Enhancing nutritional security through bio-fortification of Major food crops

Introduction

Bio-fortification augments staple food crops with indispensable micronutrients and offers a sustainable intervention to counteract widespread micronutrient deficiencies (Bouis and Saltzman, 2017). Bio-fortified crops provide nutrient-rich options and are a cost-effective investment (Pfeiffer and McClafferty, 2007). Agronomic bio-fortification improves plant growth and nutrient profiles and plays a vital role in fighting poverty (Hornick, 1992) [36] . However, further research is necessary to enhance crop yield and nutritional content. Cereals, such as rice, wheat, maize and millet, hold historical significance and contribute to energy, carbohydrates, protein and other nutrients (Ram and Mishra, 2010). Despite their nutritional importance, cereals frequently exhibit micronutrient insufficiencies, which are particularly pronounced in low-income nations. Bio-fortification of cereal crops using molecular approaches like marker-assisted breeding, genetic engineering and gene editing has successfully developed nutrient-enriched rice and maize varieties. Molecular bio-fortification has become an indispensable instrument in tackling undernutrition and realizing food security targets by 2030 (FAO, 2020) [24] . Rapid population growth and limited natural resources have created a burden on the world impacting food security and nutritional quality. Bio-fortification, a genetic approach to breeding nutrient-enriched crops, offers a costeffective solution to hidden hunger. By leveraging genetic and genomic tools, such as highthroughput phenotyping and marker-assisted breeding, bio-fortification aims to combat malnutrition and achieve sustainable food security goals. Integration of genomics and computational tools enables precise trait selection, paving way for a healthier and more sustainable future (Saltzman et al., 2013; Mayer et al., 2008; Beyer et al., 2002).

Nutrition Silent Crisis: Unveiling the Global Scarcity of Vital Micronutrients

Insufficient nutrient availability for plants affects their food synthesis system, posing a severe issue for humans who rely on crops for sustenance. Micronutrient inadequacies in staple cereals such as wheat and rice have escalated into a worldwide public health emergency, affecting more than 2 billion individuals. This leads to compromised physical and mental development, increased disease susceptibility and issues like mental health disorders, blindness and reduced productivity. In the global landscape of malnutrition, developing countries emerge as the primary arena, out of a staggering 792.5 million undernourished individuals worldwide, a substantial 780 million can be attributed to developing countries (McGuire, 2015). A considerable fraction of the global population is burdened by multiple nutritional deficiencies, encompassing iron (Fe) deficiency (60%), zinc (Zn) deficiency (30%), iodine (I) deficiency, along with communal deficiencies of calcium (Ca), copper (Cu) and magnesium (Mg), while 15% are affected by selenium (Se) deficiency (Kaur et al., 2020) [40] . Approximately half of the global population is afflicted by micronutrient malnutrition, particularly attributable to inadequate dietary consumption of selenium (Se), zinc (Zn) and iodine (I) owing to restricted dietary diversity (Mao et al., 2014). Crops represent the principal repository of essential nutrients, delivering sustenance and metabolic energy to the human population. Cereal crops, constituting three main dietary staples, function as the predominant food source for roughly four billion people, supplying approximately 60% of the plant-derived caloric intake of humanity (Frison, 2016) [6] .

I. Enhancing crop nutrition through effective practices of agronomic bio-fortification

India is a country with highly diverse climatic and soil conditions with high heterogeneity in soil spread across different states (Table 1). Agronomic bio-fortification, realized through economically efficient and temporally optimized agronomic interventions, enhances crop nutritional composition and physiological status (Hefferon, 2015) [35] . By deploying physical measures, such as judicious fertilizer application, rotational cropping and soil fertility management, the micronutrient quality of crops and rhizospheric soils can be substantially improved (Cakmak and Kutman, 2018) [13] . Strategies such as balanced NPK and S fertilizer application, combined with agrotechnical measures including tillage, organic amendment and soil amelioration, contribute to augmented crop productivity and elevated nutrient content (De Valença et al., 2017) [19] . Micronutrient supplementation, encompassing zinc fertilization and foliar spray techniques, further improves crop nutritive quality (Lawson et al., 2015). By combining micronutrient management strategies with improved highyielding cultivars, agronomic bio-fortification can substantially elevate the nutrient density and productivity of crops, thereby promoting human health (Zuo et al., 2004). Agronomic bio-fortification functions as a pivotal mechanism to augment the dietary intake of targeted minerals directly from biofortified crop sources, particularly benefiting nutritionally vulnerable communities (Hefferon, 2015) [35] . Organizations like HarvestPlus, Biofort, CIMMYT and IITA play a significant role in developing and disseminating bio-fortified crops at the global level (Cakmak and Kutman, 2018) [13] . By guaranteeing the presence of essential micronutrients at concentrations below 1 mg/day, agronomic bio-fortification elevates food quality and contributes to improved human health outcomes (Cakmak 2010). The incorporation of NPK fertilizers, organic amendments and high-yielding cultivars reinforces the effectiveness of agronomic bio-fortification, culminating in elevated nutritional value and crop yield (De Valença et al., 2017) [19] . This approach paves the way for producing cost-effective bio-fortified crops and making vital microelements more accessible for human consumption. [20] . This method significantly enhance micronutrient accumulation across diverse crops, with the extent of improvement varying depending on the application method, crop type and nutrient involved (Table 2).

