Keywords

1 Introduction

Using their inbuilt biochemical processes, plants have capabilities to synthesize majority of dietary micronutrients, excluding vitamin D (cholecalciferol, ergocalciferol) and cobalamin (B12) compared to developed nations; crops such as rice, wheat, cassava, and maize are the major sources of energy and proteins in the developing nations which are inadequate to meet the minimum daily requirements of other essential nutrients. Moreover, the nutrients are not evenly distributed in all parts of the plant (Zhu et al. 2007). For instance, rice leaves are rich in provitamin, while the same is lacking in edible grain. Therefore, attention should be given to enrich the dibble portions of the grains with a specific nutrient or combination of more than one by a process commonly known as biofortification.

Biofortified food crops are characterized by enriched nutrients with enhanced bioavailability of essential micronutrients, which are often preferred by the consumers over food supplementation owing to traditional consumption pattern and trade. Biofortification differs from industrial fortification/supplementation as the latter depends upon external addition of micronutrients, whereas the former relies upon changing biosynthesis or physiological capacity of food plants in order to produce or accumulate desired level of specific essential nutrients. The common approaches practiced for generating biofortified crops involve fertilizer application, conventional breeding, and transgenic technology. Implementation of these above-stated approaches results in micronutrient-dense staple crops, with dense minerals and vitamins. Owing to today’s global scenario of increasing nutritional insecurity, biofortification is gaining popularity as it targets the staple food consumed predominantly by the sizeable population inhabiting relatively in the remote areas. Further, developing fortified seeds represents onetime investment which can be consumed year after year and also shared between the communities across the globe (Dwivedi et al. 2012).

Biofortification process pursues various strategies such as optimizing the concentration of antinutrients, mobility of micronutrients within plants, binding capacity/sink strength of seeds, and concentration of metabolites that promote micronutrient absorption (Cakmak et al. 2010). Application of these strategies involves identifying target nutrients suitable for such biofortification. To date, targets for biofortification have been diverted toward essential amino acids (methionine), fatty acids, vitamins A and E, lycopene, flavonoids, Fe, and Zn. In this chapter, we outline the characteristics of the minerals and nutrient elements and evaluate the progress made so far toward enhancement of these targets.

2 Antioxidants

Fruits and vegetables supply passable amount of antioxidants such as anthocyanins, carotenoids, vitamins C and E, and polyphenolics such as quercetin (Shehanaz 2013). These antioxidants protect human from various kinds of reactive oxygen species (ROS). Carotenoids along with other similar compounds are generated via general isoprenoid biosynthetic pathways in plants.

2.1 Anthocyanins

These compounds are colored (red, purple, or blue) pigments that belong to a water-soluble flavonoid family. It has been seen that tomato berry contains negligible amount of anthocyanins. Significant efforts have moderately enhanced its level mainly in peel parts which is normally destroyed during processing. However, the trait of resulting purple fruit was transferred to several commercial cultivars via backcrossing without compromising growth and yield (Hirschi 2008). Further studies have supported the fact that such transgenic tomato powder has extended the life span of tumorigenic mice. Blackberries and raspberries are among the best sources of dietary anthocyanins, but both are expensive and are consumed in smaller quantities than tomatoes (Crozier et al. 2009). Possibly, these engineered tomatoes could contribute substantially to the antioxidant levels of human diets.

2.2 Carotenoids

2.2.1 Lycopene

Lycopene is a carotenoid which is poorly absorbed by the human body from fruits because of its fat-soluble nature. Also, its absorption varies across its cis or trans form. Furthermore, ambiguity prevails concerning the enhanced use of lycopene in foods and its subsequent benefits to human body. Lycopene is the major carotenoid pigment found in tomatoes which has received tremendous attention due to its potential cancer chemopreventive property. Its consumption mostly shields from prostate cancer (Kucuk et al. 2002; Miller et al. 2002). The studies in these lines have produced some inconsistent results, which is soberly clarified by problems with the bioavailability of lycopene from different sources (Giovannucci 2002). Consumption of fruits and vegetables rich in lycopene protects breast and colorectal cancers. While many of the studies conducted on a few animal models have assured a protective reaction of lycopene, its mechanism has been resolved to an appreciable extent (Terry et al. 2002a; Cohen 2002). Heber and Lu (2002) have reported that similar to other dietary carotenoids, lycopene might work in various ways to defend cancer.

