Biofortification of Crops Using Biotechnology to Alleviate Malnutrition

Abstract

Malnutrition affects millions of people around the world, and the vast majority are found in developing countries. Malnutrition increases childhood mortality, amplifies poor outcomes during pregnancy, and is responsible for a variety of health disorders ranging from anemia to blindness. Biofortification of crops using biotechnological approaches such as genetic modification and genome editing holds promise as a powerful tool to combat malnutrition. This chapter describes progress that has been made in the development of biofortified staple crops to address malnutrition.

1. Introduction

Micronutrients play a variety of roles in metabolism and homeostasis. Micronutrient deficiency, also known as malnutrition, can result in the increased incidence of many diseases and metabolic disorders. To improve nutritional status through a balanced and enriched diet, the quantification and bioavailability of vitamins and minerals must be determined. The analysis of micronutrient content can enhance nutritional quality and improve nutritional status [1].

The level and composition of micronutrients vary significantly among crop varieties. Globally, cereals, roots, and tubers represent major staple food staples. While these crops are rich in carbohydrates, they may have very low quantity or poor-quality proteins and micronutrients [2]. In Asia, people who depend on rice are more prone to vitamin A deficiencies due to the lack of this micronutrient. This in turn makes them more susceptible to a number of health problems such as blindness [3]. Similarly, over 20 different dietary minerals are considered essential for human health. Global-level deficiencies in iron (Fe), zinc (Zn), and iodine (I) are most common as they have a significant negative impact on public health.

Since the concentrations of most vitamins in the edible parts of the plants are frequently low, one research goal has been to identify biochemical pathways involved in the synthesis, translocation, and accumulation of micronutrients in plant tissues [4]. Further understanding of these mechanisms would enable us to manipulate these pathways and improve their micronutrient content through metabolic engineering [5]. Although these strategies have demonstrated some degree of success, issues such as appropriate nutrient levels, bioavailability, ready adaptation by farmers, and acceptance by consumers must be addressed [6].

For the past several years, food supplementation has been the main strategy used for vitamin and mineral fortification. This strategy has a number of weaknesses, such as the decreased bioavailability of micronutrients after food processing. Biofortification has been considered an alternative solution and can be achieved via (i) an agronomic approach, (ii) conventional plant breeding, and (iii) genetic engineering [2, 7, 8, 9, 10]. In the following chapter, the micronutrient biofortification of edible crops by genetic engineering will be examined.

2. Uptake and bioaccumulation of minerals by plants

Minerals can accumulate in various ways and are stored in different compartments/organelles by plants species. These in turn can be affected by growing conditions as well as through interactions with other mineral nutrients [11, 12]. For example, iron is an essential element for plant metabolism, growth, and development [13]. Iron can be absorbed by the roots in the form of Fe²⁺, then becomes oxidized to Fe³⁺, is chelated by citrate, and then is transported to the top of the plant [14]. Zinc, another essential nutrient for plant growth and development, accumulates preferably in the vacuoles of the epidermal leaf cells as electron-dense deposits [15, 16].

3. Mineral biofortification using transgenic plants

Biofortification of crops using modern biotechnology techniques has been under exploration. Transgenic crops with increased accumulation of important minerals such as iron, zinc, and calcium within edible tissue are under development. Simultaneously, research into transgenic crops with reduced concentrations of antinutrients such as phytate has been developed. Antinutrients reduce the bioavailability of minerals by interfering with their absorption in the gut [9].

