Global incidence of micronutrient malnutrition across the world population is estimated to be above 2 billion, with a greater percentage of this statistic being resource poor households (Cashman, 2020). Children of pre-school age, adolescent women and reproductive women in general are at high risk of suffering from micronutrient deficiency health related conditions (Bouis et al., 2020). Inadequate essential micronutrients quantity, 'hidden hunger' can culminate into various physical, mental, social and economic ills such as increased morbidity, disability,   stunted   and   physical   growth  and  reduced national socio-economic development (Ekholuenatale et al., 2020). High mortality amongst children of school going age and complications in pregnant women due to micronutrient deficiencies in low to medium income countries has also been reported (Ahsan et al., 2017; Castrogiovanni and Imbesi, 2017). The World Health Organization (WHO) and Food and Agricultural Organization (FAO) report of 2003, listed Zinc, Iron, Iodine and Vitamin A deficiencies as the most prevalent deficiencies in developing countries which lead to various notable   health   conditions.  There  is  however  growing concern in deficiency of folates, Vitamin D, Thiamine (B1), Lysine, Tryptophan, and Selenium (Strobbe et al., 2017; Holick, 2017; Cashman, 2020; Motukuri, 2019; Ligowe et al., 2020).

The prevalence of micronutrient deficiency in resource poor communities is largely attributed to dysfunctional food systems, depending heavily on inherently micronutrient poor staple cereal crops (maize, wheat, rice, sorghum, millets) with limited access to diverse foods which supply a variety of micronutrients (Conti et al., 2021). Global food systems have always been largely a direct function of the output from agricultural systems which largely focused more on increasing yield output with little regard to the quality of the produce. This has contributed significantly to the rise of unprecedented consequences such as micronutrient malnutrition amongst resource poor communities (Davis et al., 2019). Studies show that apart from food security, nutritional security is also equally important and as such, a robust and sustainable approach to agriculture production, which addresses both the energy as well as nutrition and health requirements, is thus required (Conti et al., 2021).

FOOD FORTIFICATION STRATEGIES TO COMBAT MICRONUTRIENT DEFICIENCIES

Developed economies have made significant strides in combating malnutrition through various interventions which include, fortifying processed foods, supplementation, and diversifying diets (Mannar et al., 2018). However, in low to middle income countries, these strategies have low feasibility due to the associated high cost factor. As such, remediation approaches that can reach low-income rural populations with limited access to commercially marketed fortified foods are a strategic solution. The following sections focus on highlighting the merits and demerits of the common food fortification strategies.

Industrial fortification

This is the enrichment of processed food products with specific essential nutrients during processing. It can be done either as a commercial product enhancement strategy by the food processors or as a policy influenced intervention stipulated by governments. Industrial fortification normally targets basic food commodities such as sugar, cooking oil, milk, butter, maize meal, table salt and wheat flour, due to their widespread and regular consumption in almost all households (Kumar et al., 2019). This malnutrition combating strategy is fairly effective owing to its wide reach of consumers; however it also notably has a number of downside factors. In cases of inefficient quality monitoring, systems, there is a huge risk    of    exposing   consumers   to   mineral   toxicity   if excessive doses of the micronutrients are added (Maqbool and Beshir, 2019). Another major concern also comes on the cost implication of the industrial fortification process which is factored into the fortified products thus transferring the cost burden to consumers (Lalani et al., 2019). Consequently, this makes industrially fortified food products more expensive than unfortified products. This has greater disadvantages for resource poor communities in developing countries who cannot bear such costs.

Dietary supplements

Dietary supplements are normally acquired through nutritional intervention programs by governments, non-Governmental Organizations (NGOs) or direct purchases from pharmacies (Marra and Bailey, 2018). Several supplements, supplying micronutrients such as Zinc, specific vitamins like Vitamin A and C as well as protein, creatine and other amino acid supplements are available on the markets for open purchase (Goredema-Matongera et al., 2021). The positive impact of government or NGO driven dietary supplementation programs in Africa, for instance, cannot be ignored but however an evaluation of dietary supplementation as a strategy to reduce micronutrient deficiencies suggests low sustainability. Like industrial “biofortification”, overdosing on dietary supplements can lead to toxicity in consumers (Dwyer et al., 2018). The intervention is also limited by the absence of critical supporting infrastructure or health services like pharmacies or clinics in malnutrition prone regions. The socio-economic environment in most of the malnutrition prone regions is also not favorable to enable regular supply of supplements more so pricing of the supplements is beyond the reach of target consumers (Garg et al., 2018).

Dietary diversification

Consuming diets based on a variety of food sources decreases the risk of suffering from micronutrient deficiency related illnesses. This strategy is one of the most efficient, long-term solutions to militate against malnutrition. Consumption of a balanced diet supplies the body with all the nutrients it requires in sufficient quantities. Some of the micronutrient rich foods include animal based food sources like eggs, milk, red meat, vegetables, fruits, fish and sea food (Aakre et al., 2020; Wallace et al., 2016; Manwaring et al., 2016). Despite the benefits, adequate dietary diversification is beyond the financial capacity of many households in low to medium income countries (Manwaring et al., 2016). A number of donor funded organizations have tried to bridge this gap by introducing programs that promote establishment of nutritional gardens in rural areas; however the extent of coverage is still  not  wholesome.  Such  programs  would also require the support of governments through their agricultural extension services to maintain the nutritional gardens if NGO funding pulls out of those areas (Singh et al., 2016).