Various genetic modification (GM) techniques were used to increase the levels of lycopene in tomato plant. Overexpression of phytoene synthase gene, a key enzyme in the carotenoid pathway from the bacteria Erwinia uredovora in tomato plants, resulted in 1.8- to 2.2-fold enrichment of lycopene and β-carotene and a twofold to fourfold rise in the total carotenoid levels (Fraser et al. 2002). Furthermore, overexpression of a yeast S-adenosyl methionine decarboxylase has increased lycopene levels in tomato fruit significantly (Mehta et al. 2002). Processed tomato such as pastes mixed with fats enhances lycopene absorption significantly. An artificial formulation made by entrapping lycopene within whey proteins was explained in a recent report by Tucker (2003). This lacto-lycopene has shown a similar bioavailability to lycopene in tomato paste (Richelle et al. 2002).

2.3 Vitamin

Biofortification of crops with vitamin A and vitamin C can increase the Fe absorption in the intestine (Garca-Casal et al. 2003). Folate plays a significant role in preventing neuronal disorders in developing fetus. However, it is hard to hold it due to its high water solubility, for example, in rice kernels during boiling (Shrestha et al. 2003). Some of the important biofortified vitamins have been discussed below.

2.3.1 Vitamin A

Rice is a staple food crop of the global population. Overdependence on rice has led to vitamin A deficiency (VAD) which directly affects nearly 250,000–500,000 children every year. Vitamin A-based malnutrition in children can be prevented by improved vitamin A nutrition (Wegmuller et al. 2003). Provitamin A-containing rice could significantly decrease VAD (Zimmermann and Qaim 2004). To decrease the rancidity of rice during storage, oil-rich aleurone layer is removed during milling process, and the remaining endosperm lacks vitamin A. Genetic engineering-based enrichment of rice grain with provitamin A has increased its level significantly (Ye et al. 2000). The immature rice endosperm can produce the intermediate compound geranylgeranyl diphosphate (GGPP) which is then used to generate phytoene by expressing the enzyme phytoene synthase (Cunningham and Gantt 1998). The synthesis of β-carotene from phytoene requires three additional enzymes such as phytoene desaturase, β-carotene desaturase, and lycopene β-cyclase. The important concern is the bioavailability of the engineered β-carotene to humans. Although the conversion factor to vitamin A for synthetic β-carotene and β-carotene in fruits is approximately 6:1, however, the conversion factor in case of vegetables like spinach may be as low as 24:1 due to the poor release of the carotenoids during digestion (Institute of Medicine 2000).

2.3.2 Vitamin E

Another vital dietary antioxidant that occurs in various isomeric forms in plants is vitamin E (tocopherol), with α- and γ-tocopherol being the most abundant. The α-tocopherol is considered as the most beneficial dietary form; however, it is found less dominantly in many foods than γ-tocopherol. Tocochromanols consist of four tocopherols and four tocotrienols that constitute vitamin E (Cahoon et al. 2003; DellaPenna and Pogson 2006). In the plants, vitamin E content can be increased using nutritional genomics (DellaPenna 2007; Shintani and DellaPenna 1998). Furthermore, the content and types of tocochromanols can be altered by coexpression of several genes related to the tocochromanol biosynthetic pathway. This can increase the vitamin E content of seed oil by tenfold (Henry and Qi 2005). Efforts have been made to extend this technology to soybean, maize, and canola (DellaPenna 2007). An added benefit of this work may upsurge the shelf life of foods (Mehta et al. 2002). It is believed that foods with greater levels of vitamin E are protected from oxidative pressure which could enhance agricultural productivity and improve storage.

2.4 Other Antioxidants

Polyphenolics such as flavonols are other important antioxidants which have been enhanced in tomato. In the normal fruit, flavonoids are present in the skin in small amount. Bovy et al. (2002) have increased levels of the kaempferol in a tissue that would normally be lacking this compound in the flesh of the tomato fruit by expression of two transcription factors, LC and C1, from maize.