Constraints of agronomic bio-fortification

The efficacy of agronomic bio-fortification is substantially dependent on the mode of fertilizer administration. Distinct fertilizer delivery methods can differentially influence micronutrient concentrations in grain tissue. Excessive micronutrient fertilizer input may suppress crop productivity and adversely impact plant physiology. Notably, micronutrient fertilizer application can enhance grain yield without proportionately elevating the micronutrient content. Among the various delivery approaches, foliar application is generally regarded as the most effective modality. Nevertheless, in cereal crops, precise timing of foliar fertilizer application relative to developmental stage is critical to attain desirable outcomes, as off-stage application confers limited benefit.

II. Advancing bio-fortification in crops through Molecular Techniques

Bio-fortification strategies targeting specific nutrient traits in cereal crops rely on molecular methodologies. These include whole-genome sequencing of crop species, genomic-level modifications such as sequence editing or targeted mutagenesis and transcriptomic analysis of the modified genotype. This pipeline identifies promising variants exhibiting transgressive segregation for the target nutritional trait and their phenotypic stability is subsequently evaluated. Validation at both the molecular and phenotypic levels is conducted to confirm consistent expression of the target trait.

A. Genomic approaches 1. Genome Sequencing/Resequencing

Whole-genome sequencing of diverse crop and non-crop plant species has uncovered novel genetic variations in nutritional traits. Such variants can be introgressed into elite crop backgrounds using marker-assisted breeding platforms.

Rice was the pioneering crop to undergo complete genome sequencing under the International Rice Genome Sequencing Project in 2005. Subsequently, more than 3000 distinct rice accessions have been resequenced globally, facilitating the identification of unique genes and QTLs (quantitative trait loci) associated with micronutrient accumulation. This has markedly broadened our understanding of micronutrient variability across crop genotypes.

QTL mapping

Liu (1997) illustrated that QTLs are genetic regions that consist of multiple genes with small individual effects, which collectively influence quantitative traits. Unlike qualitative traits, the variation in quantitative traits is continuous rather than discrete. Gande et al. (2014) [27] further highlighted the importance of QTLs concerning the functional aspects of micronutrient uptake, transport and accumulation in grains. They emphasized the coordinated role of specific genes/QTLs under conditions of deficient or excessive micronutrient levels, which can be utilized to enhance the concentration of respective micronutrients. Sequencing and analysing these QTLs allow for techniques like knock-out gene or overexpression analysis, which provide valuable information about the genes or sequences of interest. Once the QTLs are sequenced, the information can be used to design functional markers. This facilitates QTL mapping through fine mapping or their utilization in breeding programs, such as marker-assisted selection (MAS) to improve the trait.

Micro RNA discovery

MicroRNAs (miRNAs) were identified relatively recently and have since attracted considerable scientific attention among plant molecular biologists. Systematic investigations have progressively elucidated their biological functions, expression patterns and structural characteristics, particularly with respect to metabolic regulation and posttranscriptional gene silencing. Unlike genomic DNA, miRNAs lack exonic sequences and do not encode functional proteins. Rather, they operate primarily as regulatory intermediaries, facilitating post-transcriptional mRNA cleavage and translational repression in gene expression modulation. Kehr (2013) and Fischer et al.

(2013) [25] have reported the involvement of miRNAs in nutrient uptake and transport. Agarwal et al. (2015) [1] , provide evidence of miRNAs playing a potential role in modulating zinc and iron homeostasis and the associated signal transduction cascade under micronutrient-deficient conditions in rice.

