Biofortification of maize with zinc and iron not only enhances crop growth but also improves grain quality

Hidden hunger is an emerging challenge for scientists, especially connected to the agriculture sector because over two billion people are facing it globally. This issue is more common in developing countries which have less access to a diverse diet due to their low income. Different potential practices are introduced to minimize the pressure of malnutrition but agronomic biofortification is being considered best practice to improve the contents of micronutrient in grains. A field based study was executed to explore the impact of zinc sulphate (ZnSO 4 ) and iron sulphate (FeSO 4 ) on productivity and grain quality of maize crop. Sole and combined application of ZnSO 4 and FeSO 4 either via soil or/and plant foliage not only enhanced the yield attributes of maize crop but grain quality was also improved. Soil supplementation of ZnSO 4 (10 kg ha -1 ) produced maximum plant height and cob weight. Combined treatment of ZnSO 4 (10 kg ha -1 ) and FeSO 4 (12 kg ha -1 ) through soil produced more grains per cob, 1000-grain weight, biological and grain yields. Foliar applied 0.1% ZnSO 4 and 0.3% FeSO 4 produced highest chlorophyll contents. Foliar treatment of 0.1% ZnSO 4 and 0.3% FeSO 4 improved the concentration of zinc and iron in grains, respectively. Combined treatment of 10 kg ha -1 of ZnSO 4 and 12 kg ha -1 of FeSO 4 through soil improves the yield attributes while foliar spray of 0.1% ZnSO 4 and 0.3% FeSO 4 enhances quality parameters. Overall, foliar spray approach is more applicable regarding nutrients availability for optimum development and growth of crop and improved grain quality.


Introduction
Micronutrient malnutrition is affecting rural populations residing in developing countries with less access to a diverse diet due to their less purchasing power (Tsakirpaloglou et al., 2019;Kumar et al., 2019). Globally, over two billion people are facing hidden hunger (micronutrients deficiency), and it is an emerging challenge for scientists, especially connected to agriculture sector (WHO, 2016). In human, malnutrition is mainly caused by vitamin A, Zn, Fe, selenium (Se) and iodine (I) deficiency (Haider and Bhutta, 2009;Hess and King, 2009). Particularly Fe and Zn metal deficiencies, are affecting over 50% of world community because they depend on cereal crops, chiefly rice, maize and wheat for their regular diet (Ramzan et al., 2020). Both Zn and Fe are indispensable nutrients for biological systems in plants, humans, and animals (Broadley et al., 2007;Failla, 2003). In human, Fe is needed for haemoglobin formation (oxygen transport), psychomotor development, and resistance to infection (Stoltzfus, 2001). Its clinical deficiency is also associated with pallor (anaemia), dizziness, reduced work capacity and reduced intellectual performance and pregnancy-related issues, i.e., low birth weight and mortality (Lynch, 2003;CDC, 2010). In humans, Zn is needed for activation of over 300 enzymes, maintenance of sensory functions, physical growth and development, immune system, and neurobehavioral development (Gibson, 2012;Levenson and Morris, 2011). Many health impediments, like poor physical growth, damage to DNA, central nervous, gastrointestinal, epidermal, reproductive, skeletal and immune systems may occur due to Zn deficiency (Hambidge and Walravens, 1982;Prasad, 2006). In Pakistan, more than 40% mothers and one-third children are under Zn malnutrition, with a higher rate in rural communities (MINH, 2009). Worldwide, maize is growing under wide range of soil and climatic conditions for grain and fodder production (Ranum et al., 2014). Maize ranks third vital cereal crop after wheat and rice that contributes almost 0.5% grand domestic production (GOP, 2019) Maize is also considered as a staple food for greater than 200 million people, and it can be expected that globe population will be eight billion in 2025 (USDA, 2008;Lutz et al., 2001). It is known as a queen of cereals because of high monetary value as it is also treated on commercial scale to make a variety of products for the consumption of human, livestock and poultry industries (Harris et al., 2007). Due to highest yield potential among cereals, it can be paramount crop to overcome global nutrition (Tariq and Iqbal, 2010). Among different possible agricultural approaches to conquer the malnutrition, the agronomic biofortification is top ranked approach to improve the grain Fe and Zn contents (Borrill et al., 2014;Hassan et al., 2019;Cakmak et al., 2010). It is achieved through the application of micronutrient to crop foliage directly and/or soil (De Valença et al., 2017;Zahra et al., 2020). It can be implemented easily being more sustainable and economical as compared to other techniques including genetic engineering (Cakmak, 2008). Ngozi (2013) reported that agronomic biofortification of the crops is an emerging practice to overwhelm malnutrition particularly metal deficiencies in the developing world. Considering the above mentioned rationale, the current study was designed to study the following objective; i) to assess the either sole and/or combined influence of Zn and Fe on quality, growth and yield of maize crop, ii) to compare the efficiency of two application approaches, i.e. foliar and soil application.