4. Transgenic crops biofortified with iron and zinc

Rice is one of the most well-studied cereals for mineral biofortification. Rice (Oryza sativa) is a staple of a large proportion of the world’s poor and is deficient in several essential micronutrients. Transgenic rice plants have provided a model system to enhance the amount of bioavailable iron and zinc that is found in the edible seed (endosperm) of cereals. Plant scientists discovered that metal transporter proteins found in many crop species can be used for multiple metal substrates, including iron, zinc, and even cadmium. These metal substrates can be taken up from the soil and into the roots. Researchers found that loss of function mutants of these transporter proteins creates a loss of uptake of all three of these metals into plant cells [17]. Ferritin, the iron storage protein, can assist in metal accumulation in plant tissue. Masuda et al. demonstrated an increase in accumulation of ferritin as well as an increase in iron translocation via the overexpression of the iron (II)-nicotianamine transporter OsYSL2 within rice endosperm. Transgenic lines generated higher levels of both iron (6-fold in the greenhouse and 4.4-fold in the paddy) and zinc (1.6-times), demonstrating that introduction of multiple genes involved in iron and zinc homeostasis could improve iron biofortification more than the introduction of a single gene. Later, Masuda et al. [18] increased iron and zinc accumulation through increased iron uptake and transport using the ferric iron chelator, mugineic acid. Transgenic plants that were generated expressed the ferritin gene from soybean (SoyferH2), and are driven by two endosperm-specific promoters, in addition to the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT-A and HvNAAT-B), and a mugineic acid synthase gene (IDS3) (to increase mugineic acid production in rice plants). These transgenic plants were tolerant of iron-deficient soil and displayed increased iron accumulation by 2.5-fold. Under iron-sufficient conditions, transgenic rice lines increased iron accumulation by 4-fold as much as lines that had been cultivated in either commercially supplied soil (iron-sufficient conditions) or calcareous soil (iron-deficient conditions). Transgenic lines expressing both ferritin and mugineic acid biosynthetic genes displayed signs of iron-deficiency tolerance in calcareous soil, and the iron concentration in polished T3 seeds increased by 4 and 2.5 times, respectively, compared to nontransgenic lines grown in normal and calcareous soil. Recently, Li et al. [19] have identified a zinc transporter protein family (ZIP) for taking up divalent cations in plants. The researchers found that by overexpressing the ZmZIP5 protein, iron and zinc levels were increased in seeds of rice plants. Similarly, Beasley et al. [20] constitutively expressed the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat. This brought about the upregulation of nicotianamine (NA) and 2′-deoxymugineic acid (DMA), which are important for iron and zinc transport and nutrition. Transgenic plants accumulated higher concentrations of Fe and Zn in wheat grain endosperm and iron bioavailability was increased in white flour milled from field-grown CE-OsNAS2 grain.

There are other ways for iron deficiency to be addressed using transgenic plants. For example, Sharma and Yeh [21] used an ethyl methanesulfonate (EMS) mutant in Arabidopsis that is tolerant of iron-deficient soil and demonstrated the accumulation of 4–7 times higher amounts of iron than wild type in roots, shoots, and seeds. This mutant presented a dominant “Metina” phenotype that constitutively activates the Fe regulatory pathway by optimizing Fe homeostasis and thus may be useful in Fe biofortification. Similarly, Qiao et al. [22] found that the wheat gene encoding the cell number regulator (CNR) protein showed enhanced tolerance to Zn, and overexpression of TaCNR5 in Arabidopsis increased Cd, Zn, and Mn translocation from roots to shoots. This indicates that heavy metal tolerance characteristics can be used as a tool to biofortify cereal grains with micronutrients.

Since the same molecular machinery is utilized for transporting iron and zinc into plants, increasing iron content in rice also brings about increased zinc accumulation. As an example, Aung et al. [23] generated a transgenic line of rice commonly eaten by consumers in Myanmar, where approximately 70% of the populace is iron deficient. This line overexpressed the nicotianamine synthase gene HvNAS1 to enhance iron transport, the Fe(II)-nicotianamine transporter gene OsYSL2 to transport iron to the endosperm and the Fe storage protein gene SoyferH2 to increase iron accumulation in the endosperm. The rice plants were shown to accumulate over 3.4-fold higher iron concentrations, in addition to 1.3-fold higher zinc concentrations compared to conventional, nontransgenic rice. The results of this study indicate that transgenic rice biofortified for increased iron content could address both iron as well as zinc micronutrient deficiency in the Myanmar population.

Paul et al. [24] generated transgenic high-yielding indica rice that expressed the soybean-derived ferritin gene. Transgenic plants produced over 2.6-fold higher levels of ferritin than their nontransgenic counterparts, even in the fourth generation of rice plants. Upon milling, transgenic rice grains provided 2.54-fold and 1.54-fold increases in iron and zinc content, respectively. Similarly, the iron transporter gene MxIRT1 taken from apple trees was utilized by Tan et al. (2015) to generate transgenic rice plants that exhibited an increase in iron and zinc of threefold, in addition to a decrease in cadmium concentration. Cadmium is thought to compete with iron and zinc for transport and accumulation in the rice endosperm and, thus, lower levels of cadmium to reduce toxicity in the rice seed.

Improvements in iron and zinc biofortification have also taken place using other approaches. Trijatmiko et al. [25] demonstrated that plants expressing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes possessed enriched endosperm Fe and Zn content. A Caco-2 cellular assay illustrated that increased iron and zinc levels found in these rice plants were bioavailable. Transgenic plants generated by Banakar et al. [26] expressed high levels of nicotianamine and 2′-deoxymugenic acid (DMA). These plants were able to accumulate up to 4-fold more iron and 2-fold more zinc in rice endosperm, in addition to lower levels of cadmium compared to wild-type plants.

