7.1 Agronomic Biofortification
Agronomic biofortification involves the application of mineral fertilizers to soil or crops to increase the concentration and bioavailability of specific nutrients in the crops (Adu et al., 2018). Initially, agronomic practices were done to improve the health of crops and increase yield. However, the importance of nutrition has been highlighted over the years; hence agronomic practices have been expanded to improve the nutritional qualities of crops (de Valença et al., 2017; Malik & Maqbool, 2020; Wakeel et al., 2018). Changes in climate conditions and rapid depletion of soil nutrients is an indication of the need to improve and expand agronomic practices to include improving the nutritional qualities of crops (Siwela et al., 2020). Agronomic biofortification focuses on improving solubilization and mobilization of minerals (de Valença et al., 2017; Sheoran et al., 2022). The effectiveness of agronomic interventions depends on the soil composition, the solubility and mobility of minerals, the ability of crops to absorb minerals, and the accumulation of bioavailable minerals in non-toxic levels in the edible parts of the crops (Sheoran et al., 2022; Singh et al., 2016; Umar et al., 2019). Agronomic biofortification mainly covers minerals and not vitamins because vitamins are synthesized in the crops. Hence, agronomic biofortification cannot be used as a single strategy in eliminating micronutrient deficiencies and should complement other strategies for effective biofortification (Jha & Warkentin, 2020; Sheoran et al., 2022; Wakeel et al., 2018). The use of fertilizers for agronomic biofortification must be performed carefully as an improper application of fertilizer can have unanticipated, and sometimes severe, consequences for the environment and crops. By contrast, a balanced fertilization strategy is both economically more beneficial and environmentally more sustainable. Additionally, soil microorganisms play a crucial role in the soil ecosystem and are highly sensitive to fertilization. A deficient fertilization regime results in nutrient deficiency and subsequent modifications of the microbial community of the soil. Unbalanced fertilizations can have detrimental effects on soil biological health over the long-term (Shahzad Aslam et al., 2012; Sheoran et al., 2022).
Mineral fertilizers are mostly applied to the soil or leaf of crops where the former is more common and applicable and when nutrients are required in higher amounts. Foliar application is more economical and applicable when symptoms of nutrient deficiencies in crops are visible (de Valença et al., 2017; Singh et al., 2016), when mineral elements are not translocated and accumulated in adequate amounts in the edible parts of the crop (de Valença et al., 2017; Jha & Warkentin, 2020). Foliar application tends to be more effective than soil applications by increasing micronutrient contents rather than just promoting yield (de Valença et al., 2017; Melash et al., 2016; Singh et al., 2016), whilst the latter promote yield with minimal effects on improving nutritional quality. Foliar application is dependent on several factors including the type of fertilizer, characteristics of crops, time of application and environmental conditions (Alshaal & El-Ramady, 2017; Praharaj et al., 2021). Agronomic biofortification of crops with minerals such as Fe and Zn require certain adjustments. Due to their low mobility, adding metal chelators to the fertilizer is essential (Wakeel et al., 2018). Foliar application of FeSO4 has proven effective for Fe biofortification (Dodake et al., 2022; Pal et al., 2021). For I2, potassium iodate has been effective as seen as in countries like China (Krzepiłko et al., 2019; Mao et al., 2014; Wakeel et al., 2018). Inorganic fertilizers such as ZnSO4, ZnO and Zn-oxy-sulphate are suitable for Zn agronomic biofortification. Just like Fe, foliar application of Zn chelators such as ZnEDTA is highly effective (Bruulsema et al., 2012; Cakmak & Kutman, 2018; Rehman et al., 2020). Se is agronomically fortified as selenate which is converted into organic selenomethionine in the crop. Both foliar and soil applications are suitable for Se biofortification, but dependent on soil type and timing of the application (Galić et al., 2021). However, foliar applications are costly and could easily be rinsed off by raining water (de Valença et al., 2017; Galić et al., 2021). The characteristics of the leaf play an important role in absorbing nutrients during foliar applications. Nutrients from foliar application penetrate the cuticle to leaf cells and are transported to other parts through the plasmodesmata. The age, structure and permeability of the leaf affect nutrients absorption (Alshaal & El-Ramady, 2017). Foliar application is mostly effective during the flowering and early milk phases than booting and elongation phases of the developmental stages of crops. The flowering and early milk stages are among the earliest phases where absorption of nutrients for fruit formation begins hence, foliar application of nutrients at this stage would contribute greatly to increasing the micronutrient contents of the fruits (Melash et al., 2016; Zaman et al., 2018). This was experienced during Zn agronomic biofortification of wheat using foliar application; and it was attributed to enhanced phloem mobility and active photo-assimilation allocation to reproductive silk organs that enhanced remobilization of nutrients (Melash et al., 2016; Praharaj et al., 2021; Zaman et al., 2018). Also, environmental conditions such as time of the day, humidity, temperature and wind speed affect the efficiency of foliar applications (Alshaal & El-Ramady, 2017). Warm and moist conditions in the early morning and late evening promote permeability of nutrients whilst low relative humidity and high temperature evaporate water from sprayed solution, leading to concentration of minerals on surfaces and intend reduces permeability (Alshaal & El-Ramady, 2017). Other strategies that are used for agronomic biofortification include coating and priming of seed with mineral fertilizers. These strategies aid in promoting crop yield and development but have minimal effects on the nutritional qualities of crops (de Valença et al., 2017; Sheoran et al., 2022).
