and life cycle completion in humans are supported by Hunt [39]. Boron actively accumulates in bones instead of soft tissues, so it is involved in the calcification and maintenance of bones and has a positive influence on the central nervous system, maintaining structural integrity and cell membrane functions [37]. B compounds have anti-inflammatory, and antioxidant properties [40]. The deficiency of B causes several pathological disorders such as Cancer and several forms of osteoarthritis and osteoporosis [41].

In a bid to cater the food needs of an ever-increasing human population, food quality and human health have been overlooked [42]. To overcome micronutrient deficiency for quality food production, two approaches are suggested in agriculture. A sustainable and long-term solution to the problem is the development of cultivars with high micronutrient concentration in the sink by breeding and genetic engineering; however, this approach takes too much time and expertise for the solution. The other well-known strategy is agronomic bio-fortification by soil and foliar application of these micronutrients [43]. To combat Zn deficiency in cereals, several foliar sprays of dilute Zn solutions are recommended from booting to grain initiation stage, due to partial phloem mobility and possible leaf burning by the toxicity of excessive Zn [44]. Hussan et al. [45] suggested that soil application of Zn can enhance the yield but the foliar application is a better option to enhance Zn concentration in grains.

A significant progress has been made to control micronutrient deficiencies through supplementation, but some of these approaches are too costly and time consuming. A substantial approach has been adopted to invest $192 million for the biofortification of Fe in beans, especially in the food system of Latin America, East Africa, and South Asia [46]. Harvest-Plus and Pan Africa Bean Research Alliance (PABRA) has claimed a “Nutrition success story” in biofortification of Bean with Fe, by citing a trial with human efficacy [47]. Due to the limited resources of rural families which cannot change their traditional eating habits biofortification is the only approach to be believed to have the ability to target these families [48]. Keeping in view the above facts and the need for biofortification in mungbean, the current study was thus planned to enhance the concentration of Zn, Fe and B in grain and mungbean productivity by hypothesizing that these micronutrients will play a key role in several metabolic processes, enzyme activation, crop growth and development and will also enhance the concentration of these nutrients in source which will ultimately be remobilized to sink.

2  Materials and Methods

2.1 Experimental Details

To evaluate the impacts of Zn, Fe and B on mungbean, three different experiments were carried out with soil and foliar application (% solution w/v) of each nutrient individually at the research farm of Cereals and Pulses Section of Agronomic Research Institute, Faisalabad using randomized complete block design (RCBD) and replicating each experiment thrice. The first experiment comprised of Zn with five treatments viz. control (no Zn application), soil application of Zn at 4.125 kg ha−1, soil application of Zn at 8.25 kg ha−1, foliar application of Zn at 0.3% at flowering initiation, and foliar application of Zn at 0.3% at one week after flowering initiation. The second experiment comprised of Fe with five treatments viz. control (no Fe application), soil application of Fe at 2.5 kg ha−1, soil application of Fe at 5.0 kg ha−1, foliar application of Fe at 0.2% at flowering initiation, and foliar application of Fe at 0.2% at one week after flowering initiation. The third experiment comprised of B with five treatments viz. control (no B application), soil application of B at 0.55 kg ha−1, soil application of B at 1.1 kg ha−1, foliar application of B at 0.1% at flowering initiation and foliar application of B at 0.1% at one week after flowering initiation.

The field was cultivated twice at field capacity, rotavated, planked, and again cultivated once for seedbed preparation. Mungbean was sown in 60 cm apart rows with help of a hand drill on 30.05.2019 and 04.05.2020 during the first and second year of study, respectively. NPK at 23:58:30 kg ha−1 was applied to the crop at sowing as a basal dose. Soil application of micronutrients was done with first irrigation and foliar application was done at flower initiation using 300 liters of solution per hectare.

Soil analysis of the experimental site was done before sowing and the results are presented in Table 1.

2.2 Weather Conditions during the Research

Weather data for both years was collected from the meteorological observatory of Agronomic Research Institute Faisalabad and are presented in Fig. 1.

Figure 1: Weather data during 2019 and 2020

2.3 Data and their Collection/Analysis Procedures

Five plants from each treatment were selected and tagged at physiological maturity. After taking plant height with the help of a meter rod from the base of the soil to the tip of plants, they were cut from the base. Primary branches and the number of pods were counted per plant and averaged. Five pods were removed from each plant, their length was taken with help of a measuring scale and the numbers of seeds were counted in each pod and averaged. The crop was harvested at harvest maturity, dried, and threshed to get the seed yield. Three samples of 1,000 seeds were collected, weighed with help of a weighing balance and averaged thereof to calculate the 1,000-seed weight. Seed samples were collected and dried for grain analysis. After that, seeds were ground, sieved and used for dry ashing and wet digestion. Seed B contents were measured by dry ashing [49] and subsequent determination was done by colorimetry using Azomethine-H [50]. Seed Zn and Fe contents were determined on Atomic Absorption Spectrophotometer after wet digestion [51] of seed samples. Methods of B, Fe, and Zn determination are mentioned in ICARDA Manual by Estefan et al. [52]. Firstly, grain samples were dried at 60°C in an oven. Later, after being grounded, grains were passed through a 1 mm sieve before wet digestion. Next, the contents of B, Fe and Zn were determined based on nitric–perchloric acid digestion method.