Experimental particulars
The present field-based trial was conducted to explore the impact of Zn and Fe on grain quality and yield of maize crop. Seeds of maize hybrid (Soni dharti-626) were collected from Sohni Dharti International Seed Company, Sahiwal-Pakistan. Seeds were sown at research area of Agronomy Farm, Universsity of Agriculture, Faislabad-Pakistan during crop growing season of 2018. Site soil was ploughed to a depth of 30 cm and ridges of 30 cm in height and 75 cm of spacing were prepared. Two seeds were planted on the top of all ridges with space of 25 cm between hills. Every experimental unit consist of 4 ridges that were 3 m in length. The experimental soil of area, under study, was sandy loam having following properties; pH 8.1, EC 1.45 dS m -1 , organic matter 0.81%, total nitrogen (N) 0.08%, phosphorus contents (P) 8.2 ppm, potassium contents (K) 200 ppm, DTPA-Extractable iron (Fe) 2.54 mg kg -1 , zinc (Zn) 0.45 mg kg -1 .

Crop husbandry
After almost 2 weeks of sowing, thinning was done to maintain the plant population and a basal dose of NPK fertilizer (230:145:92) was applied by using urea, DAP and sulphate of potash fertilizer. ZnSO 4 and FeSO 4 were used as sources of micronutrient. At sowing, one-third of nitrogen and all of the phosphorus, potash, ZnSO 4 and FeSO 4 doses were applied as a basel dose. The remaining dose of nitrogen was supplemented in equal splits with 1 st and 2 nd irrigation while foliar application of ZnSO 4 and FeSO 4 at silking and grain filling stage. Experimental units were weeded twice in the course of the growing duration. To study the above mentioned objectives, following treatments were applied: Biochemical analysis and quality attributes Arnon (1949) method was used for the determination of the chlorophyll pigments (a and b). Atomic Absorption Spectrophotometer was used to measure the amount of Zn and Fe in maize grains according to method described by AOAC (1990). The total soluble protein was measured according to Bradford method (1976).

Agronomic parameters
Data of yield and its attributes were recorded at crop maturity. Plant population was counted at harvesting (m -2 ). Ten plants were selected randomly from each experimental unit to record the data of plant height. Plant height of individual plant was measured with a meter rod from base to top and then averaged. Number of grain rows per cob, cob weight and its length, and grains per cob were determined manually from the selected cobs. For biological and grain yield, plants harvested from a row of one meter length and converted in to per hectare and weighed with weighing balance. Harvest index (%) was estimated by dividing the grain yield with biological yield.

Statistical analysis
A randomized complete block design (RCBD) having three replications was adopted. Statistical package (Statistix 8.1) was used to analyse and evaluate the collected data. ANOVA (analysis of variance) technique proposed by Fisher was followed to observe the statistical differences among the variance and means of treatments.
( Fig. 2b). All the treatments either sole or combined through foliar spray or soil application reduced the protein contents in maize grain (Fig. 2c). Highest protein contents were observed in those grains which received no exogenous treatment.

Figure-2: Influence of foliar and soil supplied ZnSO 4 and FeSO 4 on Fe contents (a) Zn contents (b) and total soluble proteins in maize grains (c).
The number of leaves plant -1 were not significantly increased by external treatments of Zn and Fe (Table  1). Soil applied Zn @ 10 kg ha -1 produced maximum plant height as compared to other treatments (Table  1). Cob length was not affected by exogenous application of Zn and Fe (Table 1) while cob weight was significantly affected. Soil supplemented Zn @ 10 kg ha -1 produced highest cob weight ( Table 1) that was statistically at par with soil supplementation of Zn and Fe @ 10 and 12 kg of ZnSO 4 and FeSO 4 per hectare. Number of grain rows were not affected by treatment application (Table 1). Data regarding number of grains cob -1 , 1000-grain weight, biological and grain yield and harvest index are presented in table 2. Application of 10 kg of ZnSO 4 and 12 kg of FeSO 4 through soil produced maximum grains cob -1 , 1000-grain weight, biological as well as grain yield, however, harvest index was statistically nonsignificant (Table 2).