Agronomic biofortification has been used effectively in several countries to combat micronutrient deficiencies and promote agricultural productivity. The effect of agronomic biofortification of selected underutilized vegetables in Ghana has been assessed (Adu et al., 2018). Increasing application rate of K fertilizer increased fruits and vegetables weight. Also, the application rate of K fertilizer and the type of K fertilizer synergistically affected K concentration in the fruit. The highest fruit K concentration was reported to be 2316 mgK/kg DW and this was a 140% increase with respect to the control (no K fertilizer application). In another study that assessed the influence of irrigation and fertilizer application on β-carotene yield and productivity of OFSP in South Africa (Laurie et al., 2012), the total storage root yield increased by 2–3 folds and β-carotene content increased from 133.7 µg/g to 151.0-153.1 µg/g when 50–100% fertilizer was applied, compared to no fertilizer application.
Agronomic biofortification is simple and yields results rapidly in the short term (Galić et al., 2021; Wakeel et al., 2018). However, mineral fertilizers used in agronomic biofortification is costly which increases the prices of biofortified crops making it inaccessible to poorer populations (Cakmak & Kutman, 2018). Also, agronomic biofortification is highly dependent on farmers. Application of mineral fertilizers is a regular activity hence may be omitted by farmers if they do not gain profits from the process (de Valença et al., 2017; Singh et al., 2016; Umar et al., 2019). Application of mineral fertilizers repeatedly may also cause accumulation, leading to toxicity (de Valença et al., 2017; Jha & Warkentin, 2020). In addition, increasing demand for mined minerals such as Se may cause exhaustion and negative impact on the environment (de Valença et al., 2017; Umar et al., 2019).
7.2 Plant Breeding
Plant breeding involves producing genetically different or new varieties of crops with improvements in essential micronutrients (Dhaliwal et al., 2022; Shahzad et al., 2021; Sheoran et al., 2022). Biofortification through plant breeding aims at improving the concentration and bioavailability of minerals in crops by utilizing the genetic differences between crops of similar species (Marques et al., 2021; Sheoran et al., 2022). Plant breeding initially focused on promoting yield and improving agronomic traits of crops however, recent plant breeding techniques have been geared towards promoting both the nutritional quality and agronomic traits of crops (Dhaliwal et al., 2022; Stangoulis & Knez, 2022). Plants breeding techniques should focus on introducing genotypes that would enhance the uptake, transport and redistribution of minerals to improve the efficacy of the biofortification (Lal et al., 2020). In order to achieve this goal, there is the need to enhance mineral mobility in the phloem vessels responsible for redistributing and remobilizing these minerals (Lal et al., 2020). The translocation and redistribution of Zn from the shoot to fruits or edible portions of crops has been a challenge due to the low mobility of Zn in phloem vessels, leading to lower Zn concentrations in the edible portions as compared to the leaves or root system (Buturi et al., 2021; Lal et al., 2020). Plants have been bred using three main techniques - conventional, molecular and mutation breeding techniques (Sheoran et al., 2022; Singh et al., 2016).