Bio-fortification

Amongst the many available agricultural tools that can be implemented to increase micronutrient concentrations in food crops, “biofortification” is the first agricultural tool that has been implemented at a global scale with notable success (Bouis et al., 2013). “B

iofortification” of cereal crops is the deliberate and targeted enrichment of grain micronutrient density, through conventional and molecular plant breeding; transgenic, micro-flora and fertilization techniques. “Biofortification”  is considered cost effective, long term and sustainable approach to address malnutrition in resource poor communities (Sharma et al., 2016). 

Agronomic “biofortification”  involves the application of specific fertilizers to the soil or directly to foliage to increase the mineral concentration available for uptake by the plant. Various studies with different minerals have proven that increasing mineral concentration available for uptake by plants translate to increased levels of the micronutrient in edible portions of the crop (Zhang et al., 2018; Wang et al., 2016; Joy et al., 2017). Successful implementation of agronomic “biofortification” is however affected by several factors which include availability of the mineral in a form that can be available for uptake by the plant roots, physiologically related interferences to efficient translocation of the mineral in the crop plant, and remobilization of minerals from vegetative tissues into storage tissues (Rehman et al., 2021). The cost of fertilizers is also another huge obstacle to the successful implementation of agronomic “biofortification” as a mitigation measure against malnutrition (Bouis et al., 2020).

Recent novel discoveries on the role of certain bacteria strains that solubilize metals such as zinc, making them more available for uptake by plant roots have presented an additional approach towards “biofortification” (Mumtaz et al., 2017). Inoculating crop seeds with such microbial strains have the potential of not only increasing micronutrient densities in edible portions but also can significantly enhance soil fertility and crop yield (Hussein et al., 2018). 

Genetic “biofortification” on the other hand involves the use of both conventional and transgenic breeding techniques to introgress genes that promote high micronutrient accumulation into elite genotypes or manipulate the crop genetic makeup using genome editing tools in order to express proteins that enhance micronutrient accumulation (Ludwig and Slamet-Loedin, 2019). Genetic “biofortification” of staple crops presents a sustainable, low cost and durable solution to delivering micronutrients in the diets of malnourished populations.

ADVANTAGES OF GENETIC BIOFORTIFICATION

Cost effective and inexpensive

Developing bio-fortified staple crops is a one-time investment, with very low recurrent costs (Sharma et al., 2016). Upon release, bio-fortified germplasm can be shared across communities and considering the global extent of cultivation of staple crops such as maize, sorghum and wheat impacts are far reaching.

Bio-fortified varieties can be grown sustainably

Seed of released bio-fortified crop varieties, especially self-pollinating crops, can be retained and grown without losing the trait. HarvestPlus programs supported by partnerships with country Agricultural Research Institutions have supported the development and promotion of bio-fortified crops. The focus has mainly thrived to reach marginalized communities and seed supply to ensure access to bio-fortified germplasm by vulnerable communities (Sharma et al., 2016; Yadav et al., 2020).

Breeding for higher micronutrient density in some crops has no yield penalty

Several character association studies performed on some bio-fortified crops, have revealed absence or no correlation between micronutrient accumulation in edible portions and yield (Welch and Graham, 2004; Velu et al., 2012; Kumar et al., 2015). Thus breeding for high micronutrient density can be done successfully without reducing the yield output that is expected for a given genetic background. Where yield penalty exist, exploration of other crop improvement tools such as genetic engineering can be exploited with minimal linkage drag.

STRATEGY FOR SUCCESSFUL GENETIC BIO-FORTIFICATION

Success in genetic “biofortification” depends on increasing the existence of sufficient genetic variability for the nutrient in question in the final edible portion. Where variability is non-existent within the crop gene pool techniques such as mutation breeding, gene editing and genetic engineering can be employed depending on the likelihood for successful commercialization of the final product.  A  pipeline  approach  of fitting “biofortification” objectives within regular crop improvement programs can be a sustainable way of ensuring consumer acceptance. Integrating an additional “biofortification” trait to farmer preferred varieties can be useful for enhanced adoption. If the sensory attributes of the crop are not affected and preparation methods are the same bio-fortified crops can thrive.

Bio-fortification of maize

Maize (Zea mays) remains one of the most targeted cereals for “biofortification” owing to its widespread cultivation as a staple crop and key source of diverse products in developing regions of sub Saharan Africa (SSA), South America and South Asia. The wide native genetic variability in micronutrient concentration enables elite breeding lines of the crop to be nutritionally enhanced through plant breeding approaches. Furthermore, the cultivation of maize is possible across a broad agro-ecosystem coverage which further justifies its continued inclusion in “biofortification” programs.

In SSA, 30% of the total calories in cereal based diets of 20 countries are provided by maize grain. High dependency on the cereal pushes its daily per capita consumption in the region up to 450 g/person/day (Ekpa et al., 2019). Consumption of maize in SSA is primarily as boiled, roasted green mealies or alternatively as a thick porridge prepared from ground maize meal and served with an accompanying relish. The dependency on consumption of maize in these poor and marginalized regions coupled with the inherent low essential micronutrient density characteristic of normal maize (Cakmak and Kutman, 2018), puts such consumers at greater risk of suffering various micronutrient deficiency related conditions.

Extensive work on “biofortification” of maize has been done by the International Maize and Wheat Improvement (CIMMYT) in partnership with HarvestPlus whose mandate is to improve nutrition and public health through development of bio-fortified food crops. Most of the interventions in bio-fortifying maize have targeted improvement of proteins (lysine and tryptophan), zinc and pro-vitamin A grain concentration (Goredema-Matongera et al., 2021).

