Agronomic bio-fortification of wheat (Triticum aestivum L.) to alleviate zinc deficiency in human being

Abstract

Worldwide, 40% population consumes wheat (Triticum aestivum L.) as a staple food that is low in zinc (Zn) content. Zn deficiency is a major micronutrient disorder in crop plants and humans worldwide, adversely impacting agricultural productivity, human health and socio-economic concern. Globally, the entire cycle of increasing the Zn concentration in wheat grains and its ultimate effect on grain yield, quality, human health & nutrition and socio-economic status of livelihood is less compared. So the present studies were planned to compare the worldwide studies for the alleviation of Zn malnutrition. Zn intake is affected by numerous factors from soil to crop, crop to food and food to humans. The post-harvest fortification, diversification in dietary habits, mineral supplementation and biofortification are various possible approaches to enhance the Zn concentration in food. The wheat grains Zn is influenced by the Zn application technique and time concerning crop developmental stages. The use of soil microorganisms mobilize unavailable Zn, and improve Zn assimilation, plant growth, yield and Zn content in wheat. Climate change can have an inverse impact on the efficiency of agronomic biofortification methods due to a reduction in grain-filling stages. Agronomic biofortification can improve Zn content, crop yield as well as quality and ultimately, have a positive impact on human nutrition, health and socioeconomic status of livelihood. Though bio-fortification research has progressed, some crucial areas are still needed to be addressed or improved to achieve the fundamental purpose of agronomic biofortification.

Introduction

Wheat (Triticum aestivum L.) known as the “King of cereals” is a member of the Poaceae family, the most widely grown crop on the planet. Globally, more than 40% population consumes wheat as a staple food (Giraldo et al. 2019). Due to its adaptive attributes to varied climate and environmental stresses, wheat is contributing greatly to overcoming the hurdle of food security (Muslim et al. 2015). In India, wheat stands second in consideration to area and production after rice (FAOSTAT 2021) and it provides 20% of daily food calories and proteins (Ramadas et al. 2019). In some countries of South Asia such as India, Pakistan, Bangladesh and Nepal, wheat accounts for providing 70% of the daily calorie intake in the rural population (Zhang et al. 2010). About 90% of the matured wheat grain is composed of starch, proteins and cell wall polysaccharides whereas phenolics, minerals, terpenoids and vitamins are minor constituents of wheat grain (Shewry et al. 2013).

Micronutrient malnutrition is a serious health issue especially in developing countries (Wakeel et al. 2018) as they are highly important for humans as well as plants for attaining normal growth and development. A normal human body requires varied quantities of different micronutrients such as zinc (Zn), iron (Fe) and selenium (Se) but cereals are unable to fulfil this demand. The consumption of cereals that are deficient in Zn, Fe, iodine, folate and vitamin A can cause invisible health problems termed ‘hidden hunger’ mainly among women and children (Jawaldeh et al. 2019; Black et al. 2013). Zn deficiency is a major micronutrient disorder in crop plants as well as humans globally, adversely impacting agricultural productivity and human health (Rashid et al. 2019). The average Zn content in wheat is only 28.48 mg kg⁻¹ (Wang et al. 2020). However, the recommended Zn requirement for men and women is small (11 and 8 mg day⁻¹, respectively) (De Groote et al. 2021). To meet the daily requirement of the human body, the Zn content in wheat should be raised to 45 mg kg⁻¹. According to the world health organization (WHO), the recommended daily Zn consumption of infants (7–12 months), children (1–10 years), grown-ups (11–51 years) and pregnant women is 5, 10, 15 and 20–25 mg day⁻¹, respectively (Wang et al. 2020). It is estimated that around 2000 million people in Asia and 400 million people in Sub-Saharan Africa can be affected due to the low concentration of Zn in cereals (IRRI 2006). The bioavailability of Zn is reduced due to the immoderate consumption of monotonous wheat which is low in Zn and high in phytate. In humans, severe Zn deficiency is reported in South Asia and the African continent (Fig. 1). Another reason for a reduction in the bioavailability of micronutrients (Zn, Fe and Mg) is phosphorus which is present in the form of phytate in wheat bran (Onipe et al. 2015).

Fig. 1.

Overlapping of human Zn deficiency with soil Zn deficiency in world (adapted from http://www.omex.com)

Zn is a very important micronutrient for human health (Chen et al. 2017). It is estimated that around 3000 proteins, corresponding to 10% of human proteomes are Zn-dependent (Kambe et al. 2015; Krezel and Maret 2016). Zn deficiency hinders the production of proteins which causes physiological issues such as growth retardation, susceptibility to infectious diseases, abnormal immune system, and impaired learning (Wessels and Rink 2019). The Zn deficiency is responsible for 14.4% of deaths due to diarrhoea, 10.4% of deaths due to malaria and 6.7% of pneumonia among children aged between 6 months to 5 years in Africa (Berhe et al. 2019). The deficiency of Zn affects birth outcomes in pregnant women (Terrin et al. 2015) and causes a reduction in the linear growth of children. Zn plays an important role in the metabolic processes of more than 300 enzymes in the body (Lu et al. 2021), immune defence, growth, bone health, and cognitive function (Huang et al. 2015) and acts as a neuromodulator in the central nervous system (Goldberg and Lippard 2018). Due to its immune-modulator and antiviral effect, it is regarded as a potential supportive treatment in COVID-19 therapy (Skalny et al. 2020) In biological systems, it exists as a divalent cation (Zn²⁺) which is a strong electron acceptor and does not cause oxidant damage to cells. Various scientists reported the significance of Zn in the human body in different organ systems (Table 1).

Table 1.