2.4 Statistical Analysis

All the recorded data were analyzed statistically using Fisher’s analysis of variance technique and the difference between treatments’ means was compared using Tukey’s Honestly Significant Difference (HSD) test [53]. Correlation (Pearson) analysis was also performed for all parameters.

3  Results

3.1 Effects of Zn Application on Performance of Mungbean

Data in Table 2 shows that all the yield and yield components were enhanced by the application of Zn either through soil or foliar application during both years of study. The maximum number of primary branches, plant height, pods per plant, pod length, seeds per pod, test weight, and grain yield were recorded when Zn was applied into the soil at 8.25 kg ha−1 followed by soil application of Zn at 4.125 kg ha−1 and foliar-applied Zn at 0.3% while the minimum was recorded with control (no Zn application). In 2019, the number of primary branches was statistically more for soil-applied Zn at 8.25 kg ha−1 as compared with foliar application of 0.3% Zn at one week after flowering initiation. During the second year of the experiment, there was a non-significant difference between soil-applied Zn at 4.125 and 8.25 kg ha−1 and 0.3% foliar Zn spray for the number of primary branches at flowering initiation and one week after the flowering initiation stage. Plant height was also recorded as statistically at par when soil application of Zn was done at 8.25 and 4.125 kg ha−1 and foliar application of 0.3% Zn at one week after flowering initiation during both study years. The minimum values for the same parameter were recorded for the control without Zn application and foliar application of 0.3% Zn at flowering initiation. Similarly, Zn application at 4.125 and 8.25 kg ha−1 recorded a higher number of pods per plant with a non-significant effect between these rates. Pod length was also similar (p ≤ 0.05) for soil application of Zn at 4.125 and 8.25 kg ha−1 during both years and foliar application of 0.3% Zn in 2019. Furthermore, minimum values for pod length were recorded for the control treatment which was statistically the same as the foliar application of 0.3% Zn at both stages. The numbers of seeds per pod were statistically same for both soil and foliar application of Zn during both years of study. Soil application of Zn at 4.125 and 8.25 kg ha−1 produced maximum but statistically same values for test weight and seed yield followed by foliar application of 0.3% Zn and control without Zn application during both study years. Foliar application of 0.3% Zn produced maximum but statistically similar grain Zn concentration to soil application of Zn at 8.25 kg ha−1 during both study years followed by soil application of Zn at 4.125 kg ha−1 (Fig. 2). The minimum values for Zn concentration were recorded for the control without Zn application. A strong correlation of all parameters was found with grain yield however there was a non-significant correlation for grain Zn contents with all other parameters (Table 2A).

Figure 2: Effect of zinc (Zn) application rates on Zn contents in the grains of mungbean. T1, control; T2, soil application of Zn at 4.125 kg ha−1; T3, soil application of Zn at 8.25 kg ha−1; T4, foliar application of Zn at 0.3% at flowering initiation (FI) stage; T5, foliar application of Zn at 0.3% at one week after FI. The values are means ± SE, n = 3. Significant differences are shown by lowercase letters (at p ≤ 0.05)