Conventional breeding is the most common and accepted form of plant breeding biofortification (Garg et al., 2018; Sheoran et al., 2022). Conventional breeding enhances improvement in the nutritional qualities of crops without compromising the other agronomic traits of the crop (Dhaliwal et al., 2022; Sheoran et al., 2022; Stangoulis & Knez, 2022). Biofortification through conventional breeding involves crossing crops with genotypic characteristics of high nutrient density and other agronomic traits to produce new varieties with desirable nutrient and agronomic traits (Garg et al., 2018). It requires identifying the biodiverse varieties of crops, assessing traits and amounts of target nutrients in these varieties, and determining the effects of growing conditions on the stability of these traits (Shelenga et al., 2021). Currently, about 299 varieties of biofortified cops via conventional breeding have been released in over 30 countries (Dhaliwal et al., 2022). A typical crop biofortified through conventional breeding is OFSP which has been biofortified with pro-VitA and with increased yield traits (Dhaliwal et al., 2022; A. Kumar et al., 2021; Marques et al., 2021). Quality Protein Maize (QPM) is also a product of conventional breeding (Sheoran et al., 2022; Singh et al., 2016). Other recent examples of conventionally-bred biofortified PBFs include biofortified wheat varieties, “Zincol” and “Akbar-2019” released in 2015 and 2019, with enhanced Fe and Zn contents, Fe-biofortified beans and, pro-VitA-biofortified cassava and maize (Marques et al., 2021; Shahzad et al., 2021; Van Der Straeten et al., 2020).
Mutation breeding differs from conventional breeding such that, differences in genetic traits among crops are created by introducing mutations through chemical treatments or physical methods such as irradiation (Sheoran et al., 2022; Singh et al., 2016). Mutation breeding has been recently adapted to biofortify resistant chickpea mutants like Pusa-408 (Ajay), Pusa-413 (Atul), Pusa-417 (Girnar), and Pusa-547, developed at I.A.R.I., India. Crop improvements via mutation in Pusa-547 include: thin testa, attractive bold seeds, better cooking quality and high yield performance (Zakir, 2018). Unlike conventional breeding, differences in genetic traits among crops are created by introducing mutations through chemical treatments or physical methods such as irradiation (Sheoran et al., 2022; Singh et al., 2016).
Biofortification through molecular breeding involves the identification of the location of a gene responsible for improving the nutritional quality of crops and attaching markers to that specific gene. With the aid of the marker, the desirable traits can then be bred into generations of the crop using conventional breeding (Jha & Warkentin, 2020; Sheoran et al., 2022). Molecular breeding can be used to determine if a desirable trait is present or absent in a specific crop during developmental stages. Hence, it is more rapid as compared to other forms of plant breeding (Sheoran et al., 2022; Singh et al., 2016). Molecular or marker-assisted breeding has been used to develop several varieties of maize with improved pro-VitA contents which can provide 25–50% of the estimated average requirements for VitA for women and children (Saltzman et al., 2013; Sheoran et al., 2022). These varieties have been released in countries such as Zambia, Nigeria and India. Also, it has been reported that several rice varieties have been bred to produce a variety with high Fe and Zn contents and improved agronomic traits (Garg et al., 2018).
Plant breeding is sustainable and less costly as compared to other biofortification strategies (Garg et al., 2018; Jha & Warkentin, 2020), and financial investments occur only at the research and development stages. Also, unlike agronomic biofortification, plant breeding has little to no impacts on the environment (Marques et al., 2021). Consumers generally accept crops that are biofortified through conventional plant breeding and easy to obtain regulatory approval as compared to genetically modified (GM) foods (Marques et al., 2021). Plant breeding may take a longer time to develop varieties with both desirable nutrient densities and agronomic traits (Carvalho & Vasconcelos, 2013; Marques et al., 2021). Also, there may be limited genetic variations among crops, making it impossible to biofortify these crops via plant breeding (Garg et al., 2018; Sushil Kumar et al., 2019; Shahzad et al., 2021) and may not be successful for all nutrients. For instance, breeding varieties of rice with improved VitA content initially proved to be challenging, but recent advances in omics technologies have provided the opportunity to practically biofortify varieties of rice with pro-VitA (Sushil Kumar et al., 2019). Also, crops such as banana that are propagated by vegetative means are not suitable for conventional plant breeding (Sushil Kumar et al., 2019).
7.3 Genetic Engineering/Transgenic Approach
Plant breeding relies heavily on the genetic variations among crops and this may prove to be a hindrance to biofortification through plant breeding when genetic variations are limited (Dhaliwal et al., 2022). Unlike plant breeding, genetic engineering is not limited to crops of related species. Genetic engineering has demonstrated to be a viable solution to this problem (Garg et al., 2018; Malik & Maqbool, 2020) and has been shown to effectively biofortify crops such as banana and rice, which cannot be subjected to conventional plant breeding (Dhaliwal et al., 2022; Kawakami & Bhullar, 2018; Sushil Kumar et al., 2019). Genetic engineering provides the platform for introducing nutrient or agronomic traits new to specific crop varieties by applying plant breeding and biotechnology principles (Melash et al., 2016; Mir et al., 2020) and when employed in biofortification, it identifies and characterizes suitable genes which could be introduced into crops to translate into desirable nutritional qualities (Garg et al., 2018). It utilizes genes from vast array of species, including bacteria, fungi and other organisms. Certain microorganisms enhance the uptake of nutrients by plants. Genes from these microorganisms can be genetically engineered into crops to enhance nutrient absorption, transportation, concentration and bioavailability (Mir et al., 2020; Singh et al., 2016). Fluorescent pseudomonas is a bacterium that enhances plant Fe uptake. Plants growth-promoting rhizobacteria and mycorrhizal fungi enhance the absorption of minerals from the soils and promote plants growth. Genes from bacteria and Aspergillus species have been used to adjust the lysine and phytate contents of crops such as rice and wheat, respectively (Sheoran et al., 2022; Singh et al., 2016).
