Biofortification of vegetables: Strategies, challenges and future prospects

Introduction

Micronutrient malnutrition affects over half the global population and is a major challenge. By 2050, with a population exceeding 9 billion, sustainable food production is crucial. Plant breeders have mainly worked to increase crop yields in recent decades to feed poor populations. However, this focus ignored the nutritional quality of vegetables, leading to many varieties with low nutrition. Now, there is greater awareness that nutritious food is key to fighting malnutrition. Malnutrition is a serious global problem, especially in poorer countries, caused by diets that lack essential nutrients (Yadava et al., 2018) [36] . For good health, people need various minerals from food, especially vegetables. Lack of certain minerals can harm health. Since plant-based diets provide these minerals, it is important to eat balanced and nutritious foods regularly. One effective strategy to alleviate this issue is biofortification, which involves breeding essential nutrients directly into food crops. This approach offers a cost-efficient, sustainable, and long-lasting solution to deliver higher micronutrient levels, especially benefiting rural populations in developing countries who often have limited access to diverse and nutrientrich diets (Kumari et al., 2022) [18] . This process of increasing the nutrient content of crops through breeding or genetic engineering is known as biofortification (Neste et al., 2006) [22] . Biofortification improves the nutritional quality of staple crops by increasing nutrient content or availability through farming techniques, breeding, or biotechnology. While biofortified crops may have lower nutrient levels than supplements or industrially fortified foods, they help boost micronutrient intake across populations at all life stages. (Bouis et al., 2010) [6] . Biofortification is a form of food fortification that involves enhancing the concentration and bioavailability of essential nutrients in food crops during their growth and development. Biofortification of vegetable crops is the process of increasing essential micronutrients and vitamins, including trace elements, in vegetable varieties to improve the nutritional quality of the food supply and promote public health. This natural enhancement offers a sustainable and efficient means to provide nutrient-rich foods, particularly in remote rural regions where access to commercially fortified products is scarce.

As a result, biofortification is a vital strategy to combat micronutrient deficiencies and enhance overall nutrition (Singh et al., 2021) [33] .

Nutritional Significance of Vegetables

Vegetables are a rich source of essential nutrients-vitamins, minerals, fiber, and bioactive compounds that support overall health, prevent chronic diseases, and maintain physiological functions. Unlike fruit, which often contains higher sugars, vegetables contribute micronutrients and fiber with low calorie density, making them essential for managing weight and disease risk (Table 1.)

Table 1: Sources of nutrients from vegetables (Gomati et al., 2017) [14] Vitamin C Ascorbic acid is abundant in vegetables like bell peppers, broccoli, Brussels sprouts, kale, cabbage, and tomato. Vitamin C supports collagen synthesis (crucial for skin, bone, and blood vessels), enhances iron absorption, acts as an antioxidant, and slows aging processes. Broccoli, yellow/red sweet peppers, kale etc. are good sources of vitamin C.

Vitamin K

Vitamin K, especially phylloquinone (K1), is vital for blood clotting and bone health. Dark leafy greens such as kale, spinach, swiss chard, and collard greens are excellent sources of vitamin K, essential for blood clotting and bone metabolism. Biofortification enhances vitamin K content through breeding, fertilization, or genetic approaches to combat deficiencies. For instance, optimized nitrogen fertilization in spinach increases K levels, improving its nutritional value and supporting public health. Spinach biofortified via nitrogen management showed higher vitamin K1 content (Booth et al., 2003) [5] . [11] .

Vitamin B Complex

Vegetables-A rich source of minerals

Vegetables are a rich source of essential minerals vital for human health. They provide key minerals such as potassium, magnesium, calcium, iron, and zinc, which support various bodily functions including muscle contraction, nerve transmission, bone health, and immune function. Vegetables can be enhanced to contain higher levels of essential minerals such as iron, zinc, calcium, and selenium (Table 2). Regular consumption of a variety of vegetables helps maintain mineral balance and supports overall health, making them a crucial part of a balanced diet. [34] .