Role of Zn in human organ systems

Organ Systems	Role of Zn	References
Cardiovascular	A cardio protective role reduces risk of atherosclerosis	Choi et al. (2018)
Integumentary	Skin health, wound healing and act as antioxidant	Ogawa et al. (2018)
Reproductive	An important role in the formation and maturation of spermatozoa for ovulation and fertilization	Kumar and Singh (2016), Baltaci et al. (2019)
Nervous	The modulator of neuronal excitability, an important role in neuronal metabolism and modulator of synaptic activity	Gower-Winter and Levenson (2012), Prakash et al. (2015)
Respiratory	Reduces the incidence of viral and respiratory tracts infection	Wessels et al. (2020)
Endocrine	Thyroid hormone metabolism and activity of insulin	Baltaci et al. (2019)

Zn also plays a vital role in biological metabolism along with the normal growth and development of plants. It plays a significant role in stress tolerance (Hassan et al. 2020), pollen tube formation (Xie et al. 2021), gene expression (Lilay et al. 2019), chloroplast development (Hansch and Mendel 2009), auxin and enzymatically driven metabolism (Hacisalihoglu 2020; Rudani et al. 2018) in plants. Zn is a structural and catalytic protein cofactor of enzymes with major structural functions in the protein sites (Naik and Kumar 2020). The DNA binding of transcription aspects and interactions among proteins are driven by “Zn finger” proteins (Sinclair and Kramer 2012). and its deficiency symptoms are visible on the lower leaves of plants (Vadlamudi et al. 2020). In the last four decades, agriculture research has mainly focused on increasing food production with high-yielding crop cultivars which led to considerable Zn depletion from the soil (Behera et al. 2009). Soils having DTPA-extractable Zn lower than 0.5 mg kg⁻¹ are generally known to have low available Zn for optimum plant growth (Costerousse et al. 2017) and its deficiency is observed more in calcareous soils (Recena et al. 2021). In the world, more than half of wheat is cultivated on Zn-deficient soils which leads to an insufficient supply of Zn nutrients for human health (Yu et al. 2021). Zn-deficient soils produce wheat cultivars with a low amount of Zn as there is a reduction in absorption and accumulation of Zn in wheat grains. The overlap of Zn deficiency in soils and humans indicates the directly proportional relationship of soil Zn deficiency to Zn uptake by humans (Fig. 1).

The socioeconomic status of countries plays an important role in the well-being of their population. The deficiencies of various micronutrients such as Zn are related to the socioeconomic status of regions with more prevalence in weak socio-economic classes. The provinces of India with low socio-economic index (n = 1655) constitute around 43.8% low plasma Zn prevalence among children (Kapil and Jain 2011). Although preventative Zn supplementation has been shown to reduce childhood mortality and morbidity, there are currently no large-scale preventive Zn supplementation programmes (King et al. 2015). In regions of low socioeconomic status, people cannot afford money on Zn-supplemented food. In developing countries, regular consumption of cereal-based foods results in a low dietary intake of Zn. Widespread poverty, high food prices and cultural preferences are the major factors of the low dietary intake of Zn (Bouis and Welch 2010). Keeping in view, the importance of wheat in daily energy consumption by a large population, its production with improved and better distribution of Zn content becomes crucial. Instead of using supplementation and food fortification approaches to alleviate Zn deficiency, agronomic bio-fortification can provide an efficient and cheaper way to provide Zn-enriched food to low socioeconomic status countries. The entire cycle of increasing the Zn concentration in wheat grains and its ultimate effect on grain yield, quality, human health & nutrition, climate change and socio-economic status of livelihood is less compared. The widespread poverty and high food prices of Zn-supplemented foods highlight the need for low-cost Zn-enriched cereals particularly wheat which can be achieved through agronomic biofortification. The present studies were undertaken to study agronomic Zn biofortification approaches across the globe to alleviate Zn deficiency in humans without impairing the wheat quality.

The path of bioavailability of Zn

The bio-fortification of wheat with Zn is a sustainable approach as it is consumed as a staple food in developing nations (Velu et al. 2014; Zou et al. 2012). Besides the application of Zn nutrient to improve its content, increased grain Zn concentration may not always accumulate equally to its enhancement (Tako et al. 2008). Zn follows a path from the soil to the crop/food and then into the human body. The success of agronomic bio-fortification to alleviate Zn deficiency depends upon several factors which include the availability of Zn in soil solution, allocation of Zn within plant parts and its re-translocation into the harvested food (Gupta et al. 2016).

Movement of Zn from soil to crop

The various soil factors viz., pH, soil aeration, moisture, organic matter and elemental interactions affect the bioavailability of Zn to crop plants. Soils in many cereal-growing areas have several chemical and physical issues that diminish Zn solubility and hinder root absorption (Fig. 2). The rhizosphere can be changed through the excretion of organic acids or H⁺ ions by some plants to enhance nutrient availability and uptake (Adeleke et al. 2017). The soluble, exchangeable and organically bound Zn pools form the potentially bioavailable portion of Zn. In plants, Zn is absorbed by roots in the form of Zn²⁺ from the soil solution. The pH of soil regulates the bioavailability of Zn to plants, as with the increase in pH, its availability decreases. When soil pH is above 8, calcites bound the Zn more strongly, making it less available to plants. Increased soil pH stimulates Zn adsorption to soil constituents (e.g., metal oxides, clay minerals) and lowers adsorbed Zn desorption.

Fig. 2.

Zn dynamics influenced by soil factors in the rhizosphere (adapted from Gupta et al. 2016)

A very minor gain in Zn enhancement through soil fertilization of Zn fertilizer in wheat reported by Zou et al. (2012) when experiment was conducted in seven countries across different soils and environmental conditions. The movement of Zn through diffusion is impaired by soil moisture as well as low organic matter content which results in reduced accumulation of Zn in crop grains (Rengel 2015). Soil management strategies such as the application of soil amendments like organic manures can improve the Zn bioavailability. The soil microflora has a considerable positive impact on Zn bioavailability as they take part in the biogeochemical cycling of nutrients. Nearly all metal-microbe interactions, for instance, oxidation, reduction, solubilization and complexation play a significant role in the movement of nutrients (Gupta et al. 2022). Soil microbes such as bacteria, fungi, mycorrhiza and cyanobacteria could help in the precise nutrient acquisition by plants. The symbiotic association between Arbuscular mycorrhizal fungi and roots help in increasing the bioavailability and nutrient uptake of scarce soluble nutrients like Zn (de Valençaa et al. 2017). Nitrogen fertilizers such as ammonium sulphate and ammonium nitrate in addition to supplying nitrogen enhance soil acidity which results in the desorption of Zn (Alloway 2008). Therefore, it is important to visualize the soil conditions to know about Zn bioavailability in the soil.