3.2 Effects of Fe Application on Performance of Mungbean

Fe application increased yield and yield components of mungbean during both study years (Table 3). Our results showed that Fe application at 5.0 kg ha−1 recorded the maximum number of primary branches and pods per plant which was statistically similar to 2.5 kg ha−1 while the minimum was recorded for the control without Fe application and foliar application at 0.2% Fe at both growth stage during 2020 and both years, respectively. However, in 2019, there was a non-significant difference in the treatments. Soil application of Fe at 2.5 and 5.0 kg ha−1 and foliar application of 0.2% Fe produced statistically similar plant height while minimum values were recorded for the control treatment and foliar application of Fe at 0.2% at one week after flowering initiation during the first study year. However, in 2020, all treatments recorded statistically similar values for plant height. Similarly, for pod length seeds per pod, there was a non-significant difference among the treatments in 2019, however, Fe application at 5.0 kg ha−1 increased the same parameter in 2020 followed by soil-applied Fe at 2.5 kg ha−1. While minimum pod length and seeds per pod were recorded for control treatment which was statistically at par with foliar application of 0.2% Fe at both stages. Results also showed that maximum test weight was recorded when for soil application of Fe at 5.0 kg ha−1 which was statistically similar (p ≤ 0.05) to soil application of Fe at 2.5 kg ha−1 followed by foliar application of Fe at 0.2%. The minimum value of test weight was recorded for the control treatment. As compared with control, Fe application either by the soil or foliar spray enhanced grain yield during both years, however, there was a non-significant difference among the treatments. The minimum grain yield was recorded for the control treatment during both study years. Foliar application of 0.2% Fe at flowering initiation recorded maximum grain Fe content followed by foliar spray of 0.2% Fe one week after flowering initiation, soil application of Fe at 5.0 and 2.5 kg ha−1 (Fig. 3). The minimum Fe content was recorded for the control treatment during both study years. Furthermore, as shown in Table 3A, a correlation also exists between the collected parameters with grain yield; however, there was a non-significant correlation found with grain Fe contents.

Figure 3: Effect of iron (Fe) application rates on Fe contents in the grains of mungbean. T1, control; T2, soil application of Fe at 2.5 kg ha−1; T3, soil application of Fe at 5.0 kg ha−1; T4, foliar application of Fe at 0.2% at flowering initiation (FI) stage; T5, foliar application of Fe at 0.3% at one week after FI. The values are means ± SE, n = 3. Significant differences are shown by lowercase letters (at p ≤ 0.05)

3.3 Effects of B Application on Performance of Mungbean

Application of B significantly influenced the growth and yield traits in mungbean except for plant height during both years and the number of primary branches in 2019 (Table 4). In 2020, the number of primary branches was statistically similar among soil applied application of B at 0.55 and 1.1 kg ha−1 and foliar application of B at 0.1% solution at flowering initiation and one week after flowering initiation, while minimum values were recorded for the control. Soil application of B at 1.1 and 0.55 kg ha−1 produced higher but statistically similar pods per plant and pod length during both study years followed by the foliar application at 0.1% while the minimum value was recorded for the control treatment. In 2019, maximum seeds per pod were recorded when B was applied to the soil application at 1.1 kg ha−1, however, the values were statistically similar with soil-applied B at 0.55 kg ha−1 and foliar application at 0.1%. During the second year of study, maximum and statistically similar seeds per pod were recorded for the foliar spray of B at 0.1% at flowering initiation and one week after flowering initiation and soil application of B at 0.55 and 1.1 kg ha−1, while the minimum was recorded for the control treatment. Maximum test weight and grain yield were recorded for soil application of B at 1.1 kg ha−1 which were statistically at par with soil application of B at 0.55 kg ha−1, followed by foliar application of 0.1% B at flowering initiation and one week after flowering initiation. The minimum values of these traits were recorded for the control treatment during both years of study. Maximum grain B concentration was recorded for foliar application of 0.1% B at flowering initiation followed by its spray with the same concentration one week after flowering initiation and soil application of B at 0.55 and 1.1 kg ha−1. The minimum grain B concentration was recorded for the control treatment without B application (Fig. 4). A correlation exists between grain yield and other collected parameters but not with grain B contents (Table 4A).

Figure 4: Effect of boron (B) application rates on B contents in the grains of mungbean. T1, control; T2, soil application of B at 0.55 kg ha−1; T3, soil application of B at 1.10 kg ha−1; T4, foliar application B at 0.1% at flowering initiation (FI) stage; T5, foliar application of B at 0.1% at one week after FI. The values are means ± SE, n = 3. Significant differences are showed by lowercase letters (at p ≤ 0.05)

Means not sharing a letter in common differ significantly at 5% probability level.

4  Discussion

The application of macro and micro-nutrient is essential for all field crops for better yield and quality [54]. Micronutrients (Fe, B, Zn and Se) [55] not only play a significant role in crop growth and development but also stimulate the human immune system. In our experiment, the application of Zn either through soil or foliar, significantly improved primary branches, plant height, pods per plant, pod length, seeds per pod, test weight and grain yield of mungbean. A positive correlation was also recorded among the recorded traits. However, soil application enhanced all these parameters more than foliar application. This increase might be attributed to the role of Zn in the synthesis of protein, lipids, carbohydrates, nucleic acid, nitrogen metabolism, cell division, photosynthesis [24], and all other metabolic processes of cells [56,57]. Better growth and yield of mungbean might also be attributed to the participation of Zn in several physiological processes during crop development [58], resistance to abiotic stress [59], nitrogen use efficiency [60], photosynthesis and protein synthesis [61]. The presence of Zn in cell triggers several enzymes involved in cell processes which ultimately enhance crop growth [62–64]. Zn plays a key role in auxin production which is involved in the enhancement of quantity and size of cell, resulting in taller plant height [65]. It has been reported by several scientists that the application of Zn at different levels enhanced the grain yield of mungbean [46,66–69]. Usman et al. [70] reported that Zn-fertilization enhanced grain and biological yield by enhancing the number of grains per pod and test weight of mungbean.