Iodine (I)

Iodine is an essential micronutrient that plays a vital role in human metabolism. A deficiency in iodine can result in serious thyroid disorders and impair the proper synthesis of thyroid hormones (Dai et al., 2004) [4] . These hormones are crucial for regulating various metabolic processes. The recommended daily allowance (RDA) of iodine ranges from 40 to 200 μg per day, depending on age, sex, and physiological conditions. Consuming iodine-rich vegetables such as spinach, potatoes, and seaweed can help meet dietary requirements.

Types of Biofortication (Strategies for biofortification of plant-based foods)

Biofortification can be achieved through three main strategies: agronomic biofortification, biofortification through conventional breeding and biofortification through genetic engineering.

Agronomic biofortification

Agronomic biofortification involves the application of micronutrient enriched fertilizers to enhance the nutrient density of crops. It is an efficient and rapid approach to improve crop nutritional quality, and the consumption of such biofortified crops contributes to better human micronutrient intake and overall nutritional status (Cakmak and Kutman, 2018) [28] . Agronomic biofortification primarily depends on fertilizer application methods, as well as the solubilization and translocation of mineral elements from the soil to the edible parts of the. The micronutrients enhance nutritional quality and macronutrients such as nitrogen (N), phosphorus (P), and potassium (K) play a crucial role in supporting plant (Bhardwaj et al., 2022) [3] . The application of micronutrients through fertilizers can enhance soil micronutrient availability and help correct deficiencies in both plants and humans. However, in many cases, the applied micronutrients become quickly immobilized in the soil, limiting their uptake and translocation to the edible parts of the plants. One major benefit of agronomic biofortification is that it is not limited to a specific crop. Micronutrient fertilizers and their application methods can be easily adjusted and used across different crops. On the other hand, genetic and transgenic biofortification are crop-specific processes, which makes expanding biofortified traits to multiple crops slow, costly, and labor-intensive

Targeted vegetables crops for biofortification through agronomic practices

Field pea is well known for its high protein content and can be biofortified with zinc through foliar application, either alone or in combination with soil-based zinc treatments.

(Poblaciones et al., 2016) [26] . The uptake of Copper (Cu), Manganese (Mn), and Zinc (Zn) in common beans was enhanced through the application of both organic and synthetic fertilizers (Ram et al., 2016) [28] . Chickpea biofortification through mycorrhizal association has been effective in addressing iron and zinc deficiencies (Pellegrino and Bedini, 2014) [24] . [32,30] . Biofortification through plant breeding aims to improve the concentration and bio accessibility of minerals in crops by utilizing genetic differences among varieties of the same species. Initially, plant breeding focused on increasing yield and enhancing agronomic traits. However, recent breeding techniques are now geared. Conventional breeding is the most common and widely accepted method of plant breeding for biofortification. It enhances the nutritional quality of crops without compromising other important agronomic traits. Biofortification through conventional breeding involves crossing crop varieties that possess high nutrient density with those that have desirable agronomic traits, to produce new varieties with improved nutritional and agronomic characteristics (Garg et al., 2018) [13] . This process requires identifying biodiverse crop varieties, assessing the traits and levels of target nutrients, and evaluating the effects of growing conditions on the stability of these traits (Shelenga et al., 2021) [31] . Mutation breeding differs from conventional breeding in that it generates genetic variation by inducing mutations through chemical agents or physical methods such as irradiation. In contrast, conventional breeding relies on naturally existing genetic diversity within or between plant populations. Mutation breeding introduces novel genetic variations by directly altering the plant's DNA, thereby expanding the pool of traits available for selection.

Biofortification through conventional breeding

Biofortification through molecular breeding involves identifying the genomic location of genes associated with improved nutritional quality, along with molecular markers closely linked to these genes. These markers enable the precise selection and incorporation of desirable traits into crop varieties through conventional breeding methods, thereby accelerating the development of nutritionally enhanced cultivars (Jha and Warkentin, 2020) [17] . 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 compared to other forms of plant breeding. Conventional plant breeding approaches can benefit not only large populations but also people living in remote rural areas with limited access to commercially marketed fortified foods (Bouis et al., 2010) [6] . This approach requires a onetime investment in plant breeding, after which the crops can be grown and multiplied by farmers over the years at virtually zero marginal cost. Recurrent costs remain low, and germplasm can be shared internationally without negatively affecting productivity or health. As a result, biofortification enjoys broad public acceptance (Nestel et al., 2006; Winkler, 2011) [22,35] . Targeted vegetables crops for biofortification through conventional plant breeding: Sweet Potato: The primary goal of biofortification programs in sweet potatoes is to replace white-fleshed varieties, which are low in provitamin A, with orange-fleshed varieties rich in beta-carotene. Harvest plus has set a target beta-carotene concentration of 32 μg/g in sweet potato roots, although some varieties already contain up to 100 μg/g (Nestel et al.,