Movement of Zn from crop to food

The incidence of Zn deficiency in human populations appears to be due to a lack of Zn intake through diet (Ram et al. 2015). Localization and concentration of Zn in wheat grains have remarkable significance in supplying Zn to humans. Instead of using whole grains, the different parts of the wheat grain are used in different foods. Generally, grains have three parts viz., embryo, endosperm and aleurone layer. Zn is localized mainly in the aleurone layer and embryo parts of wheat grain. On the other hand, it is present in very small amounts in endosperm which constitutes the major part of wheat flour. The endosperm contains about 10 mg Zn kg⁻¹ whereas the embryo and aleurone layer may have up to 100 mg Zn kg⁻¹ (Morgan 2021). Generally, wheat grain is consumed after milling which results in the removal of Zn-containing parts and results in low Zn content which resides in the endosperm. Wheat grain is consumed in the form of wheat flour which has around 12 mg Zn kg⁻¹ (Wang et al. 2020). The conditions depicted above cannot help in alleviating the Zn deficiency. Due to this, fortification strategies are necessary to increase the Zn content in wheat flour, especially in developing countries (Brown et al. 2010).

Movement of Zn from food to human

There can be either food or host-related factors that affect the absorbance of micronutrients from the food (de Valençaa et al. 2017). The bioavailability of micronutrients is influenced by their chemical form, amounts as well as interactions between nutrients and the composition of the dietary matrix (Ostrenga 2018). Certain host-related factors such as an individual’s health, nutrient status, age, genotype and physiological state impact the micronutrient bioavailability from foods for uptake by the human body (Malik and Maqbool 2020). Infections and parasites reduce the absorbance of Zn from food and cause malnutrition (Astiazarán-García et al. 2015). Due to the high phytic acid content in cereals, (Gupta et al. 2015), Zn-phytate acid complexes are formed in the intestine ultimately reducing the intestinal absorption of Zn (Maares and Haase 2020). Furthermore, several other inhibitors such as calcium and polyphenols markedly influence the bioavailability of Zn. In a nutshell, Zn bioavailability is affected by various factors during different stages from soil availability and plant uptake to human absorption. These stages must be considered to alleviate the hidden hunger through effective agronomic bio-fortification.

Wheat bio-fortification with Zn

Agricultural systems and biofortified food

Traditionally, vitamins and nutrients which are present in less concentration in cereals are supplied by nutrient supplements. These supplements are available throughout the world having Zn as one of the important nutrients (NIHODS 2021) but it has not achieved the goals that were set by the international health organization because of funding-related problems, the purchasing power of poor people, defaulted healthcare systems and lack of awareness about the significance of consuming micronutrient supplements (Garg et al. 2018). Moreover, our agriculture system has been designed to increase crop yield (Shah and Wu 2019) and productivity instead of promoting human health by improving all required nutrients in the grains. Borrill et al. (2014) stated that postharvest fortification, diversification in dietary habits, mineral supplementation and bio-fortification are various possible approaches to overcome hidden hunger. The bio-fortification of cereals such as wheat to enrich the Zn content could be the most desirable method to fight ‘hidden hunger’ (Dapkekar et al. 2018).

Genetic biofortification versus agronomic biofortification

Biofortification of staple cereals with micronutrients by using agricultural methods, such as plant breeding and agronomic biofortification appears a useful, cost-effective and long-term technique to address micronutrient deficiencies in human populations (Zou et al. 2019). Among these methods, agronomic bio-fortification of Zn is considered a method of choice to enhance Zn content in wheat grain because it can improve Zn content as well as crop yield in a short-term period (Akhter et al. 2020). The application of micronutrient-containing mineral fertilizer (blue circles) to the soil and/or plant leaves (foliar) to improve the micronutrient content of the edible section of food crops is referred to as agronomic bio-fortification. (Fig. 3 adapted from de Valençaa et al. 2017). On the other hand, the breeding method is costly and takes a long time to develop Zn-rich cultivars (Chattha et al. 2017). Furthermore, improving Zn content in wheat grain by breeding and transgenic strategies reduces the crop yield (Fan et al. 2008) since these have inverse correlations (McDonald et al. 2008). Despite having Zn-rich genotypes in future, agronomic bio-fortification could not be disregarded under poor Zn soils. Therefore, an effective complementary approach could be performed through agronomic and breeding bio-fortification to get Zn-rich wheat (White and Broadley 2009; Velu et al. 2014). The agronomic bio-fortification is aimed at supplying micronutrients that can be directly absorbed by the plants through the foliar application with nutrient-containing material such as fertilizers or the improvement of the solubilization and mobilization of mineral nutrients in the soil, is considered to be the simplest method used to increase the levels of microelements in crops (Szerement et al. 2022). The agronomic bio-fortification is accomplished by the application of Zn-containing fertilizers to the soil and foliage of the crop. Additionally, seed priming (a low-cost technique of soaking seeds in a solution containing nutrients) is also an important strategy as seed reserves are the key source of nutrients during the early seedling stage. It is the most commonly used strategy for experiencing the effects of high-Zn seed on germination as well as seedling development (Rashid et al. 2019). Agronomic biofortification is likely to be more economical, sustainable and easily implementable as compared to breeding and other methods (Ramzan et al. 2020). Ngozi (2013) stated that agronomic bio-fortification is an evolving way to reduce micronutrient malnutrition. Providing nutritious, healthy and safe food in an affordable, sustainable and sufficient way to the whole world is the main long-term goal of agronomic bio-fortification (Saltzman et al. 2013). Moreover, a combination of Zn-containing fertilizers with pesticides represents a useful and cost-effective method to address the Zn deficiency problem in human populations (Ram et al. 2016, 2022).

Fig. 3.

The application of micronutrient-containing mineral fertilizer (blue circles) to the soil and/or plant leaves (foliar) to improve the micronutrient content of the edible section of food crops is referred to as agronomic bio-fortification (adapted from de Valençaa et al. 2017)