Epigenetics

Epigenetics refers to the collective impact on gene expression resulting from cellular components such as chromatin structure, histone modification and DNA methylation. In their study on stress signalling and pathways, Chinnusamy and Zhu (2009) [16] highlighted how gene expression can be regulated through epigenetic modifications in DNA sequences upon exposure to stress signals. This regulation plays a role in developing stress tolerance or resistance mechanisms in crop plants. Duan [4] Maize Protein Vivek QPM 9 41% higher tryptophan, 30% higher lysine MAS Gupta et al., 2013 [33] Protein Pusa HM-9 Improved Improved lysine content ( Limitations in enhancing nutritional content through molecular approaches 1. Presence of Nutrient Inhibitors: The presence of antinutrients in millets, such as phytic acid, can hinder the absorption of essential minerals like zinc, calcium and iron. These anti-nutrients, while having beneficial functions, can also interfere with the expression and availability of other micronutrients.

Trade-off between yield and micronutrient improvement

Breeding lines and cultivars of pearl millet that have been selected for improved yield may exhibit a negative or insignificant correlation with micronutrient content. This is because the focus of selection was primarily on yield, rather than specific micronutrients. However, there is a positive relationship between iron and zinc, so breeding for high-iron pearl millet may inadvertently lead to increased zinc content as well.

Challenges in Hybridization:

Artificial hybridization is necessary for developing mapping populations, but it can be challenging in self-pollinated millets like foxtail millet, finger millet and Kodo millet. These millets have small flowers, making emasculation and pollination procedures difficult and time-consuming.

Consideration of micronutrient bioavailability

while gene/QTLs for nutrient deficiencies can enhance their expression, it is crucial to also consider the bioavailability of the micronutrients. Simply increasing their expression does not guarantee improved nutrient absorption and utilization.

Complex Interactions with the Environment

The interaction between QTLs and environmental factors, along with the additive gene action of multiple loci, complicates marker-assisted selection. The influence of climate and the complex genetic architecture pose challenges in accurately predicting and selecting desired traits.

III. Bio-fortification in crops through transgenic approach

Transgenic methodologies represent a feasible alternative for generating bio-fortified crops particularly when inherent genetic variability for nutritional traits is limited within crop germplasm. These methods facilitate the transfer and stable expression of beneficial genes across taxonomically distant plant species without evolutionary constraints. Furthermore, transgenic approaches become indispensable when a particular micronutrient is naturally absent in a crop, as they permit direct fortification with the target nutrient. A thorough comprehension of gene function and the capacity to engineer plant metabolic pathways have been pivotal in transgenic crop development. By introducing heterologous genes from diverse sources, it is feasible to elevate micronutrient concentrations and their bioavailability in edible tissues while simultaneously suppressing antinutritional factors that impede mineral absorption. Transgenic techniques additionally enable targeted redistribution of micronutrients among plant organs, enrichment of their levels in consumable portions and optimization of biosynthetic pathways. When certain crop species are inherently deficient in a specific micronutrient, transgenic interventions emerge as the only viable strategy to enhance the nutritional profile of these crops (Perez-Massot et al., 2013). Additionally, the absence of phylogenetic barriers in genetic engineering permits the design and deployment of synthetic gene constructs. These innovations in transgenic technology hold considerable promise for mitigating micronutrient deficiencies, particularly among underprivileged populations in developing nations, by increasing the levels of vitamins, minerals, essential amino acids and essential fatty acids in food crops (Garg et al., 2018) [28] .

1.Transgenics:

Due to the limited success and complexity of achieving nutritional targets through conventional breeding, alternative approaches that are target-specific have gained attention. One such option is transgenics, although it raises ethical it has yielded significant achievements. Several researchers have reported the increase in storage iron (Fe) in rice seeds ranging from 1.5 to 3.7 fold by introducing the ferritin gene (OsGluB1 proSoya-ferH1) through transgenic technology in various studies (Paul et al., 2012). Overexpression of NAS genes (OsActin1 Pro-HvNAS1 and 35S Pro-HvNAS1) has resulted in two-to four-fold enhancement of Fe translocation, as reported by different researchers (Johnson et al., 2011) [38] . Similarly, the overexpression of "OsNAS2" and "OsZIP4" genes has been shown to enhance Iron and zinc (Zn) levels (Ishimaru et al., 2007). Wirth et al. (2009) reported a six-fold increase in NAS and a 1.3-1.5-fold increase in ferritin transgenes. Transgenic approaches offer an efficient method to improve complex traits within a shorter timeframe and with high precision. However, there is a need for a legitimate framework to address the use of transgenes in nutritional improvement programs to address potential complications.