3 Folate

Folate is also known as tetrahydrofolate (THF) which is not synthesized by animals. The recommended dietary allowance for folate ranges from 400 to 600 μg per day for pregnant women (Rosemary and Lisa 2013). Its deficiency leads to megaloblastic anemia (Scott et al. 2000). Other significances are the initiation of hyperhomocysteinemia which is a risk factor for cardiovascular disease (Stanger 2004), misincorporation of uracil in DNA, and chromosomal damage (Lucock et al. 2003). Furthermore, folate deficiency leads to abnormal DNA methylation arrays associated with carcinogenesis (Choi and Friso 2005; Ulrey et al. 2005). Plants are the ultimate source of folate for animals. Cereals such as maize, wheat, and rice contain extremely low levels of folate (USDA National Nutrient Database for Standard Reference. Release 9; http://www.nal.usda.gov/fnic/foodcomp/search/). To reduce the risk of folate deficiency, fortification of cereal foods with synthetic folic acid has been effected in the USA and other countries (Food and Drug Administration 1996). By contrast, the third world countries lack a solid infrastructural platform to enable effective preventive methods in the form of fortification, supplementation, or educational campaigns (Sachs 2005). Therefore, biofortification of staple crops together with conventional public health practices appears as the probable option to meet the folate deficiency especially in developing countries.

Folate consists of three different components such pteridine, para-aminobenzoic acid (PABA), and glutamate. It is synthesized from these precursors within the mitochondria, while these components are synthesized in unlike compartments within the plant cell (DellaPenna 2007). Bioavailability of folate from various cereals and legume foods depends on a number of factors such as the food matrix, the polyglutamyle conjugation, etc. (McNulty and Pentieva 2004).

The polyglutamylation of folate reduce the bioavailability, because dietary folates need to be deglutamylated by the intestinal conjugase before efficient uptake by intestinal (Rebeille et al. 2006). Recently, it has been exposed that the ratio of monoglutamate to polyglutamate in natural folate derivatives has no clear impact on the intestinal absorption (McKillop et al. 2006). This signifies that the amount of the intestinal conjugase is sufficient to remove the polyglutamate tail without affecting the rate of absorption. This proposes that other factors have an impact on bioavailability, such as entrapment of folates in the food matrix, making them inaccessible to the conjugase that is tethered to the intestinal cell membranes. Therefore, one likely strategy to increase bioavailability could be to boost the levels of the plant conjugase activity of gamma-glutamyl hydrolase (GGH), which would be released from the vacuole following maceration and facilitate folate release within the food matrix before digestion. Other strategy is the use of FBPs. One must also consider the fact that the presence of folate binding proteins (FBPs) in the food matrix, although mechanistically unclear, can lead to a decrease in folate absorption (Witthoft et al. 2006).

4 Essential Amino Acids

Most crops lack one or more essential amino acids, for example, cereal grains are deficient in lysine and threonine, whereas legumes are in methionine and cysteine (Hartwig et al. 1997; Newell-McGloughlin 2008; O’Quinn et al. 2000; Sautter et al. 2006). As the mainstream population of the world depends on cereals and legumes for their diet, plant biologists have used various approaches to augment anticipated deficient amino acids in these plants (Rapp 2002). For example, expression of storage proteins that comprise high levels of required amino acids has raised lysine content in rice and wheat (Christou and Twyman 2004) and some essential amino acid content in potatoes (Egnin and Prakash 1997). However, attempts to raise sulfur-containing amino acids have not been as helpful (Dinkins et al. 2001).

To address this issue, synthetic proteins have been expressed in cassava to match the amino acid requirements for humans. The inadequate availability of free amino acid pools within the edible portion of plants suggests us to alter the amino acid content of target crops. In higher plants, synthesis of threonine, lysine, and methionine are under complex feedback regulation (Hesse et al. 2004). Therefore, studies now focus on developing feedback-insensitive enzymes for these pathways (Newell-McGloughlin 2008). This has significantly improved the free lysine levels in maize (from 2 % to almost 30 %) (Sofi et al. 2009). Expression of these altered enzymes has also improved lysine content in canola and soybean which has significantly improved the production of tryptophan levels in grains. A fundamental concern with each of these manipulations is to ensure that the total amount and composition of storage proteins are not changed.