Quality protein maize (High Lysine and Tryptophan Content)

Maize kernels have a large portion of zein proteins in the endosperm which is deficient of essential amino acids. As a result nutritional enhancement efforts for the crop commenced as early as the 1960s with development of Quality Protein Maize (QPM) by CIMMYT after the discovery of the mutant opaque-2 (o2) gene (Listman et al., 2019).

Genetic variability for grain lysine and tryptophan content in maize germplasm

Any crop improvement program primarily depends on the amount of genetic variability present for the given trait of interest. The extend of diversity enables effective selection and generation of an improved population with a higher population mean as compared to the mean of the original population. Singh et al. (2020), in a study to evaluate the protein content, lysine and tryptophan content in maize inbred genotypes, reported existence of broad diversity in lysine and tryptophan content in QPM germplasm. The concentration of lysine in QPM ranges from 3.3 to 4.0 g/100 g of total endosperm protein compared to normal maize grain, QPM has lysine and tryptophan content that is 30 and 55% higher (Nuss and Tanumihardjo, 2011).

Genetic basis for high grain lysine and tryptophan production and breeding efforts in maize

The genetic basis for QPM breeding is based on homologous recessive alleles of the mutant opaque-2 gene which is normally homozygous dominant or heterozygous (Tripathy et al., 2017). The homozygous recessive genotype confers production of increased levels of the amino acids tryptophan and lysine than conventional maize (Maibvisira et al., 2018). However, initial efforts in breeding QPM revealed that, expression of the opaque-2 mutant produced a soft chalky endosperm which was undesirable among consumers. It was however discovered that breeding for the presence of the mutant gene together with its accompanying genes produced a hard endosperm preferred by consumers. The ultimate success of QPM breeding thus requires a multi-pronged approach that manipulates 3 different genetic systems which are: (i) replacing the normal gene with the mutant gene on the opaque-2 locus, (ii) modifier genes that enhance the expression of the opaque-2 gene relative to lysine and tryptophan content, and (iii) modifier genes that induce development of a hard endosperm (Prasanna et al., 2020).

BREEDING STRATEGIES FOR QPM

Different breeding strategies have been employed to manipulate and improve the performance of specific maize genotypes with regards to grain lysine and tryptophan content. The most utilized strategy is hybridization done with the objective of exploiting heterosis. Using this breeding method, open pollinated varieties (OPVs) or hybrids can be produced (Tripathy et al., 2017). The preponderance of additive gene action for Quality Protein trait enables development of QPM OPVs through recurrent selection. This can be accomplished by crossing a recurrent parent which  must  be  an  improved OPV and a trait donor parent with high lysine and tryptophan content followed by successive backcrosses with the recurrent parent to retain most of its original genome (Goredema-Matongera, 2021). Another breeding approach for production of QPM is hybrid development. The success of hybrid development largely depends on heritability of the trait, gene action governing expression of the trait as well as combining ability of different genotypes upon crossing (Goredema-Matongera, 2021). A study by Machida et al. (2010) reported significant GCA and SCA for tryptophan/protein content (Quality Index) from a diallel cross of QPM inbred lines. This indicates the possibility of producing hybrid QPM through exploiting the non-additive gene effects that can contribute significantly to increasing heterosis for the quality protein trait.

There is also evidence of current research on use of recent breeding tools such as Marker Assisted Breeding (MAB) and genome editing for producing QPM. MAB significantly increases the efficiency of the selection process since it is based on the operational principle where presence of a specific trait linked marker confirms presence of a trait (Maqbool et al., 2021). Tandzi et al. (2017) reported 3 simple sequence repeats (SSR) markers and these are phi057, phi112 and umc1066. As such introgression of the o2 gene to non-QPM genetic backgrounds can be made possible with relative ease and efficiency using the highly polymorphic markers. According to Goredema-Matongera et al. (2021), to date a number of QPM varieties have been released across the world for instance HQPM-5 (India), Obatanpa (Ghana), ZS261 (Zimbabwe), BHQP542 (Ethiopia), Q623 (South Africa), and Yanrui-1 (China).

Zinc bio-fortified maize

Maize grain is rich in calories but deficient in some essential micronutrients that include zinc (Ignjatovic-Micic et al., 2015).

Consumers who subsist on maize as a staple crop are thus at risk of suffering from zinc deficiency if other sources of zinc are not available. The maize zinc “biofortification” drive has been largely driven by CIMMYT and International Institute of Tropical Agriculture (IITA) in partnership with other private and public sector organizations, for widespread alleviation of the micronutrient deficiency in communities that subsist on cereal based diets was initiated (Prasanna et al., 2020).

Genetic variability for grain zinc content in maize germplasm

HarvestPlus set 33 μg/g as the breeding target for maize grain zinc content that would address deficiencies assuming 90% zinc retention after processing and 25% bioavailability    (Andersson  et  al.,  2017).  The  baseline grain zinc level in maize is 20 μg/g thus a target increment of 13 μg/g would achieve the breeding target (Akhtar et al., 2018). Several screening procedures have revealed sufficient grain zinc variability in maize that can suffice to attain the breeding target. Mageto (2020) synthesized data from 23 reports on grain zinc screening studies done since the year 2000 on over 3000 genotypes that included landraces, inbred lines, hybrids, core accessions, improved genotypes, and QPM inbreds from various agro-ecological regions across the world and found grain zinc variability spanning a range of 3.8 to 95.6 μg/g. Such wide variability affirms the practicality of breeding for high grain zinc in maize through selective breeding.