Agronomic bio-fortification and grain Zn concentration of wheat

Agronomic bio-fortification is a quick way to improve the wheat grain Zn content. The Zn enrichment in wheat can be accomplished through the soil, foliar and soil + foliar applications (Ram et al. 2015; Cakmak and Kutman 2018). Rehman et al. (2020) concluded that the agronomic bio-fortification technique has proven successful in Pakistan in terms of increasing grain Zn content, agricultural productivity and profitability. The wheat endosperm is often ingested as a main component of wheat-based foods. It has less phytate concentration than the aleurone layer of wheat that is removed during the milling processes. So, increasing Zn concentration in low phytate-containing endosperm improves the Zn:PA molar ratio which is an important indicator of Zn bioavailability (Gupta et al. 2015). Rehman et al. (2020) reported that Zn application through soil, foliage or seed treatment methods improves the Zn concentration in whole grain as well as in the different seed parts such as endosperm, aleurone layer and embryo. It further reduces the phytate accumulation which ultimately enhances the Zn bioavailability. The efficacy of agronomic bio-fortification depends upon the application methods viz., soil, foliar and seed treatment methods that might be up to 3–4 times increase than the control (Gupta et al. 2016). Ramzan et al. (2020) observed the difference in Zn content due to the adoption of different application methods in which foliar application demonstrated the highest efficiency. The data of an experiment conducted across 14 locations in seven countries by Zou et al. (2012) revealed the efficiency of agronomic bio-fortification through different methods in which it was observed that foliar application of Zn showed better results than soil Zn application. However, at some locations, a combination of foliar and soil application showed better outcomes in consideration of Zn content. The foliar Zn application alone resulted in an 83.5% rise in grain Zn concentration but soil Zn application was less effective. In soil application, a poor correlation was observed between DTPA-Zn content of soil and grain Zn content which might be due to unsuitable soil conditions which reduce the nutrient mobilization to plant roots and ultimately lower the subsequent absorption. Rahman and Schoenau (2020) stated that soil-applied Zn is adsorbed by various compounds which diminish its mobility in the soil–plant system.

Likewise, Hassan et al. (2019) reported the highest grain Zn content accumulated in foliar-applied Zn treatment over the soil, seed priming and control which was significant. In addition, foliar Zn application at the rate of 0.5% solution illustrated better results (70.7% improvement in Zn content over control) followed by soil application, seed priming and control. The significant increase in grain Zn due to foliar Zn spraying would make a significant contribution to boosting human Zn dietary intake. Likewise, Wang et al. (2015) verified that foliar application-based Zn is more phloem mobile and readily translocated into developing grains in wheat. However, when the target is improvement in grain Zn content as well as grain yield, the soil + foliar application method is recommended (Velu et al. 2014).

To provide additional benefits in terms of seed vitality and seedling vigour at early growth stages, seed priming with Zn plays a crucial role because high Zn content at early stages ensures good root growth and contributes to better protection against soil-borne pathogens (Cakmak 2012a). Praharaj et al. (2021) stated the benefits and limitations of different application methods (Table 2). Moreover, different types of wheat such as bread wheat (Triticum aestivum L.), triticale (Triticale hexaploide Lart.) and durum wheat (Triticum durum Desf.) and their cultivars show varied responses to different methods of Zn bio-fortification. There is already evidence of significant heterogeneity in Zn utilization efficiency among wheat varieties (Lu et al. 2020). Due to the effect of genotype and environment interactions, Zn concentration improved from 15 to 35 ppm in some genotypes (Bhatt et al. 2020). The various studies on the effect of agronomic biofortification on grain Zn concentration are given in Table 3. Zn concentration increased more than two fold in all wheat cultivars over control, among which the highest was reported in durum wheat (133.9%) (Dhaliwal et al2019). There might be a difference in Zn concentration due to the initial seed Zn content that determines the seedling vigour and growth at the early stages.

Table 2.

Advantages and disadvantages of various application methods

Application methods	Advantages	Limitations
Soil application	Induces the Zn efficiency of the soil Succeeding crops may benefit from the residual effect	High fertilizer requirement Due to poor soil conditions, plant availability may be reduced
Foliar application	Reduces the need for fertilizers Negative soil features do not affect it	Crop requirements are not met during the seedling stage The foliar approach cannot be used to provide a very high amount of fertilizer
Seed priming	Lowers fertilizer requirement Suitable for high-stress situations	This method cannot be used to apply a larger amount of nutrition since a high concentration of priming fluid can harm germination

Source: Praharaj et al. (2021)

Table 3.

Effect of agronomic bio-fortification of Zn on grain Zn concentration in wheat

Wheat type	Application method	Application rate (ZnSO4·7H2O)	Grain Zn concentration in control (mg kg−1)	% increase in Zn concentration in treatment over control	References
Bread wheat	Foliar application	0.5%	31.8	112.3	Dhaliwal et al. (2019)
Triticale	Foliar application		30.5	117.7
Durum wheat	Foliar application		27.7	133.9
Bread wheat	Seed priming	0.3%	30.7	13.4	Chattha et al. (2017)
	Soil application	10 kg Zn ha−1	30.7	29.6
	Foliar application	0.5%	30.7	70.7
Bread wheat	Foliar application	0.1%	30.3	11.9	Jalal et al. (2020)
		0.2%		12.5
		0.3%		27.7
		0.4%		29.0
Bread wheat	Foliar application at tillering, booting and milking stage	0.2%	18	170.3	Kiran et al. (2021)
Bread wheat	Soil + foliar application	11.5 kg Zn ha−1 and 0.4%	18.5	78.5	Afzal et al. (2017)

It was observed that plants grown from high intrinsic Zn content seeds have better performance under abiotic stresses (waterlogging, drought and salinity). Moreover, this type of plant has lower oxidative damage, higher seed yield and more grain Zn concentration when compared to low Zn seeds (Faran et al. 2019). Barut et al. (2017) recorded the different responses of three wheat varieties concerning grain Zn concentration under different methods of Zn application (Fig. 4). Practicing agronomic bio-fortification using a high Zn seed-containing cultivar improved the grain Zn concentration and productivity but it also decreased the Cd uptake in Cd-contaminated soil concerning plants grown through low Zn seeds (Qaswar et al. 2017). So, agronomic bio-fortification with Zn in wheat retards the accumulation of heavy metals in the sink. As a result, cultivar selection is an important step to consider not only to maintain but also to increase Zn-use efficiency which also requires taking into account the climate, soil type, organic matter content and soil fertility.

Fig. 4.

Variation in Zn accumulation by three wheat cultivars in consideration to different application methods (adapted from Barut et al. 2017)

The variation in concentration of foliar-applied Zn illustrates different results for grain Zn accumulation. Jalal et al. (2020) reported that the highest grain Zn content was shown by 0.4% foliar Zn concentration which was at par with 0.3% foliar Zn concentration. Similarly, Zhang et al. (2012) and Pascoalino et al. (2018) observed that grain Zn content differs at different levels of foliar Zn concentration. The difference was reported in grain Zn content, and flour Zn content due to variations in the concentration of foliar-applied Zn (Fig. 5). So, it is important to consider the optimum Zn concentration to obtain the most efficient results. When comparing the numerous types of Zn fertilizers that were examined, the application of Zn as ZnSO₄ was the most successful in raising grain Zn. The various wheat herbicides, insecticides and fungicides can be blended with ZnSO₄ without reducing the effectiveness of foliar spray for increasing grain Zn concentration, according to preliminary investigations. Farmers may be more inclined to apply ZnSO₄in their fields if the cost and time of application are reduced (Velu et al. 2014).