Other crop species have also been studied for iron and zinc biofortification using biotechnology. Tan et al. [27] improved iron levels in the pulse crop chickpea (Cicer arietinum L.) by increasing iron transport and storage through a combination of chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes. Transgenic chickpea plants that overexpressed these genes illustrated a doubling of NA concentration, suggesting an increase in iron bioavailability. Pearl millet was examined by Manwaring et al. [28] for iron and zinc biofortification by improving the currently available gene pool. High iron and zinc-biofortified pearl millet would be advantageous for poor regions of the world where soil management or supplementation programs are ineffectual. Narayanan et al. [29] have expressed the iron sequestering Arabidopsis AtVIT1 gene in cassava plants to increase iron storage in the crop’s roots. Iron concentration also increased in stem tissues and accumulated in plant cellular vacuoles.

5. Calcium-biofortified transgenic plants

The calcium content of crops can also be increased using biotechnology. These advances hinge on improved knowledge of how soluble calcium ions found in the soil are transported and accumulate in plant tissue [30]. Calcium plays a significant role in general cell signaling; how calcium transporters are expressed can thus influence a plant’s ability to withstand stress, ward off pathogens, and can influence the nutritional status of animals and humans. Park et al. [31] have generated transgenic tomato, potato, lettuce, and carrots expressing high levels of calcium transporters. One of these calcium transporters, known as a short cation exchanger (sCAX1), can increase calcium transport into plant cell vacuoles [32]. Enhanced calcium absorption has been demonstrated in animal models that were fed transgenic carrots. Similarly, Sharma et al. have examined the potential of finger millet, an orphan crop with high calcium content, by studying the mechanisms behind calcium uptake, transport, and accumulation in grain. It has been reported that climate change may act detrimentally on mineral accumulation in different crop species; this could limit their further availability from food crops for both humans and animals [33].

6. Bioaccumulation of vitamins in plants

Vitamins such as β-carotene and folic acid are critical for human health. The development of microbial biochemistry facilitated the understanding of the biosynthetic pathways involved in vitamin production in plants. All vitamins that are required in the diet are synthesized by plants with the exception of ascorbic acid (vitamin C), which is specifically synthesized by eukaryotic cells [5, 34, 35]. Often biosynthesis is compartmentalized within various organelles. With greater comprehension of the metabolic pathways involved in vitamin production, plants can be developed with high levels of vitamin accumulation.

7. Vitamins and transgenic biofortification strategies of edible crops

GM technology also has the potential to reduce the global burden of malnutrition and hidden hunger. Vitamin- or mineral-enriched GM foods (GM biofortified foods) are considered to be the next generation of GMOs. Non-GM biofortified crops have been widely developed and commercialized, but the applied conventional breeding techniques may be inadequate for crops with a low level or absence of a certain micronutrient [36]. A recent review has summarized successful R&D efforts in the field of GMOs with increased micronutrient content in staple crops [37].

8. Vitamin-biofortified rice

The well-known example of GM vitamin biofortification is Golden Rice, enriched with pro-vitamin A (β-carotene) [38, 39], followed by vitamin B9 (folate)-enhanced rice [40, 41]. Conventional breeding techniques could not be applied due to the absence/low content of vitamin A in rice grain. For Golden Rice, daffodil, and Pantoea genes were used to increase pro-vitamin A levels within rice endosperm [39]. The most recent version of Golden Rice has been improved further for a 23-fold increase in carotenoids [38]. Similarly, folate-biofortified rice has been generated by overexpressing Arabidopsis genes in rice endosperm. A fourfold increase in folate concentrations in rice was accomplished using this strategy [41] and in the process, folate stability for long-term storage was improved (Blancquaert et al., 2015).

Fifteen simulation analyses confirmed the positive impact of GM biofortified crop consumption on dietary intake and nutritional outcomes in humans [42]. The vast majority of these studies also confirmed that a regular portion of the targeted biofortified crop would provide the daily micronutrient requirements. For example, the recent simulation analysis of Golden Rice in Asia [43] indicated that it could reduce the prevalence of dietary vitamin A inadequacy by up to 30% (children) and 55–60% (women) in Indonesia and the Philippines, and up to 71% (children) and 78% (women) in Bangladesh.