Genetic and molecular basis for grain zinc accumulation in maize

The ultimate goal of zinc breeding is to increase zinc uptake and transport efficiency, increase the amount of bioavailable zinc and reduce concentration of anti-nutritional factors or increase concentration of promoters (Mulualem, 2015). Zinc accumulation in maize grain is reported to be a polygenic trait (Gupta et al., 2015). Apart from the genetic effects, it is also significantly dependent upon the integrated action of several other factors such as environmental effects, genotype × environment interaction, micronutrient availability in the soil, translocation and partitioning to different plant parts and uptake by roots (Hussain et al., 2018). This implies differential zinc concentration among genotypes depending on where they are grown.

Combining ability studies have revealed that the inheritance of zinc accumulation in maize grain is largely controlled by additive than non-additive gene effects (Fan et al., 2014; Fasahat et al., 2016), with heritability of maize grain zinc reported to be in the range of 59 to 76% (Fan et al., 2014; Cheah et al., 2020; Mageto et al., 2020). The extent of these genetic parameters suggests significant influence of genetic factors in determining grain zinc content as well the potential of realizing high genetic gains through selection.  

Marker Assisted Breeding (MAB) for complex traits like zinc grain accumulation in maize realizes quicker results than using conventional breeding and phenotypic characterization methods. As such, several QTL mapping and Genome Wide Association (GWAS) studies for zinc accumulation have been conducted in maize. The results from these studies however, have not been consistent with regards to gene loci, total variance explained by identified QTL and confidence levels (Mageto, 2020). Most researchers however concur that genomic regions associated with zinc accumulation in maize are located on chromosomes 1, 2, 6, 7, 9 and 10 (Zhang et al., 2017; Hindu et al., 2018; Guo et al., 2020; Mallikarjuna et al., 2020). These studies have further identified some of the important   gene   families   that   influence    maize    zinc accumulation such as Natural Resistance Associated Macrophage Protein (NRAMP), Ferritin, Cation Efflux (CE), Yellow Stripe (YS), and Zinc Regulated Transporter Protein) family (ZIP) (Goredema-Matongera, 2021).

Prasanna et al. (2020) reported the presence of 3 high utility SNPs from a total of 20 SNPs found to be associated with grain zinc. These SNPs can be used for Marker Assisted Selection (MAS) and Genomic Selection to accelerate genetic gain for grain zinc in maize.

BREEDING STRATEGIES AND PROSPECTS FOR ZINC BIOFORTIFIED MAIZE

Successful breeding of zinc dense maize depends on genotype × environment interactions as well as genetic correlation between zinc accumulation with other attributes such as concentration of other micronutrients or yield. Plant breeders can exploit the prevalence of additive gene action for grain zinc accumulation to develop improved Open Pollinated Varieties (OPVs) through recurrent selection (Goredema-Matongera, 2021). Narrow sense heritability of 72% was reported for grain zinc in maize (Hindu et al., 2018) thus with such high heritability and non-additive gene action reported for specific genotypes suggests high zinc hybrids can be developed. Plant breeders can also safely expedite the exploitation of SNP markers already identified as being strongly associated with grain zinc accumulation in maize for marker assisted or genomic selection purposes thus accelerating the breeding progress (Prasanna et al., 2020).

Pro-vitamin A maize

Yellow and orange maize contains carotenoids such as α-carotenoids, β-carotenoids and β-cryptoxanthin, which are precursors of the physiologically active Vitamin A or retinol by humans upon consumption (Kondwakwenda et al., 2019). Elevation of the carotenoids in maize, a crop that constitutes a significant percentage in the diet of communities in Sub-Saharan Africa is of paramount importance given the scarcity of cheap vitamin A sources in many developing countries.

Genetic variability for PVA content in maize germplasm

Maize grain normally contains 0.5 to 1.5 μg/g Pro-vitamin A (PVA). This amount weigh below the minimum amount required to address vitamin A deficiency (Mengesha et al., 2019). The breeding target for maize PVA was set at 15μg/g in order to provide 50% of the mean daily vitamin A requirement through normal consumption habits (Bouis et al., 2020). Initial screening studies of more than 1500 genotypes revealed considerable variability for carotenoid profiles   in   temperate,   tropical  and  subtropical  maize germplasm, with results indicating PVA content between 0 and 19 μg/g (HarvestPlus, 2014; Muthusamy et al., 2015; Muzhingi et al., 2017). Great polymorphism was also observed in alleles of genes encoding enzymes that affect concentration of grain PVA carotenoids (Kondwakwenda et al., 2019). This ultimately enables the stacking of these promoter genes into one genotype using Marker Assisted Breeding.

Genetic and molecular basis for PVA carotenoids accumulation in maize

Accumulation of PVA in maize has been largely attributed to the activity of three key enzymes in the carotenoid biosynthesis pathway, namely phytoene synthase (PSY1), lycopene epsilon cyclase (lycE), and carotene hydroxylase 1 (CrtRB1) (Owens et al., 2014). However, the contribution of each of these towards PVA accumulation varies.