Fig. 5.

Variation among Zn concentrations in wheat grain as well as in wheat flour with respect to foliar Zn application concentration (adapted from Zhang et al. 2012)

The timing of foliar Zn treatment is crucial for determining its efficiency in boosting the grain Zn concentration. Wheat grain Zn concentration might be enhanced further by optimizing the timing and solute concentration of foliar Zn treatment (Ning et al. 2019), not only in whole grain but also in the endosperm (Zhang et al. 2010). Substantial grain Zn increases most likely when foliar Zn fertilizers are administered to plants at later stages of growth (Velu et al. 2014). Foliar Zn application during reproductive growth appears to be more successful in raising grain Zn concentration than spraying Zn at an earlier growth stage. Furthermore, foliar application at later stages enhances the Zn concentration in the whole wheat grain, bran, embryo and starchy endosperm (Cakmak 2012b) (Fig. 6).

Fig. 6.

Changes in Zn concentration in the different parts of wheat grains from plants sprayed with ZnSO₄ at various phases of growth in the field (adapted from Cakmak 2012b)

As earlier discussed, the effectiveness of foliar treatment is mostly determined by crop developmental stages. As a result, understanding the stages of crop growth during which foliar micronutrient administration results in increased grain accumulation is critical. Zn application throughout the reproductive stages of the crop, such as heading and early milk stages (Cakmak et al. 2010) as well as the milky dough stage (Cakmak 2012b; Ma et al. 2017), has been shown to accumulate more grain Zn than application during booting and stem elongation stages because wheat crops mobilize more Zn during the milk stage than other stages (Ozturk et al. 2006). So, the application of foliar Zn at the optimal stage can enhance the agronomic bio-fortification efficiency which ultimately improves the Zn accumulation in grains and other nutritional attributes.

Biofertilizers also known as microbial-based fertilizers are regarded to be pivotal components of sustainable agriculture with long-term effects on soil health (Bargaz et al. 2018). According to Riaz et al. (2020), biofertilizers can be defined as formulations made up of living microbial cells (single cell or consortium), that improve plant growth by enhancing nutrient availability and acquisition. In the soil solution, a very minor portion of Zn is available as a soluble form of Zn. The remaining Zn is found as insoluble compounds and minerals (Kamran et al. 2017). The use of soil microorganisms that may mobilize unavailable Zn, and improve Zn assimilation, plant growth and yield is a conventional approach to enhancing Zn content in wheat (Wang et al. 2021; Rana et al. 2012). Plant growth is aided by plant growth-promoting rhizobacteria (PGPR) which solubilize nutrients and enhance nutrient acquisition or release biocontrol agents to protect plants against diseases (Glick 2012). PGPRs have been discovered to be potent Zn solubilizers. These bacteria help plants to grow and develop by populating the rhizosphere and solubilizing complicated Zn compounds into simpler ones, making Zn more accessible to plants. It is well-documented that Zn mobilization abilities appear to be prevalent among bacterial taxa (He et al. 2010). The bio-fortification can be enhanced by inoculating Zn solubilizing bacteria which is a potential alternative for Zn supplementation and increasing the availability of Zn by converting applied inorganic Zn into available forms for plant uptake (Kamran et al. 2017). Bacillus is one of the most studied genera since it is present everywhere in nature and has a variety of growth-promoting properties (Miljaković et al. 2020).

Gluconacetobacter sp. (Vidyashree et al. 2018), Pseudomonas sp., Bacillus sp., Burkholderia cenocepacia (Khande et al. 2017), Serratia liquefaciens and S. marcescens (Abaid-Ullah et al. 2015; Pawar et al. 2015) have been reported as Zn solubilizers on a lab scale. Bacilli strains are of particular importance because they create endospores, which allow them to withstand harsh environmental conditions, can be mass-produced and allow for easy formulation and storage. Ramesh et al. (2014) experimented to study the effect of Zn solubilizing Bacillus aryabhattai bacteria on the plant growth and bio-fortification of Zn in soybean and wheat. It was observed that different strains have different capacities to accumulate Zn and make it available to plants and MDSR 7 accumulated the highest Zn in seeds of wheat. On the other hand, accumulated phytic acid was also high in bacterial strain treatments but there was a reduction in the Phytic acid: Zn ratio which is a desirable character (Table 4). Proton extrusion and the formation of organic acids might be involved in these strains for the uptake of Zn from insoluble Zn compounds. Acids of microbial origin might cause Zn to be solubilized in a non-specific manner, hence affecting Zn bioavailability. The quantitative and qualitative content of root exudates changes due to the breakdown and the release of microbial metabolites by microbial activity (Canarini et al. 2019) which play a key role in the soil Zn cycle by transforming soil Zn.

Table 4.

Influence of Bacillus aryabhattai bacterial strain on seed Zn concentration and phytic acid content (Ramesh et al. 2014)

Bacterial strain	Seed Zn content (% increase than control)	Phytic acid content (% increase than control)	Phytic acid/Zn ratio (% decrease than control)
MDSR 7	44.5	27.5	− 11.1
MDSR 11	17.2	5.9	− 9.5
MDSR 14	42.7	33.3	− 6.3
Un-inoculated (control)	42.40 µg g−1	0.51 g kg −1	1.26

In addition to raising grain Zn content in wheat through bacterial strain inoculation, higher enzyme activities, microbial biomass, a considerable drop in rhizosphere pH and redistribution among native Zn pools resulted in improved grain yield was also found. In this context, using beneficial rhizosphere microbes as bio-inoculants to increase native Zn availability for crop assimilation and achieve the goal of low-input and sustainable agriculture (He et al. 2010) and overcome Zn malnutrition in human populations (Mäder et al. 2010) could be a viable option. In light of this circumstance, it's possible that utilizing native Zn mineralizing and solubilizing bacteria could help to alleviate Zn deficiency and boost its availability to crops.