Genome editing, also known as gene editing, corrects, introduces or deletes almost any DNA sequence in many different types of cells and organisms (Khalil, 2020). Gene editing provides an opportunity to develop GMOs without the use of transgenes; in addressing regulatory challenges associated with transgenic crops (K. Kumar et al., 2020; Mir et al., 2020). Methods such as Mega-nucleases, Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have been exploited in genome editing to produce β-carotene biofortified rice and zinc-rich wheat varieties (Jaganathan et al., 2018; Malik & Maqbool, 2020). Except for the CRISPR/Cas9 technique, these genome editing methods are complex, expensive, and labor-intensive (Malik & Maqbool, 2020). Although CRISPR/Cas9 is flexible, cost-effective, and precise, it can sometimes lead to undesired mutations when untargeted regions in the genome are involved in the editing process (K. Kumar et al., 2020; Malik & Maqbool, 2020). These off-targets can be overcome by the dimeric nuclease method, which is highly precise and specific (K. Kumar et al., 2020). According to (Gatica-Arias et al., 2019), low levels of knowledge about gene editing occur because information generated in scientific studies has not been communicated effectively to consumers (Vasquez et al., 2022).
Biofortification through transgenic approach has been greatly explored in most developed countries. The most notable example is golden rice which was developed by biofortifying rice with pro-VitA (Jha & Warkentin, 2020). This was done by expressing genes encoding phytoene synthase and carotene desaturase which are responsible for β-carotene pathway (Siwela et al., 2020). In golden rice, the expression of these genes caused an increase in pro-VitA levels by 1.6 to 3.7 µg/g DW (Siwela et al., 2020). The overexpression of Arabidopsis thaliana vacuolar Fe transporter VIT1 in cassava caused three-seven fold increase in Fe contents in the storage roots (Narayanan et al., 2019). The overexpression of Zn transporters and expression of the gene responsible for phytase activity in barley enhances the levels and bioavailability of Zn (Sushil Kumar et al., 2019; Sheoran et al., 2022).
In order to improve the efficiency of the transgenic approach as a biofortification strategy, omics technology has been introduced (Nayak et al., 2021; Riaz et al., 2020). Omics technology explains the interrelationship between genes, proteins, transcripts, metabolites and nutrients (Carvalho & Vasconcelos, 2013; Nayak et al., 2021; Riaz et al., 2020). Specific genes control the uptake, transport, concentration, and bioavailability of nutrients by crops. Hence, genomics (omics technology of genes) is important since it presents the opportunity to study these specific genes and design suitable ways of improving and inducing them into crops (Ali & Borrill, 2020; A. Kumar et al., 2021). Transcriptomics (omics technology of transcripts) aids in conducting full-spectrum analysis to identify a specific expressed gene (Lai et al., 2012; Nayak et al., 2021; Roda et al., 2020). Proteomics (omics technology of proteins) helps to understand the role of proteins in nutrient synthesis, uptake and transport pathways (Carvalho & Vasconcelos, 2013; Riaz et al., 2020). Metabolomics (omics technology of metabolites) aids in assessing metabolic pathways that control the biosynthesis of natural metabolites (Hall et al., 2008; Ranilla, 2020), while ionomics considers how minerals present in crops undergo changes in response to genetic and environmental factors (Carvalho & Vasconcelos, 2013). These omics technologies have been used in studies involving biofortification of lysine, Ca, Zn and Fe, and VitC in PBFs such as maize, finger millet, wheat and tomatoes, respectively (Jain et al., 2019). PBFs such as cauliflower, cassava, and banana have been biofortified by both transgenic and breeding approaches while barley, soybean, lettuce, canola, carrot, and mustard have been biofortified with transgenic and agronomic approaches (Garg et al., 2018). The transgenic approach has been shown to be sustainable and rapid when introducing desired traits into crops (Mir et al., 2020; Singh et al., 2016). Table 2 summarizes a selection of biofortified crops developed by transgenesis.