2006) [22] . Beans: Beans are often termed the "poor man's meat" due to their affordability and rich content of protein, iron, zinc, and essential vitamins. Mineral biofortification of common beans is crucial to combat micronutrient deficiencies in lowincome populations. This process involves several stages, including germplasm screening, studies on trait inheritance and plant physiology, assessment of mineral bioavailability, and the development of improved biofortified varieties suitable for widespread cultivation and consumption (Blair, 2013) [4] .

Biofortification through genetic engineering

Genetic engineering is the preferred approach when there is limited or no genetic diversity for essential nutrients within crop species (Perez-Massot et al., 2013) [25] . This method allows for the transfer and overexpression of desirable heritable traits from unrelated plant species or even nonplant organisms, such as bacteria, into staple cropsregardless of their taxonomic or evolutionary relationship. Through genetic engineering, specific genes can be directly introduced into elite cultivars to enhance nutritional content in two main ways: by modifying nutrient uptake and utilization pathways, and by increasing nutrient bioavailability or reducing anti-nutritional factors (Zhu et al., 2007) [37] . This approach has utilized genes not only from various metabolic pathways found in plants but also from bacteria and other organisms. Although the development of transgenic crops requires substantial investment in the initial stages, it represents a potentially sustainable strategy capable of reaching large populationsparticularly in developing countries (Hefferon, 2016) [15] .

Targeted vegetables crops for biofortification through genetic engineering

Anthocyanin-rich tomato: To enhance anthocyanin accumulation in the fruits of the commercially cultivated tomato cultivar Arka Vikas, two transcription factors, Ros1 and Del, were introduced via Agrobacterium-mediated transformation. Their expression was driven by a fruitspecific promoter to ensure targeted activation during fruit development. The resulting transgenic fruits exhibited an average anthocyanin content of 0.1 mg/g fresh weight, reflecting 70-100-fold increase compared to the nontransgenic control fruits (Maligeppagol et al., 2013) [20] . Carotenoids-rich tomato: Lycopene, a major carotenoid in tomato, is a potent antioxidant known for its potential to prevent epithelial cancers and promote overall human health. Consequently, there is significant interest in enhancing carotenoid levels in tomato fruit through genetic manipulation. The carotenoid biosynthesis pathway begins with the production of phytoene from geranylgeranyl diphosphate (GGPP), a key step catalyzed by the enzyme phytoene synthase (PSY-1). To increase carotenoid accumulation in tomato fruit, the Psy-1 gene was constitutively expressed, leading to enhanced flux through the carotenoid biosynthetic pathway (Bergougnoux, 2014) [

Challenges and limitations of vegetable biofortification: Agronomic and Technical Constraints

Vegetable biofortification whether via breeding or agronomic means depends critically on soil conditions (pH, organic matter, texture) and environmental factors like temperature and moisture. Deficiencies or antagonistic interactions among nutrients (e.g. phosphorus reducing iron or zinc uptake) can greatly reduce efficacy (Ebbisa, 2022) [12] . Moreover, some genotypes may poorly accumulate target micronutrients under given conditions, limiting effectiveness across environments (Marques et at., 2021) [21] . Adding micronutrients via fertilizers (soil or foliar) is sensitive to timing, form of minerals, application method, and local climate. Misapplication can result in environmental pollution, farmer costs, and even crop toxicity. For instance, iodine-applied hydroponically to carrots has led to toxic accumulation; combining multiple nutrients (e.g. Zn and Se) may lead to antagonism, undermining results. Traditional fertilizer-based biofortification is expensive, labor-intensive, and may only benefit farmers able to afford multiple inputs (Ofori et al., 2022) [23] . Breeding approaches depend on genetic variability and often suffer low heritability of traits like iron or zinc content. Generating elite biofortified cultivars requires extensive trials across soils and climates and may take many years. Transgenic strategies can accelerate nutrient introduction, but face regulatory, ecological, and societal hurdles (Priyashantha et al., 2025) [27] . Enhanced micronutrient levels do not necessarily mean improved human nutrition. Antinutrient compounds phytates, polyphenols, tannins, and fiber-bind minerals such as iron and zinc, reducing. Breeding to reduce antinutrients may compromise plant metabolism or stress resistance, while some antinutrients offer health benefits, complicating removal strategies. Balancing promoters (e.g. organic acids, β-carotene) and antinutrients remains a complex task. (Bouis et al., 2010) [6] .