Phytoene synthase catalyses the condensation of 2 genanylgeranyl molecules (GGPP) into 1 molecule of phytoene. This reaction is responsible for development of yellow color in yellow maize (Babu et al., 2013). Variability in alleles of the PSY1 gene (Y1 gene) accounts for the differences in grain color in maize. A homozygous dominant (Y1/Y1) or heterezygous (Y1/y1) allelic combination results in production of high levels of carotenoids which accumulate in the endosperm of yellow maize. Conversely, a recessive combination (y1/y1) is typical of white maize (Maqbool et al., 2018).

Lycopene epsilon cyclase (lycE) catalyses the conversion of lycopene into α-carotene or β-carotene, however down-regulating the lycE gene favours progression of the β-carotene branch over α-carotene branch in the carotenoid biosynthesis pathway (Zunjare et al., 2017). Mapping analysis and allele mining studies revealed that there are 4 lycE polymorphic sites located on chromosome 8. These polymorphisms explain 58% of phenotypic variance in maize PVA accumulation (Azmach et al., 2013).

β-carotene hydroxylase (crtRB1) facilitates the conversion of β-carotene to β-cryptoxanthin (Maqbool et al., 2018). The crtRB1 locus has been shown to be the major QTL linked with β-carotene accumulation and other downstream non PVA carotenoids. Mapping analysis and allele mining studies have shown the crtRB1 locus on chromosome 10 and also existence of 3 polymorphic sites (Maqbool et al., 2018).

Various studies to evaluate combining ability and heritability of high PVA content in maize concur that PVA carotenoid content is controlled by both additive and non-additive gene action with preponderance of non-additive gene action (Babu, 2014; Halilu et al., 2016). Furthermore, Ewool and Akromah (2017) and Menkir et al. (2017), observed medium heritability for both PVA and non PVA carotenoids. However, Halilu et al. (2016) reported low  broad  and  narrow  sense  heritability for β-cryptoxanthin and α-carotene but high broad sense heritability for β-carotene. These results are suggestive of response to selection using available diverse material for PVA breeding.

BREEDING STRATEGIES FOR PVA MAIZE BREEDING

The available information on PVA genetics allows for both conventional and molecular breeding strategies and selection methods to be used for developing high PVA maize cultivars. Azmach et al. (2013), recommended backcross breeding use in the early stages of bio-fortication efforts in tropical and subtropical regions. This is because most tropical maize genotypes have low PVA carotenoids thus introgressing has to be from donor high PVA temperate parents (Menkir et al., 2017).

Recurrent selection is also another viable strategy, especially for development of OPVs, which are the most economical and sustainable option for low income regions since seed can be retained for 2 or 3 generations. This exploits additive gene effects and medium to high heritability (Suwarno et al., 2014). Recurrent selection techniques such as intra-population recurrent selection and pedigree selection have been evaluated by different researchers for improvement of PVA in maize with some significant levels of success. A study by Dhliwayo et al. (2014), found that maize PVA carotenoid levels were significantly improved after 3 cycles of S₁ recurrent selection on 3 genetically diverse maize populations.

Hybrid development is also a key alternative in breeding for PVA in maize as it exploit heterosis, that can manifest in a number of other important traits such as yield and resistance or tolerance to biotic and abiotic stresses. To unlock the heterotic potential of PVA associated genes, stable high yielding and high PVA inbred lines are developed and these are subsequently  crossed into single, 2-way or 3-way cross hybrids based on high SCA effects for PVA and other desirable agronomic traits (Kondwakwenda et al., 2019). Maqbool et al. (2018), however points out that, 3-way crosses are more ideal for PVA improved in maize due to practical implications, being economical in seed production and the relative ease of obtaining uniformity for the hybrid plants.

One of the major hurdles in the process of developing PVA bio-fortified maize is analysis, specifically partitioning and quantification of the different carotenoids present. This is because (1) maize contains different types of carotenoids, some of which are non-PVA associated (2) carotenoids form complex interactions with other biological molecules such as proteins or starch (3) like other biological molecules, carotenoids are prone to degradation (Guild et al., 2017). The practical potential for MAS in PVA breeding was exploited for identifying high PVA parental lines, tracking PVA-linked alleles during backcrossing as well as proteome quantification of PVA genes (Azmach et al., 2013; Muthusamy et al., 2014; Suwarno et al., 2014).

To date, significant progress in PVA Marker Assisted Breeding (MAB), a pedigree breeding scheme using MAS to target selection of the 2 superior alleles was used to develop donor germplasm with >20 µg/g. This was done by selecting seeds which were homozygous for the favorable alleles at the CrtRB1-3’TE and LcyE3’Indel polymorphisms from segregating F₂ and F₃ populations (Zunjare et al., 2018; Ortiz-Covarrubias et al., 2019). CIMMYT and IITA developed Single Nucleotide Polymorphism (SNP) markers associated with the favorable alleles and this has increased the efficacy of using MAS for PVA breeding (Prasanna et al., 2020). 

Bio-fortification of rice

Rice (Oryza species) is a major staple food for many Asian countries and feeds more than half of the world’s population. Like the majority of cereals rice is deficient in micronutrients iron and zinc, which causes various disorders that are prevalent in many developing countries (Bashir et al., 2013). In rough rice, the micronutrients are present but reduce significantly during postharvest processing which is crucial to suit consumer needs and allow for long term storage of grain without deterioration. Dehulling and debranning reduces the micronutrients that are confined in the aleurone layer, pericarp and embryonic tip which predisposes communities that are dependent on rice as a major food to iron and zinc deficiency. Accordingly, any “biofortification” efforts in rice have greater chances of success if they target the endosperm that remains after processing.