Table 2
Some examples of biofortified crops produced by transgenesis
Crop
|
Gene/Protein
|
Target and Country
|
Status1
|
Reference
|
Soybean
|
Phytoene synthase crtB
|
β-carotene
|
Released
|
(Mir et al., 2020; Pierce et al., 2015)
|
FATB1-A and FAD2-1A
|
Reduced linoleic acid, Vistive Gold® (USA)
|
Monsanto
(K. Kumar et al., 2020)
|
Maize
|
Aspergillus niger phyA2
|
Phytate degradation; BVLA4 3010 (China)
|
Released
|
Origin Agritech
(Chen et al., 2008)
|
Corynebacterium glutamicum cordapA
|
Lysine; Mavrea™ YieldGard (Japan and Mexico)
|
Monsanto
Renessen LLC
(K. Kumar et al., 2020)
|
Lysine; Mavrea™ Maize (LY038)
(Australia, Colombia, Canada, Japan, Mexico, New Zealand, Taiwan, USA)
|
Ferritin and lactoferrin
|
Iron
|
Research
|
(Drakakaki et al., 2005; Malik & Maqbool, 2020)
|
Rice
|
C1 and R-S
PAL, F3’H, ANS, CHS and DFR
|
Flavonoids
|
Research
|
(Ogo & Takaiwa, 2017; Shin et al., 2006)
|
OsIRT1
|
Zinc
|
(Lee & An, 2009; Mir et al., 2020)
|
Maize psy1, Pantoea ananatis bacterium crtI, and E coli strain K-12 pmi
|
Provitamin A rice line GR2E (Australia, New Zealand, Canada, USA)
|
Released
|
ISAAA Database 2019
(K. Kumar et al., 2020)
|
Cassava
|
Ferritin and FEA1
|
Iron
|
Released
|
Biocassava Plus
(Sayre et al., 2011)
|
Arabisopsis ZAT and ZIP
|
Zinc
|
Phytoene synthase crtB and DXS
|
β-carotene
|
ASP1 and Zeolin
|
Protein
|
Sweet potato
|
Crtl, CrtB, CrtY, LCYe
|
β-carotene (South Africa, Mozambique, Bangladesh and other African countries)
|
Released
|
(Malik & Maqbool, 2020; Mir et al., 2020; Tumuhimbise et al., 2013)
|
IbMYB1
|
Antioxidants
|
Research
|
(Park et al., 2015)
|
Banana
|
Phytoene synthase PSY2a
|
β-carotene
|
Research
|
(Waltz, 2014)
|
Alfalfa
|
MtlFS1
|
Isoflavonoids
|
Research
|
(Deavours & Dixon, 2005)
|
Wheat
|
Ferritin TaFer
|
Iron
|
Released
|
(Borg et al., 2012; Mir et al., 2020)
|
Silencing SBElla
|
Amylose
|
Research
|
(Sestili et al., 2010)
|
PSY, Crtl, CrtB
|
Provitamin A, carotenoids
|
(Sumanth et al., 2022; Wang et al., 2014)
|
Potato
|
nptII
|
Amylopectin component of starch; AM 04-10200 (USA)
|
Released
|
ISAAA Database (Tilocca et al., 2014)
|
Amflora TM (EH 92-527-1 (European Union)
|
Tomato
|
HMT, S3H and SAMT
|
Iodine
|
Research
|
(Halka et al., 2019; Malik & Maqbool, 2020)
|
GDP-l-galactose phosphorylase
|
VitC
|
(Bulley et al., 2012; Malik & Maqbool, 2020)
|
Barley
|
AtZIP1
|
Iron, Zinc
|
Research
|
(Malik & Maqbool, 2020; Ramesh et al., 2004)
|
1Status: Released means products are available in the marketplace; research means laboratory investigations are ongoing |
Biofortification through the transgenic approach has its limitations. The transgenic approach requires huge investments in financial, time and human resources at the research and developmental stages (Jha & Warkentin, 2020; Singh et al., 2016; Wakeel et al., 2018). Transgenic crops are not generally accepted due to concerns over GMOs (Carvalho & Vasconcelos, 2013; Wakeel et al., 2018). Also, there are several regulations governing the production of transgenic crops (Shelenga et al., 2021). Interactions among genes introduced into crops during genetic engineering may reduce the efficacy of the biofortification process (Carvalho & Vasconcelos, 2013; Garg et al., 2018). Agronomic biofortification, plant breeding and genetic engineering, including omics technology, are suitable strategies that could be used for plant-based biofortification to help reduce the occurrence of malnutrition and hidden hunger.