Agronomic bio-fortification and grain yield

Agronomic Zn bio-fortification not only increases Zn content in grains but also enhances grain yield and productivity of crops (Zulfiqar et al. 2021). Some studies have investigated that besides increasing grain Zn content, agronomic bio-fortification helps to enhance grain yield as well as economic returns (Farooq et al. 2018; Ullah et al. 2019). Dhaliwal et al. (2019) reported the effect of foliar Zn application on different parameters of bread wheat (Triticum aestivum L.), triticale (Triticale hexaploid Lart.) and durum wheat (Triticum durum Desf.) and observed that grain yield significantly increased about 4.8, 4.43 and 4.56% across the varieties of bread wheat, triticale and durum wheat in Zn applied treatments, respectively, over control. Similarly, Dhaliwal et al. (2009) reported that foliar Zn application @ 0.5% ZnSO₄·7H₂O showed improvement in yield from 54.6 to 57.4 and 52.9 to 58.6 q ha⁻¹ under durum wheat and bread wheat varieties, respectively. Ramzan et al. (2020) reported that the Zn application significantly improved the number of tillers, plant height, spike length, number of spikelets spike⁻¹, number of grains spike⁻¹ and 1000-grain weight, ultimately enhancing the grain yield and harvest index (Table 5). The average increase in yield of soil application and foliar application treated plots over control was about 18.09 and 14.11%, respectively. The reason behind this increased yield might be the availability of Zn and its role in biochemical reactions including photosynthesis. Similarly, Sultana et al. (2016) recorded the highest yield among the foliar application treatment at 0.4% concentration due to improvement in 1000-grain weight, the number of grains spike⁻¹ and spike length⁻¹. The highest increase in grain production was reported under a combination of ZnSO₄·7H₂O @ 25 kg ha⁻¹ as a soil application and 0.5% solution of ZnSO₄·7H₂O as a foliar spray (Narwal et al. 2010). The application of Zn to soils enhanced grain production by 29% in wheat (Hussain et al. 2012). Dhaliwal et al. (2012) recorded that soil application of ZnSO₄·7H₂O @ 62.5 kg ha⁻¹ and foliar Zn chelate application significantly improved the plant height and tillers m⁻². Likewise, Zn application improves yield and yield components through a variety of mechanisms including increased chlorophyll content, photosynthetic activity and auxin production, all of which contribute to improved crop growth and development (Rakesh and Jitendra 2014). Similarly, Chattha et al. (2017) reported the highest yield under the combination of soil + foliar application than control, seed priming, soil and foliar application methods (Table 5), however, results were non-significant at p = 0.05. Furthermore, this study indicates a positive correlation between grain yield and grain Zn content, previously supported by Zou et al. (2012) and Karim et al. (2012) in Pakistan and China, respectively. Hassan et al. (2019) outlined the highest grain yield of wheat under soil-applied Zn followed by seed priming, foliar application and control (no Zn application) (Table 5). This might be due to the higher availability of Zn at the early stages of the crop which helped to attain proper growth and development. So, agronomic bio-fortification of Zn in wheat improves grain yield substantially. Furthermore, considering the growing population and increasing micronutrient malnutrition, especially in cereal-based foods, Zn bio-fortification is the need of the hour.

Table 5.

Effect of various Zn application methods on grain yield of wheat

Cultivar	Control (q ha−1)	% change over control in grain yield of wheat				References
		Seed priming	Soil application	Foliar application	Soil + foliar
-	32.6	–	18.1 (10 kg Zn)	14.1 (0.5% ZnSO4)	–	Ramzan et al. (2020)
Faisalabad-2008	31.4	7.3 (0.3% ZnSO4·7H2O)*	34.1 (50 kg ZnSO4)	23.9 (0.5% ZnSO4·7H2O)	45.5 (50 kg ZnSO4 + 0.5% ZnSO4·7H2O)	Chattha et al. (2017)
Punjab-2011	41.5	8.4 (0.3% ZnSO4·7H2O)	23.9 (50 kg ZnSO4)	17.6 (0.5% ZnSO4·7H2O)	34.7 (50 kg ZnSO4 + 0.5% ZnSO4·7H2O)
Millat-2011	35.9	10.9 (0.3% ZnSO4·7H2O)	28.1 (50 kg ZnSO4)	23.7 (0.5% ZnSO4·7H2O)	46.5 (50 kg ZnSO4 + 0.5% ZnSO4·7H2O)
-	55.3	10.1 (0.3% ZnSO4·7H2O)	19.9 (10 kg Zn)	4.0 (0.5% ZnSO4·7H2O)	-	Hassan et al. (2019)

^*Figures shown in parenthesis are Zn application rates

Agronomic bio-fortification and wheat grain quality

To fulfil the requirements of a burgeoning population, the maintenance of wheat grain quality is highly important. Wheat grain quality is determined by its total protein content and protein components, which has a key role in flour processing quality and the market value of flour products. Zn has a considerable effect on monomeric protein structure, the formation of high molecular weight glutenin subunits and the proportions of glutenin and gliadin in total proteins, which ultimately affects the wheat flour quality (Liu et al. 2015). The zn application could enhance the nitrate reductase and glutamine synthetase activity which affects protein content in wheat grains. Sharma et al. (2008) reported higher protein content with foliar-applied Zn. Similarly, Nadeem et al. (2020) reported higher wheat grain protein content with the Zn application through various methods over control. The highest protein content was found from soil Zn application and seed priming during the first and second year, respectively. However, they observed the highest grain fat (1.04 and 1.05%) with seed priming and soil application of Zn during both years, respectively. Dapkekar et al. (2018) stated that higher grain Zn content leads to an increase in grain protein content as it is essentially required during protein biosynthesis. Secondly, the increase in protein content is possibly due to enhanced nitrogen uptake because Zn and N have a synergistic effect. Some studies related to the effect of agronomic bio-fortification of Zn on wheat grain quality are presented in Table 6. Liu et al. (2015) conducted a pot experiment and reported an increase in the protein content of wheat grains up to the application of 10 mg Zn kg⁻¹ of soil. Application of Zn changed the gliadin, glutenin, albumin and globulin contents in flour which might be the result of Zn as disulfide bonds. Zhang et al. (2012) stated that Zn is closely related to cysteine, cysteine Zn proteome of Zn binding ligand ratio is 28%, the highest percentage for all amino acids. Esfandiari et al. (2016) reported an increase in the hectolitre weight of wheat due to the foliar application of Zn at the soft dough stage and the booting + milking stages in comparison to the control. Furthermore, the application of Zn reduced the phytate concentration of wheat grain which might be due to the less phosphorus uptake especially in soil application methods and its translocation with in the plants (Rehman et al. 2018). It can be concluded from the experiments that agronomic biofortification of Zn has a positive impact on different wheat grain quality parameters.