The drive to biofortify rice to address zinc and iron deficiencies commenced following the HarvestPlus prepared “Biofortification” Index which identified a number of Asian countries that were critically zinc deficient (Bouis et al., 2020).

HarvestPlus in partnership with International Rice Research Institute (IRRI) have made significant strides in developing zinc biofortified rice varieties for target countries like Bangladesh, Philippines, India, and Indonesia. Significant research and breeding efforts to improve rice micronutrient quantities have also been done to improve iron and carotenoid concentration with a number of varieties released to date. Target values for improved grain iron and zinc in rice that can reach 30% of human Estimated Average Requirement (EAR) were set at 13 and 28 μg/g, respectively (Bouis et al., 2011).

Genetic variability of zinc and iron concentration in rice

Unlike most staple cereals with elevated zinc and iron traits available within the species gene pool, there is limited variation for these two in rice. Some wild rice relatives  such  as  O. latifolia,  O. nivara, O. officinalis, O.barthii, O. longistaminata, and O. rufipogon have significant iron and zinc with potential use in pre-breeding. The major impediment is linkage drag during introgression that compromises yield (Majumder et al., 2019). Despite the existence of synergism between iron and zinc in rice grain, only zinc “biofortification” has gained more traction and several varieties are already released in Asia (Calayugan et al., 2021). The success of zinc “biofortification” came from the use of aus germplasm in conventional breeding programs. However, there is need for multi-environment evaluation of breeding materials given the significant genotype-by-environment interaction for zinc.  On the contrary, there is no high iron germplasm within the rice germplasm which means any improvement in this micronutrient will require use of recombinant DNA technology, gene editing or mutation breeding (Matres et al., 2021).  In rice micronutrient biofortification several mutants and products of genetic engineering show great potential but commercialization has been limited due to prohibitive GM legislative frameworks in the targeted developing countries.

Genetic and molecular basis for zinc and iron uptake and accumulation in rice grain

Numerous studies have shown that zinc and iron uptake and accumulation in grain is controlled by several genes. Characterization of gene families in zinc homeostasis have identified gene families such as OsNAS, OsTOM1, OsDMAS, OsSAMS and OsNAAT which increase metal uptake by roots through their involvement in biosynthesis, transport and secretion of phytosiderophores (Kavakani et al ., 2018; Raza et al., 2019; Dixti et al., 2019; Yang et al., 2020). Equally important are the ZIP family genes responsible for transport of zinc and ferrous iron as well as the OsYSL family proteins which are actively involved in long distance transport of metals such as zinc and iron in the phloem and their subsequent influx into the seed endosperm (Swamy et al., 2016).

Genetic characterization of several rice genes for grain zinc have revealed significant phenotypic co-efficient of variation (PCV), genotypic co-efficient of variation (GCV), genetic advance as well as broad and narrow sense heritability (Sala et al., 2015; Ajmeera et al., 2017; Umarani et al., 2017; Dhakal et al., 2020; Prasannakumari et al., 2020; Rathod et al., 2020). Studies on combining ability have also shown significant additive gene effects for grain zinc (Zhang et al., 1996; Sharifi, 2013). In other studies, heterosis and transgressive segregants have also been identified in rice breeding populations by Babu et al. (2012) and Stangoulis et al. (2017).

BREEDING STRATEGIES FOR HIGH GRAIN IRON AND ZINC IN RICE

There is substantial and useful  genetic  variation  in  zinc content sufficient to be exploited in a breeding program to develop biofortified rice. On the contrary, genetic variability for iron is limited thus most of the available literature on conventional breeding of these essential metals focuses on zinc breeding. The narrow genetic variability coupled with significant losses of the micronutrient realised during polishing disqualifies selective breeding as a viable option for iron content improvement in rice (Boy et al., 2017). However, with regard to zinc content improvement in rice, Balakrishnan et al. (2020) suggested use of the advanced backcross method for genetic dissection of wild rice, and for developing high zinc introgression lines, as an attractive approach for efficient use of wild rice accessions. Narrow sense heritability high, single plant selection can be utilized as an effective approach for improving nutrient content(Calayagan et al.,2020).Furthermore, use of multiple crosses involving several donors and recipient parents such as 3-way or 4-way crosses or reciprocal crosses to enhance zinc levels and yield potential is also a viable alternative.  Meng et al. (2016) highlighted the use of Multi-parent Advanced Generation Inter-cross (MAGIC) as a viable method to pool genes for high zinc as well as providing a good resource for selecting high zinc lines and transgressive segregants. Heterosis can also be exploited to develop high grain zinc and high yielding hybrids since reports show that there is satisfactory heterosis in rice (Naik et al., 2021).

Every breeding effort must ensure that essential agronomic attributes such as high yield are not lost as other traits are introgressed. Calayagun et al. (2020), reported a positive correlation between zinc content and grain weight, thus implore that yield related traits must be considered during selection. Despite contrasting results on the relationship between yield and zinc in rice, most of the recent studies have found no significant relationship between the 2 traits thus ensuring the possibility of developing zinc rich and high yielding varieties (Trijatmiko et al., 2016; Jaksomsak et al., 2017; Anusha et al., 2021).

Iron biofortified pearl millet

Pearl millet (Pennisetum glaucum) is a crop of economic significance in the semi-arid and arid regions of Africa and India where it is consumed as a staple crop by some communities (Govindaraj et al., 2020). Its grain zinc content is comparatively higher than in other cereals such as rice and wheat, however major concern is on the grain iron content produced by most pearl millet genotypes. This prompted the need to undertake crop improvement efforts focused on raising iron content in OPVs and hybrids for Africa and India, respectively (Boy et al., 2017).