Consumer and Farmer Acceptance

Biofortified vegetables may differ in color, flavor, or texture-e.g. orange-fleshed sweet potatoes. In some regions, consumers prefer familiar traits,may reject biofortified variants based on cultural preference or perceived quality differences. Studies indicate that acceptance hinges on preserving sensory characteristics and educating consumers about benefits (https://en.wikipedia.org/wiki/Biofortification?utm_source = www.web). Farmers do not want adopt novel biofortified varieties if they are not agronomically competitive-i.e. yield, disease resistance, or marketability must match existing cultivars. Without clear advantages and incentives, uptake is limited. Limited seed availability and infrastructure challenges also impede last-mile delivery to smallholder farmers (Marques et al., 2021) [21] .

Policy, Regulatory and Economic Limitations

Biofortification, especially genetically modified approachesmust navigate lengthy, inconsistent and often politicized regulatory processes across countries. For example, Golden Rice offers high vitamin A benefits but remains unavailable due to regulatory delays and resistance. Many programs lack consistent governmental policies to scale biofortified crops. In Ethiopia and Sri Lanka, insufficient funding, weak lab infrastructure, fragmented awareness campaigns, and poor public-private coordination slow progress (Bachewe et al., 2023) [1] . Donor and policymaker interest can wane over time, affecting program sustainability. Biofortification could create dependence on proprietary seed or fertilizer suppliers, potentially undermining food sovereignty. Critics point out that the approach risks reinforcing reliance on large agritech sectors and oversimplifying dietary systems by focusing on staple crops instead of supporting diverse, holistic food systems. (Malezieux et al., 2024) [19] .

Environmental and Sustainability Concerns

Applying mineral fertilizers at scale raises environmental risks-soil degradation, water pollution, algal blooms, and biodiversity loss. Agronomic biofortification thus may conflict with sustainable agriculture aims, unless integrated carefully with ecological practices. Additionally, climate change is causing generalized nutrient declines in staple crops (e.g. α-tocopherol, iron, zinc), which biofortification may only partially offset.

Strategic Gaps and Program Coordination

Program effectiveness depends on coordination across breeding, agronomy, post-harvest handling, extension, and nutrition monitoring. Research from Sri Lanka and Ethiopia emphasizes need for field trials, simplified nutrient testing (e.g. near-infrared spectroscopy), clear incentives, public awareness campaigns, and monitoring frameworks to ensure sustained adoption and nutritional impact. Without multisectoral strategies, biofortification remains siloed and limited in reach (Bachewe et al., 2023) [1] .

Conclusion

Biofortification of vegetables presents a sustainable and cost-effective strategy to address micronutrient deficiencies, commonly referred to as 'hidden hunger' especially in populations relying heavily on plant-based diets. Enhancing the nutritional content of vegetables through agronomic practices, conventional breeding, and modern biotechnological tools offers a promising solution to improve public health by increasing the intake of essential nutrients such as iron, zinc, selenium, and iodine. Vegetables are an ideal target for biofortification due to their wide consumption and short growth cycles. Moreover, biofortified vegetables not only provide health benefits to consumers but also improve crop resilience, quality, and market value. However, successful implementation requires collaboration across disciplines, including plant science, nutrition, and policy-making, along with public awareness and acceptance. As global nutritional challenges continue to rise, integrating biofortified vegetables into food systems can play a pivotal role in achieving food and nutritional security in a sustainable and environmentally friendly manner.

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