Genome editing:

A genome encompasses all the DNA, genes and information necessary for an organism development and functioning. Genome editing involves manipulating gene sequences to understand their roles. To achieve this, engineered nucleases act as molecular scissors and cut specific DNA segments. These cut sequences can then be inserted, replaced, or removed as desired. Genome editing employs different types of nucleases, including TALENs (Transcription activatorlike effector nucleases), ZFN (Zinc finger nuclease) and CRISPR (Clustered regularly interspaced short palindromic repeats). Each nuclease has unique components that enable precise cutting at targeted locations. This process, with its precision and specificity, facilitates the creation of mutations or substitutions in cells. Genome editing has various applications, such as gene transfer, modifying gene expression, enhancing stress resistance, editing multiple genes and silencing genes. It is a powerful tool for controlled modification of gene sequences, including promoters and regulatory regions. Researchers widely employ this concept to investigate and improve various traits. In the context of bio-fortification, both transgenics and genome editing techniques hold immense promise in revolutionizing crop improvement and addressing nutritional challenges on a global scale. While transgenics allow the incorporation of desired genes to enhance nutrient content and distribution, genome editing offers the precision to manipulate gene sequences and optimize crop traits for improved nutritional outcomes. Through these innovative approaches, the quest for more nutritious and resilient crops takes a significant stride forward. However, careful consideration of ethical and regulatory aspects remains essential to ensure the responsible and sustainable use of these powerful tools in agriculture and food security efforts.

Various types of bio-fortification through transgenic approach i. CRISPR mediated bio-fortification CRISPR-mediated bio-fortification represents a transformative genome editing platform that has fundamentally reshaped the domains of agriculture andhuman nutrition. This advanced molecular technology equips researchers with the capability to precisely engineer the genomic sequences of organisms, including crop plants, at unprecedented resolution. By selectively targeting and modifying genes governing nutritional traits in crops, this approach unveils new avenues for substantially elevating concentrations of vital vitamins and minerals in food commodities (Table 5). A principal advantage of CRISPRmediated bio-fortification is its capacity to execute highly efficient and site-specific genomic modifications. This precision guarantees that exclusively the intended genetic loci are altered, yielding crop varieties with markedly enhanced nutritional compositions. Furthermore, this methodology differentiates itself from conventional transgenic approaches by exploiting endogenous genetic variability within a given species. This characteristic renders it a more precise and broadly acceptable technology within the scientific and regulatory communities. With CRISPRmediated bio-fortification, prospects for combating undernutrition and strengthening food security are substantially enhanced. By engineering crops with elevated concentrations of critical nutrients, investigators endeavour to beneficially influence global public health and reduce nutritional deficiencies in susceptible populations. As this technology progressively matures, its significance in determining the trajectory of agriculture and nutritional science is expected to grow considerably. ii. Gene overexpression Gene overexpression is an advanced molecular strategy deployed in modern agricultural biotechnology to amplify beneficial agronomic traits in crops. This approach entails the deliberate selection of target genes governing desirable traits, such as elevated nutritional content and driving their constitutive overexpression. To accomplish this, specialized regulatory promoters are utilized to upregulate the transcriptional activity of selected genes within the crop genome via recombinant DNA technology, the identified genes and associated promoters are stably integrated into the crop genome, enabling their consistent expression. As a consequence, the overexpression of these target genes results in a substantial accumulation of specific nutrients, generating crops with superior nutritional value and enhanced capacity to positively influence human wellbeing. Beyond its immediate nutritional benefits, gene overexpression holds the potential to elevate crop functionality and nutrient accessibility. By using this approach, crops can be tailored to possess heightened bioavailability and improved functionality of certain nutrients (Table 6). This multifaceted technique offers a promising avenue to tackle global nutritional challenges and elevate the nutritional quality of staple foods. The application of gene overexpression represents a dynamic and transformative advancement in agriculture, as it empowers researchers and farmers to create crops that align with the nutritional demands of a burgeoning world population. As scientific knowledge and technology continue to progress, gene overexpression emerges as an exciting and promising frontier for revolutionizing crop improvement and contributing to a more sustainable and health-conscious food system. iii. Foreign gene transfer Foreign gene transfer constitutes an innovative approach in modern agricultural biotechnology, directed towards augmenting the nutritional composition and mineral density of crop species. This procedure involves the identification of heterologous genes from varied biological sources, selected for their demonstrated ability to confer nutritional enhancements (Table 7). Through sophisticated recombinant genetic engineering, these exogenous genes are stably incorporated into the recipient crop genome, resulting in the generation of transgenic crop varieties. The introduction of exogenous genes into crop genomes expands the biosynthetic repertoire, enabling the production of novel enzymes or functional proteins that substantially enhance the plant capacity to synthesize and accumulate essential nutrients. Consequently, transgenic crops acquire unique biochemical attributes not inherent to their wild-type counterparts. This sophisticated biotechnological approach holds significant potential in tackling global food security concerns and fulfilling the nutritional demands of a rapidly expanding world population. While the application of foreign gene transfer has generated debate on ethical and regulatory grounds, its potential to revolutionize crop enhancement and stimulate innovation in agriculture continues to drive extensive research and exploration. [39] Limitations in Transgenic approaches:

1. Transgenic crops have the advantage of overcoming restricted genetic variation among plants in conventional breeding. However, a major limitation of transgenic methods is the low acceptance among the masses. The adoption of bio-fortified crops by farmers and communities is crucial for improving nutritional health, but low acceptance hinders widespread adoption (Babili et al., 2004).

2. Different countries have varying regulatory processes for the acceptance and commercialization of transgenic crops. The current political and economic landscape is not receptive to transgenic technology (Inaba et al., 2004) [37] . 3. Regulatory processes for transgenic crops are expensive and time-consuming (Watanabe et al., 2005). Bt Brinjal, developed by Mahyco in India, faced opposition and a moratorium on its release due to concerns raised by scientists, farmers and anti-GMO activists. Four varieties of Bt Brinjal received approval for commercial release in Bangladesh, contrasting with the situation in India. 4. The success rate of transgenic-based approaches in terms of cultivar release is low due to the time required for various stages, including target trait and gene identification, modification, expression and assessment of agronomical traits. 5. Golden Rice, a transgenic crop, has faced delays in its release due to issues with its yield and the inability to obtain government approval for dissemination.

Future Perspectives

Going forward, it is imperative to develop robust communication and outreach strategies that integrate ethical considerations pertaining to bio-fortified staple food production and consumption. Diverse countries may necessitate context-specific approaches to make bio-fortified food products acceptable and economically accessible. The public sector must assume a leadership role in formulating evidence-based policies and regulatory frameworks to foster consumer adoption of bio-fortified varieties. Furthermore, forthcoming research should prioritize integration of agronomic and genomic strategies to improve mineral translocation in crops and elucidate the physiological mechanisms governing mineral accumulation in harvestable plant organs. Such efforts will accelerate adoption of bio-fortification programs and more effectively address micronutrient insufficiencies. Despite existing constraints, bio-fortification harbours immense capacity to deliver nutritionally superior food to billions globally, advancing the goal of a world free from hidden hunger.

Conclusion

Bio-fortification represents an economically viable and ecologically sustainable strategy for enhancing crop nutritional composition and alleviating malnutrition at the global scale. It employs diverse techniques spanning plant genetic improvement, transgenic engineering and targeted mineral fertilizer application.

Nevertheless, the implementation of bio-fortification remains a complex undertaking, necessitating interdisciplinary collaboration among agronomists, plant breeders, molecular biotechnologists, genetic engineers and nutrition scientists. Although transgenic approaches are considered superior to classical breeding for crop bio-fortification, they encounter significant hurdles related to regulatory approval and public acceptance. The commercialization of transgenic biofortified rice varieties has been restricted, reflecting stringent regulatory impediments. For bio-fortification to achieve meaningful nutritional impact, the developed cultivars must be systematically mainstreamed into formal seed supply chains and assimilated into both formal and smallholder farming systems. Multi-bio-fortification, which concurrently enriches crops with several micronutrients, provides a comprehensive strategy for addressing multifaceted nutritional deficiencies. Consolidating the biofortification program demands focused research into agronomic-genomic integration and a deeper mechanistic understanding of mineral homeostatic regulation in crop plants.

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