Genetic variability of iron content in pearl millet

A    target   increment   of   +30  ppm   iron   was   set   by HarvestPlus to achieve a target level of 70 ppm iron in pearl millet grain from a baseline level of 47 ppm (Govindaraj et al., 2020). Germplasm screening by different institutions and researchers have revealed wide variability for iron across diverse populations that include landraces, inbred parents, population progenies and hybrids. To date, ICRISAT screening studies report of grain iron concentration spanning a range of 18 to 135 μg/g across the aforementioned populations (Govindaraj et al., 2019).

Genetic and molecular basis for iron accumulation in pearl millet grain

Iron concentration in pearl millet is largely controlled by additive gene effects. Studies done to evaluate the combining ability of numerous and genetically diverse breeding lines and early generations progenies have confirmed preponderance of additive gene action controlling iron content in pearl millet (Kanatti et al., 2016; Gavali, 2018; Warrier at al., 2020). The trait is also associated with high heritability thus it has the capacity to respond well to selection thereby realising significant genetic gain in improved populations (Kumar et al., 2020; Anuradha et al., 2017). Gene mapping and molecular analysis studies have revealed that grain iron accumulation in pearl millet is controlled by several gene families which include PgIZIP, PgINRAMP and PgIFER (Pujar et al., 2020; Kumar et al., 2016; Mahendrakar et al., 2020). These genes play an important role in iron metabolism at different stages of the crop development, and they provide a foundation for exploitation of iron metabolism genes in pearl millet molecular breeding programs.

BREEDING STRATEGIES FOR HIGH IRON PEARL MILLET

Iron content breeding in pearl millet can be done via the pedigree method where progeny populations can be developed from bi-parental crosses (Srivastava et al., 2022). In order to advance genetic gains in yield, composites can also be used as a base population to develop diverse hybrid parents. Development of hybrids for exploitation of heterosis is another viable breeding strategy for pearl millet (Srivastava et al., 2020). This is done by developing high iron inbred lines and later evaluating their combining ability. Currently, most pearl millet hybrids are produced through utilizing the various cytoplasmic male sterility systems available in pearl millet (Pujar et al., 2019). Pearl millet has no maternal effects for iron accumulation in grain thus high iron inbreds can be used as either males or females without affecting the outcome of the crosses (Kanatti et al., 2016).

Key to  biofortification  of  any  crop  is  the  relationship between the target micronutrient and yield. Govindraj et al. (2020) reported that iron is negatively and non-significantly associated with grain yield thus higher productivity does not necessarily lower iron levels in pearl millet grain. However, other agronomic traits of significance such as grain weight have shown positive correlation with grain iron concentration, implying the need for careful selection in the initial breeding stages.

Zinc biofortified wheat

Wheat (Triticum aestivum) has over the years played a very crucial role in the global food systems accounting for approximately 20% of the world's dietary needs (Mohammadi-joo et al., 2015). It is a fairly good adaptive crop that can thrive in various agro-ecosystems of the world thus making it one of the widely cultivated food crops (Muslim et al., 2015). Although substantially enriched with calories, wheat grain is however notably deficient in essential micronutrients such as iron and zinc and this is exacerbated by the milling process (Sharma et al., 2020).

Genetic variability for grain zinc content in wheat

A target increment of +12 μg/g was set to increase the grain zinc concentration from a baseline level of 25 to 37 μg/g in improved wheat varieties (HarvestPlus, 2014). Large scale germplasm screening for grain zinc content across wheat landraces and wild relatives by CIMMYT identified significant variability that could suffice to achieve target increments for the metal in biofortified varieties (http://HYPERLINK "http://www.cimmyt.org/germplasm-bank/"www.cimmyt.org/germplasm-bank/). More than 3000 germplasm accessions were screened and zinc content was found to be in the range of 20 to 115 μg/g (HarvestPlus, 2014). A report by the Bangladesh Agricultural Research Institute noted average zinc concentration in wheat grain spanning a range of 20 to 35 μg/g (Das et al., 2019). Furthemore, Gupta et al. (2021), reported the presence of higher Zn and Fe levels in landraces relative to cultivars. Zinc and iron levels reported by Gupta et al. (2021), reached as high as 87.29 mg/kg Zn (up to 53.3 mg/kg in wheat cultivars) and up to 122.20 mg/kg Fe (up 56.5mg/kg in wheat cultivars).

Various high zinc genotypes with potential to be used as parental lines in zinc breeding programs were identified and most are progenitors of modern high zinc hexaploid wheat (Sharma et al., 2020).

Genetic and molecular basis for grain zinc accumulation in wheat

Grain zinc accumulation in wheat is under quantitative genetic control and to date several mapping studies have identified a number of QTLs (Velu et al., 2014; Velu et al., 2017; Shariatipour et al., 2021). While the Gpc-B1 gene was identified as being highly significant in zinc homeostasis and subsequent zinc accumulation in wheat grain, a recent study by Velu et al. (2018), further asserts the significance of the zinc-finger motif transcription factor and phosphatase as playing a major role in zinc loading in wheat grain. The same study also highlights group 2 and 7 chromosomes as holding genes for nutrient uptake, translocation and sequestration of mineral elements in wheat. It must be noted however that although many genes that regulate zinc homeostasis have been characterised, their role in genotypic variation is still largely unclear (Garcia-Oliviera et al., 2018). Several studies reported zinc accumulation in wheat having high heritability thus suggesting high potential for responding to selective breeding (Velu et al., 2014, 2015; Alomari et al., 2018; Zhao et al., 2022). 