The increase in grain Zn concentration might be attributed to the remobilization of Zn reserves from vegetative parts to the grain [71]. Chen et al. [72] reported the increase in grain Zn concentration due to an increase in source Zn concentration through the soil and foliar application of Zn. Foliar application is an agronomic practice which is used for rapid biofortification of micronutrients in grains. Scientists believed that agronomic biofortification of Zn in wheat was more rapidly achieved than genetic or breeding biofortification [72,73]. Our study which indicates that Zn concentration of mungbean grains was significantly enhanced by the application of Zn mostly with foliar application of Zn than with soil-applied Zn are further supported by above-mentioned facts related to Zn. Scientists have reported the role of Fe in the respiratory electron transport chain (ETC), cell wall metabolism, photosynthesis and oxidative stress tolerance [74,75]. Fe being part of many enzymes is involved in the activation of a number of enzymes, cytochrome (involved in ETC), chlorophyll synthesis and chloroplast structure [76]. Micronutrients, especially Fe has great importance in photosynthesis and respiration as it is involved in several enzymatic activities and chlorophyll [21,77]. Moreover, it has been reported that Fe contributes in the synthesis of chlorophyll [78] and several plant growth regulators. An increase in plant height of mungbean has been reported by Khoulenjani et al. [79], however, an increase in test weight, number of grains, grain and straw yield and grain Fe concentration in wheat has been reported by Ziaeian et al. [80]. An increase in grain Fe iron concentration might also be attributed to the availability of Fe at the reproductive stage of mungbean due to foliar application. It has been previously observed that Fe application significantly enhanced the grain Fe concentration in wheat and groundnut [81,82]. Our results indicate that soil and foliar application of Fe significantly enhanced primary branches, plant height, pods per plant, pod length, seeds per pod, test weight, grain yield by soil application and grain Fe concentration by foliar application either in both years of study or in one are further supported by the above-mentioned roles of Fe in crops.

Boron plays a key role in cell wall formation, carbohydrate, Indoleacetic acid (IAA) and Ribonucleic acid (RNA) metabolism, membrane integrity, calcium uptake, translocation of sugars, flowering, pollination and ultimately growth [83–85]. The increase in primary branches, pods per plant and pod length is attributed to the role of B in cell elongation and maturation, protein synthesis, development of meristematic tissues and eventually plant growth and yield [86–90]. Jing et al. [91] reported that the application of B improves plant dry matter and pods per plant in groundnut. The foliar application of B at the flowering stage enhances the concentration of B in the source which ultimately is remobilized to sink. An increase in micronutrient concentration in flag leaf and grains was previously verified by Zeidan et al. [92] and Gomaa et al. [93]. The results of our study which indicate that application of B improved pods per plant, pod length, seeds per pod, test weight, grain yield and grain B concentration by soil application and foliar application of B, are further supported by the above-mentioned facts related to B.

5  Conclusion

This study is unique of its kind as it has addressed the vitally important issue of hidden hunger and presented a pragmatic solution for combating it as well. It was concluded that soil application of Zn, Fe and B is beneficial for better growth and yield of mungbean. However, foliar application of 0.3% Zn, 0.2% Fe and 0.1% B solution (w/v) at flowering initiation of mungbean proved to be more beneficial in enhancing the yield and grain contents of these micronutrients. A strong correlation of all parameters was found with grain yield however there was no correlation of grain Zn, Fe and B contents with all other collected parameters.

Authorship: Study conception and design: Muhammad Zafar, Siraj Ahmed, Muhammad Kashif Munir; data collection: Nawal Zafar, Muhammad Saqib; analysis and interpretation of results: Siraj Ahmed, Sadam Hussain, Aleem Sarwar, Saba Iqbal; Formal analysis: Aneela Gulnaz, Baber Ali, Mohammad Khalid Al-Sadoon; draft manuscript preparation: Siraj Ahmed, Muhammad Saqib; Funding acquisition: Aneela Gulnaz, Baber Ali, Mohammad Khalid Al-Sadoon. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgement: The authors would like to extend their sincere appreciation to the Researchers Supporting Project No. (RSP2023R410), King Saud University, Riyadh, Saudi Arabia. The authors also would like to thank Punjab Agricultural Research Board, Pakistan for funding the Research Project PARB No. 904.

Funding Statement: This research work is supported by Researchers Supporting Project No. (RSP2023R410), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.

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