Table 6.

Effect of agronomic biofortification of Zn on different quality traits of wheat

Application method	Application rate	Quality parameters	Value in control	% change in quality over control	References
Foliar application	0.5% ZnSO4.H20	Protein	9.7%	14.4	Zeidan et al. (2010)
Soil + Foliar application	5 kg Zn ha−1 + 1% ZnSO4	Glutenin	2.0%	29.5	Khattak et al. (2015)
Soil + Foliar application	7.5 kg Zn ha−1 + two foliar sprays of Zincsol	Polymeric: gliadin ratio	0.75	18.7	Peck et al. (2008)
Foliar application	2 g ZnSO4·7H2O L−1	Hectolitre weight	84.7 kg 100 L−1	1.53	Esfandiari et al. (2016)
Seed priming	0.5 molar Zn	Phytate		− 9.7	Rehman et al. (2018)
Seed coating	1.25 g Zn kg−1 seed			− 8.24
Soil application	10 kg Zn ha−1			− 11.6
Foliar application	0.025 molar Zn			− 12.9
Foliar application	0.3% ZnSO4·7H2O	Phytate	7.08%	− 6.2	Li et al. (2018)

Agronomic bio-fortification and human health

The fundamental aim of agronomic bio-fortification of Zn in wheat is transferring desired Zn content to the human body. So, considering past trends in which micronutrient enrichment of cereals is considered an important thrust area, agronomic bio-fortification is playing a crucial role in increasing micronutrient content in cereals. Qaim et al. (2007) stated the numerous factors that affect the impact the bio-fortified crops (Fig. 7). There has been an improvement in the concentration of Zn in grains through Zn-containing fertilizers which contributed to human nutrition and health in Pakistan (Joy et al. 2017) and Turkey (Cakmak 2008). It was discovered that agronomic Zn biofortification of wheat might boost Zn dietary intake in infants and children under the age of five, resulting in a 56.6% reduction in Zn deficiency-related health costs. Bouis and Saltzman (2017) stated that bio-fortified crops are produced and consumed by more than 20 million people globally. Adults consuming wheat flour from soil-applied Zn or control fields (no Zn applied) indicated that consumption of agronomic bio-fortified Zn flour significantly improved the daily dietary Zn intake of that rural population as compared to the control group (Rehman et al. 2020). On the other hand, the impact of agronomic bio-fortification on human health is still being calculated, Finley (2006) emphasized that furthering the legitimacy of agronomic bio-fortification necessitates much more research on micronutrient bioavailability, particularly metabolic pathways that influence absorption and the health advantages of various chemical forms of micronutrients. It can be concluded that there is a significant knowledge gap about the relationship between cereal micronutrient fertilization and the nutrition status of people. Systematic research is required to analyze the potential and required conditions to enhance human health with agronomic bio-fortification. This is because micronutrient malnutrition can lower human efficiency, reduce work productivity and ultimately will affect economic output at all levels, from individual to national level. Liu et al. (2017) used the DALYs (daily-adjusted life years) equation to assess the health pressure of Zn deficiency and the impact of agronomic biofortification of Zn in infants and children. In this study, current health burdens were calculated based on a study in China, which demonstrated that years of life lost (YLL) were 151 million and years lived with disability (YLD) were 202 million years, making a total DALYs value of 352 million years. After the application of 50 kg zinc sulphate heptahydrate ha⁻¹, daily Zn intake by infants and children was increased to 5.02 and 6.24 mg day⁻¹ from 4.90 and 6.0 mg day⁻¹ in control, respectively. The health impact was reported positive as 69,793 and 3,79,258 DALYs were saved for infants and children, respectively, with the application of 50 kg ZnSO₄·7H₂O ha⁻¹. Additionally, 12.7% reduction in the current health burden was reported. Similarly, Wang et al. (2016) investigated a study on the impact assessment of agronomic biofortification of Zn in wheat on human health. It was observed that agronomic biofortification of Zn saved 25.773 and 94,849 DALYs for infants and children in optimistic scenario, respectively, which reduced averaged 56.6% current health burden.

Fig. 7.

Factors influencing the impact of bio-fortified crops (adapted from Qaim et al. 2007)

Agronomic bio-fortification and environment

The micronutrient-rich fertilizers with Zn and Fe are thought to have a negligible detrimental impact on the environment as they are strongly bound in the soil and are not vulnerable to leaching losses (Udeigwe et al. 2016). However, if large quantities of these fertilizers are applied, they can accumulate over time and cause toxicity. Yan et al. (2020) stated that if Zn accumulates to high concentrations in soil, it may cause toxicity to plants and soil microorganisms. The “safe” Zn levels in soil, do not apply to all conditions because the ability of soil to bind Zn varies depending on soil pH and other soil features, for example, cation exchange capacity (Tope et al. 2014). Adoption of different precise fertilizer application strategies such as the 4 R strategy “Right source, Right amount at the Right place and Right time” can help in optimizing the nutrient use efficiency and reduce the toxicity effects (Bruulsema et al. 2012). Similarly, Wang et al. (2013) stated that to reduce the environmental risk, an investigation of the optimum dose of fertilizers is necessary that can ensure the proper growth and desirable nutrient content in soils. Moreover, the application of a desirable quantity of micronutrient fertilizers helps to alleviate its deficiency and ultimately better the crop's health status. It can be worked as a barrier against insect pests and diseases (Dimpk and Bindraban 2015) and reduced weed infestation by smothering effect. So, the use of insecticides, fungicides and herbicides can be reduced by using the agronomic bio-fortification approach, which will reduce environmental pollution.