BREEDING STRATEGIES FOR HIGH GRAIN ZINC IN WHEAT

It is recommended that zinc breeding be location or environment specific since the ultimate zinc concentration in grain is largely affected by zinc concentration in soils as shown by high genetic correlations between locations in some studies (Velu et al., 2015). The presence of additive QTLs allows for high zinc wheat varieties to be bred through conventional breeding. With the ever-increasing information on QTLs and molecular markers for zinc homeostasis in wheat, the use of Marker Assisted Selection to increase the ease and efficacy of selection for high zinc expression is another practical approach (Saini et al., 2020).

Iron and zinc biofortified sorghum

Sorghum (Sorghum bicolor L. Moench) is an important food crop in arid and semi-arid regions of Asia and Africa and is ranked the 4th most consumed cereal. Its grain is known to be rich in starch, protein, crude fibre and other micronutrients (Abah et al., 2020). However, significant amounts of iron and zinc are lost during the decorticating process which removes them from the aleurone layer and scutellum (Proietti et al., 2015). Furthermore, bioavailability of iron and zinc from sorghum is also low ~5 and ~20%, respectively (Zhao et al., 2019) largely due to the inhibitory effect of anti-nutrients such as phytates that form insoluble complexes with these micronutrients.

Genetic variability for iron and zinc in sorghum

Under the HarvestPlus Program secondary investments, a  target  increment  of  +30 μg/g  iron  and  +12 μg/g zinc was set to increase the concentration of sorghum grain iron and zinc to 60 ppm iron and 20 ppm zinc (HarvestPlus, 2014). Several screening studies for grain iron and zinc in diverse sorghum genotypes including, parental lines, cultivars, advanced breeding lines, germplasm accessions, yellow sorghum and elite lines, have revealed that the crop has wide variability for the micronutrients spanning a range of 8 to 133 μg/g and 13 to 91 μg/g, respectively (Kumar et al., 2010, 2018; Hariprasanna et al., 2014; Upadhyaya et al., 2016; Satish et al., 2016; Sen et al., 2019; Guild and Stangoulis, 2021). These results indicate the wide and significant variability that can allow for identification of genotypes that can be used as parents for high iron and zinc breeding in sorghum.

Genetic basis for grain iron and zinc accumulation in sorghum

Iron and zinc density in sorghum is controlled by both additive and non-additive gene effects and was also found to have high heritability (Phuke et al., 2017; Gaddameedi et al., 2020). Trait association studies have also revealed positive correlation between grain iron and zinc content which suggest the possibility of simultaneous improvement of the two using conventional plant breeding methods. Furthermore, no significant correlation between iron and zinc accumulation in sorghum grain and other agronomic traits including yield were observed (Kumar et al., 2015).

BREEDING STRATEGIES FOR IRON AND ZINC IN SORGHUM

Breeding for high iron and zinc concentration in sorghum can be done simultaneously, due to their highly significant positive correlation (Motlhaodi et al., 2018; Phuke et al., 2017). Iron and zinc accumulation is however under the control of different genetic effects which determine the most appropriate breeding strategy to undertake. Grain zinc concentration is largely controlled by additive gene effects while iron concentration is predominantly governed by non-additive gene effects (Kumar et al., 2015; Gaddameedi et al., 2020). This implies that high zinc sorghum lines can be developed using progeny selection in pedigree or population breeding. Conversely, iron concentration can be improved through heterosis breeding as well progeny selection. At least one parent in heterosis breeding for grain iron must possess high iron concentration whilst for zinc, both parents must be having high grain concentrations (Kumar et al., 2015). Large genotype × environment effects have been observed for iron and zinc content in sorghum thus prompting the need for multiple environmental tests for improved  genotypes  to  identify  the  highly  stable lines.

There is currently limited literature on success stories of the use of Marker Aided Breeding for iron and zinc in sorghum; therefore, there is an opportunity to develop molecular markers to expedite the breeding process for these two nutrients in sorghum.

As global food prices escalate due to Covid19 induced lockdowns, climate change and variability manifestation and conflict, alternative nutritious food will continue to be beyond the reach of many particularly in developing countries. Accordingly, there is need to enhance the nutritional value of adapted cereals to curb the nutritional deficiencies. Genetic “biofortification” will inarguably continue to present an opportunity to sustainably and economically address global malnutrition issues due to its cost effectiveness in comparison to other food fortification approaches. Use of plant breeding will be at fore given the low rate of commercialisation of products of biotechnology in many developing countries. To complement conventional plant breeding there is need for mainstreaming of Marker Assisted Breeding (MAB) in “biofortification” programs to expedite the crop improvement processes.  Moreover given the complexity in analysing micronutrients such as zinc and iron there is need to integrate cost effective, sensitive, high throughput phenotyping tools in the breeding process. Crops targeted for “biofortification” should ideally have farmer preferred traits to enhance adoption. Alternatively, “biofortification” can be integrated into the pipeline breeding approach so that all new varieties possess the key micronutrients. Overall, the process should involve the different end users so that the importance of these outputs traits are apparent and maximum benefit is derived from the crops in this era of nutrition sensitive agriculture.

The authors have not declared any conflict of interests.