A randomized trial on Golden Rice performed in the United States resulted in a high bio-conversion factor of β-carotene (3.8:1), by which 100 g of uncooked Golden Rice would provide about 80–100% of the estimated average requirement and 55–70% of the recommended dietary allowance (RDA) for adult men and women [44]. Currently, Golden Rice has been approved in an increasing number of countries, including the Philippines. Golden Rice and other GM biofortified crops [16, 42] would be highly cost-effective investments to reduce target micronutrient deficiencies such as vitamin A [45].

Recently, Endo et al. [46] devised a genome editing approach to produce β-carotene rice that is fast and direct, by making use of splicing variants in the Orange (Or) gene that cause β-carotene accumulation in cauliflower. The authors genome edited the orthologue of the cauliflower or gene in rice using CRISPR/Cas9 and were able to accumulate β-carotene, without having to introduce transgenes.

9. “Golden” bananas to combat vitamin A deficiency

Bananas are the world’s most important fruit crop and a major staple in many African countries. Banana grows in tropical climates, where vitamin A deficiency is most prevalent [47]. The vast number of different banana varieties and the highly variable distribution of vitamin A levels make them amenable for biofortification using biotechnology. Unfortunately, the cooking banana East African highland banana (EAHB) consumed in Uganda as a staple for tens of millions of people has low vitamin A levels.

As bananas are difficult to breed, genetic engineering of bananas with increased vitamin A content has been critical to improving vitamin A levels. The bulk of the research has been performed on the Cavendish banana, most popular in the Western Hemisphere. As a result, the Cavendish has been used as a model system for the EAHB. High levels of vitamin A (20 lg/g dry weight) were found in transgenic banana lines expressing phytoene synthase (derived from the fruit of the Fe’I banana found in Papa New Guinea, which only grows in small bunches) under the control of the banana ubiquitone promoter (Ubi). These transgenic lines appear as dark yellow-orange in color and can provide improved nutrition to some of the poorest subsistence farmers in Africa. Consumption of 300 g of transgenic banana could provide as much as 50% of vitamin A required per person per day. Although there is no existing regulatory framework for biotechnology that is currently set up in Uganda, early release is hoped for [48]. More recently, Kaur et al. [49] demonstrated the capability of genome editing to increase β-carotene accumulation in Cavendish banana. The authors created indels in the lycopene epsilon-cyclase (LCYε) gene to increase β-carotene content.

10. Biofortified maize, cassava, and sweet potato

Maize also produces β-carotene, and concentrations vary greatly between different varieties. Although β-carotene content can be increased using conventional breeding, genetic engineering strategies have also been implemented. Consumption of transgenic maize biofortified with β-carotene improved volunteer’s health in clinical trials held in Africa and North America [50, 51]. Moreover, chickens fed transgenic biofortified maize produced eggs that exhibited increased carotenoid content [52]. The deep orange color of biofortified maize challenges public perception for some African populaces, as orange maize is often associated with animal feed, whereas white maize is traditionally considered to be for human consumption.

The BioCassava Plus project specifically targets cassava, a staple crop in Africa that is nutritionally deficient yet is consumed by a quarter of a million sub-Saharan Africans [53]. Transgenic cassava expressing high levels of β-carotene have been demonstrated to increase vitamin A levels and improve nutritional status in feeding studies [54]. Programs such as the BioCassava Project could therefore generate cassava crops with lasting nutritional benefits.

β-Carotene biofortified sweet potato has become a priority for sub-Saharan Africa [55]. White-fleshed sweet potato was transformed with the Orange (Or) gene responsible for carotenoid accumulation, so that β-carotene and total carotenoids levels in the IbOr-Ins transgenic sweet potato were 10-fold higher compared to that of white-fleshed sweet potato [56, 57].

11. Conclusions

This chapter illustrates the ability of biofortification using genetic engineering to address micronutrient deficiencies in a variety of crops found in resource-poor nations. The current regulatory climate and anti-GMO lobbying efforts have retarded the release of GM crops that address highly prevalent vitamin and mineral deficiencies [58, 59]. Nevertheless, the proof of concept has been realized for various nutritionally enhanced GMOs [37, 60]. This has triggered an increase in the number of nutritional traits in the global GM crops pipeline over the last two decades and is expected to be further reinforced in the near future [61]. Consumer opinion on nutritious crops is hardly affected by the type of technology used to generate them [45, 62]. It is unfortunate that a significant effect of lobbying polarizes public opinion, regardless of the scientific basis of given arguments [63]. The current environment is showing signs of turning around with the approval of Golden Rice in several countries. It is anticipated that other biofortified crops will soon follow regulatory approval, and thus help to alleviate malnutrition worldwide.