Discussion
Our outcomes supported the hypothesis that the different amount of zinc and iron treatments either through soil or/and foliar significantly improves the chlorophyll contents, quality of grains, and grain yield in maize crop than control. The improvement in grain yield is associated to the cob weight, grains cob -1 and weight of 1000-grains. In this study, maximum grain yield was recorded when combined application of zinc and iron was supplemented ( Table 2). The improvement in attributes of yield of maize crop might be due to involvement of zinc and iron in biochemical processes, including photosynthesis. Outcomes of present experimentation are similar to Saleem et al. (2016), who stated the soil supplementation of Zn and Fe (each @ 30 kg ha -1 ) considerably enhanced yield and quality of grains. Similar outcomes were also described by Kanwal et al. (2010) that more Zn uptake and higher grain yield in corn were noted when Zn soil supplementation was done @ 18 kg Zn ha -1 . These findings are also in line with Mugenzi et al. (2018), who stated the collective effect of Zn and Fe enhanced the maize yield, photosynthetic capacity, and grain quality. Eteng et al. (2014) showed a considerable increase in yield of maize with the micronutrients supplementation. Globally, ~50% of the soils under cultivation of cereal have low levels of available Zn for plants (Graham and Welch, 1996). Combined treatment of Zn and Fe (soil+foliar) increased accumulation Zn and Fe contents (Fig. 2a,  b). Maximum Zn content was noted when ZnSO 4 applied @ 0.1%, and highest amount of Fe content was recorded when FeSO 4 was applied @ 0.3% (Fig.  2a, b). Highest protein contents were analyzed in control while significantly decreased when combined soil Zn and Fe supplementation was done (Fig. 2c). Our results are also in line with the findings of Cakmak et al. (2010), who stated that Zn contents were increased three times in grains by foliar and soil applied Zn, additionally noted that time and method of Zn application is so important to enhance Zn concentration in grains. By application of 0.5% ZnSO 4 and 1% FeSO 4 zinc and iron contents were also significantly improved (Pahlavan-Rad andPessarakli, 2009). Saleem et al. (2016) noted that Zn and Fe through foliage considerably enhanced the zinc and iron contents in maize grain. These findings are also in line with Zeidan et al. (2010), they concluded that zinc and iron concentrations in wheat grains were significantly improved by foliar treatment of zinc and iron. Ozturk et al. (2006) also stated that foliar spray improved the zinc content in grain at later growth stages. Sharma and Singh (1990) investigated that zinc absorption through soil to plant was improved by application of ZnSO 4 in maizestover. Foliar spray of Zn improved the availability of Zn to plants in comparison with soil application (Zhao et al., 2014). The foliar spray of Zn is more productive in enhancing grain Zn concentration of maize crop. Cakmak (2008) reported that the foliar application is better way to improve iron content in grain because clay adsorption and low organic matter reduced the flow of nutrients reported the accumulation of micronutrients in the grain with the addition of their fertilizers. Zuchi et al. (2015) investigated that an insufficient supply of other nutrients might prevent the uptake of iron and its translation to shoots and other parts, which may result in reduction of other nutrients. Among plants, nutrients interaction can be antagonistic, synergistic, Liebig-synergistic and/or zero-interactive. These interactions explained that activities of other nutrients may be affected by the supply of specific nutrient that ultimately adversely affect crop yield and growth (Rietra et al., 2017). Usually, Fe deficiency is seen in calcareous soils having high pH in arid areas (Prasad, 2003). Worldwide, it is estimated that Zn (50%) and Fe (30%) deficiencies are widespread occurring in cultivated soils (Cakmak, 2002). In Pakistan, zinc deficiencies are more extensive, about 70% soils are deficient of zinc (Imtiaz et al., 2010). Applied micronutrients and their interactions influence physiological and biochemical processes of plants, which substantially affect quality and yield of grains (Wang et al., 2015). Mugenzi et al. (2018) stated that iron and zinc application, either sole or combined had no significant influence on protein content. However, in this study protein content considerably reduced where sole or combined zinc and iron foliar or in soil was applied in comparison with control. Ramzani et al. (2017) noted that protein content increased by 64% by Fe application in comparison with control.

Conclusion
Foliar spray of 0.1% ZnSO 4 and 0.3% FeSO 4 improved the contents of zinc and iron in grains, respectively. Combined treatment of 10 kg ha -1 of ZnSO 4 and 12 kg ha -1 of FeSO 4 through soil improves the yield attributes while foliar application of 0.1% ZnSO 4 and 0.3% FeSO 4 enhances quality parameters. Overall, foliar spray approach is more suitable regarding the availability of nutrients for optimum crop growth and improved grain quality.