Agronomic bio-fortification in relation to climate change

For the last century, climate change has been a severe challenge and a burning issue across the world (Hubbe and Hubbe 2019). It has disastrous consequences on the environment, agriculture, and human population. Various climatic factors quantify the nutrient acquisition and translocation in plants (Soares et al. 2019). As a prediction, global temperature will be on the rise and water scarcity will be presiding in future which will affect nutrient uptake and crop yield (Maqbool et al. 2020). The rising temperature and shifting precipitation patterns will harm crop yields over the world (Funk and Brown 2009 and Godfray et al. 2010), hence, bio-fortification efforts will be harmed by such seasonal changes. The effects of temperature extremes due to climate change include reduced grain filling period, reduction in grain number due to excessive heat and the sterility and abortion of formed grains as a result of frost in wheat (Barlow et al. 2015). Rain-fed agriculture is dependent on rain and its timing, and intra-seasonal precipitation patterns which can decide the success or failure of crops (Sanjeevaiah et al. 2021) that might reduce the response of crops to agronomic bio-fortification. Zhao et al.(2017) stated that increased global temperature can hinder agricultural practices and crop production periods by reducing crop season. The results of agronomic biofortification can vary as a result of its effect on the duration of different growth stages and shortage in a particular stage during harsh weather conditions. Due to the rise in temperature, Zn uptake in plants is affected that ultimately proceeds to lower yield and nutritional quality of the crops (Raj et al. 2015). Besides, temperature changes, climate change can cause drought in different areas of the world. It can affect the yield and nutritional quality of the crop by decreasing nutrient uptake as nutrient uptake depends upon the amount of water present in the soil. In addition, nutrient mobilization from roots to shoots can be diminished due to the low transpiration rate under drought conditions (Silva et al. 2011). The efficacy of foliar application of Zn might be affected due to a rise in temperature and air currents which can cause the instant evaporation of water used for spray, leading to less available time for nutrient absorbance. The efficacy of biofertilizers used for Zn solubilization will be affected by increasing the temperature and reducing water content. Due to increasing global temperature, crop output would be greatly affected which may lead to higher starvation and make humans more prone to infectious diseases (Easterling et al. 2007). The net result could be a reduction in labour efficiency as well as an increase in poverty and death (Schmidhuber and Tubiello 2007). According to Wakeel et al. (2018), climate change will increase the number of undernourished people in 2080 by 5–26% or 5–10 million people in a moderate effect scenario and 120–170 million people in a more severe impact scenario. Therefore, efficient production technologies should be developed to overcome the impact of climate change.

Agronomic bio-fortification and the socio-economic status of livelihood

The agronomic bio-fortification of Zn aids in achieving desirable health standards, it has a greater impact on the socioeconomic position of people or a specific region where bio-fortified crops are consumed. Agronomic biofortification can cope with the Zn shortage by producing food enriched with Zn in soils that are Zn deficient (Cakmak 2008). Furthermore, a well-balanced diet rich in micronutrients like Zn enhances health and lowers medical and supplementation costs. The reduction in medical and supplementation costs will help in improving the socioeconomic standing of people. Furthermore, improvements in people's health make them feel better and add to their peace. The economic effectiveness of agronomic bio-fortification was evaluated by Wang et al. (2016) using the “disability-adjusted life year” to calculate the health concerns. It was found that it costs US $226 to US $594 to save one "disability-adjusted life year," when the foliar spray of Zn is done alone whereas only US $41 to US $108 is required to save one "disability-adjusted life year." When the foliar application of Zn fertilizer is combined with pesticide spray by reducing the labour cost. Therefore, it can be stated that the agronomic bio-fortification of Zn is helping people to improve their socio-economic status by reducing medical and supplementation costs and improving human productivity.

Future scope

Though bio-fortification research has progressed, some crucial areas are still needed to be addressed or improved to achieve the fundamental purpose of agronomic bio-fortification. Firstly, at the regional level, a comprehensive 4R (Right source, Right amount at the Right Place and Right time) strategy for Zn application may be created for various crops which will help to know the ideal combination for producing high grain Zn concentration. Moreover, there might be some physiological constraints of grain Zn accumulation that must be identified for different crops and suitable agronomic strategies for amending these conditions can be found to improve the grain Zn concentration. The study must be focused on the combination of agronomic and genetic methods to improve mineral transport to phloem-fed tissues which will enhance the Zn content in wheat grains. As discussed earlier, climate change can have undesirable impacts on the success of agronomic bio-fortification, different biofortification options must be investigated under stressed conditions. In addition, these effects must be noted down that will help to develop a stress-proof bio-fortification strategy. The environmental associations with agronomic bio-fortification should be undertaken as there might be the chances of Zn toxicity over a long-term application. In light of available bio-fortification technology and stakeholders that fund bio-fortification initiatives, it is necessary to refine planning, monitor, and evaluate bio-fortification programmes. Although the HarvestPlus consortium is doing a good job, indicators for evaluating the performance of bio-fortification programmes and setting priorities are required. There is a need to establish communication and marketing strategies that consider ethical values when it comes to the production and use of bio-fortified wheat. The same tactics may not be effective in various nations to make it acceptable and persuade people to pay for micronutrient-enriched food, as a result, governments should be guided to use strategies that are beneficial to their citizens. These discussed key points might be helpful to increase the Zn content in grains through agronomic bio-fortification.

Conclusion

Agronomic bio-fortification is significant to alleviate Zn malnutrition and sustain the production to meet the ever-growing global demand for food and feed. Agronomic bio-fortification of Zn in wheat could provide a low-cost, short-term solution to Zn insufficiency issues. It is the simplest and fastest way to increase the wheat grain Zn content. Furthermore, it provides a different way to reach rural impoverished people who cannot purchase mineral supplements. The Zn content in wheat grains is influenced by the application technique and time concerning crop developmental stages. Even though nutrients are present in the soil, their access to plant roots is limited due to poor soil conditions, the foliar application can aid rapid and increased absorption and heal deficiency symptoms. Zn is effectively accumulated when applied to foliage during the post-anthesis stages. Zn content in wheat grains can be increased using a combination of agronomic and genetic bio-fortification, which can help to decrease Zn-related malnutrition without impairing the grain quality.

Author contributions

Sukhpreet Singh (SS), Jagmohan Kaur (JK), Hari Ram (HR), Jagmanjot Singh (JS) and Sirat Kaur (SK). The authors confirm contribution to the paper as follows: study conception and design: SS, JK HR; data collection: SS, JS, SK; analysis and interpretation of results: SS, JK, HR, JS; draft manuscript preparation: SS, HR, JS, SK. All authors reviewed the results and approved the final version of the manuscript.

Funding

This work was supported by the Punjab Agricultural University, Ludhiana, India [NP 52].

Declarations

Conflict of interest

The authors confirm that they have no known conflicts of interest that would have appeared to have an impact on the research presented in this study.