5 Essential and Very-Long-Chain Fatty Acids

Genetically modified oil seed crops are an abundant, relatively inexpensive source of dietary fatty acids (Abbadi et al. 2004; Anai et al. 2003; Kinney and Knowlton 1998; Liu et al. 2002; Reddy and Thomas 1996; Wallis et al. 2002). Production of these lipids in vegetables could provide an easy mechanism to deliver healthier products without major dietary modifications (Damude and Kinney 2008; Newell-McGloughlin 2008). Plants are sources of the essential fatty acids such as linoleic acid and linolenic acid as well as very-long-chain polyunsaturated fatty acids (VLC-PUFAs) such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which are usually found in fish oils.

Given that many of the enzymes tangled in fatty acid biosynthesis and degradation have been characterized, there is an abundance of transgenic approaches to the modification of oil and fat content in plants (Damude and Kinney 2008; Broun et al. 1999). Examples of such modified oils include low- and zero-saturated fat soybean and canola oils, canola oil comprising medium-chain fatty acids, high-stearic acid canola oil, high-oleic acid soybean oil, and canola oil encompassing the polyunsaturated fatty acid linolenic acid (Mensink et al. 2003). Oils abundant in monounsaturated fatty acids provide improved oil stability, flavor, and nutritional qualities. Oleic acid (18:1), a MUFA, can provide more stability and health benefits than the PUFA (18:2 and 18:3). Soybeans have been manipulated to encompass more than 80 % oleic acid against normal 23 % and had a significant decrease in polyunsaturated fatty acids (Kinney and Knowlton 1998). High-oleic acid soybean oil is more resistant to degradation by heat and oxidation requiring little or no post-refining processing.

6 Mineral Biofortification

Humans require at least 22 mineral elements for their survivals (Welch and Graham 2004; White and Broadley 2005; Graham et al. 2007) which can be abounding by an appropriate diet. It is estimated that over 60 % of the world’s people are iron (Fe) deficient, over 30 % are zinc (Zn) deficient, 30 % are iodine (I) deficient, and 15 % are selenium (Se) deficient (Yang et al. 2007). In addition, calcium (Ca), magnesium (Mg), and copper (Cu) deficiencies are also common in many developed and developing countries (Frossard et al. 2000; Welch and Graham 2002, 2005; Rude and Gruber 2004; Grusak and Cakmak 2005; Thacher et al. 2006).

Fe is an essential component of many enzymes catalyzing redox reactions. More than half of the Fe in the human body is bound to hemoglobin. In the developing countries, Fe deficiency leads to anemia and is estimated that 40–45 % of preschool-age children are anemic. Approximately 50 % of this anemia results from Fe deficiency (Grillet et al. 2014). The significances of early childhood anemia include poor cognitive development (Lozoff 2007). Zn is a cofactor with diverse structural and catalytic functions in about 10 % of all human proteins. In addition, evidence has been accumulating for important regulatory roles of Zn ions in inter- and intracellular signaling (Maret 2013). A major challenge is low bioavailability of Fe and Zn from staple cereals and legumes which have absorption inhibitor such as phytic acid (Olsen and Palmgren 2014). In general, to increase mineral concentrations in edible crops (biofortification), parallel attempts are advocated such as (1) to increase the concentrations of “promoter” substances, such as ascorbate (vitamin C), β-carotene, cysteine-rich polypeptides, and certain organic and amino acids, which excite the absorption of essential mineral by the gut; (2) to reduce the concentrations of antinutrients, such as oxalate, polyphenolics (tannins), or phytate (IP6), which interfere with their absorption; (3) and to increase the mobility of Fe and Zn within the plant as well as the binding capacity/sink strength of seeds.

A recent study reported the absorption of radio-labeled Zn from maize, rice, and barley with low phytic acid mutants in comparison to cultivars with normal seed phytate concentrations. Consistently, reduced phytic acid levels resulted in enhanced Zn absorption (Lonnerdal et al. 2011). In the rice, bioavailability of iron has been targeted by introducing a gene for phytase which resulted in a 130-fold increase in the expression of this enzyme. On the other hand, dietary phytate has been associated with substantial health benefits including strong activity as an anticarcinogenic agent. Several studies reported inhibitory effects of phytate on the growth of different types of tumors. Additional protective effects include the lowering of cholesterol levels (Vucenik and Shamsuddin 2006). Thus, the nutritional benefits of low-phytate grains and seeds have to be weighed against the positive effects of phytate intake (Murgia et al. 2012). The conclusions drawn could well differ between populations depending on the relative importance of micronutrient malnutrition versus cancer incidence.

Transgenic maize plants overexpressing ferritin in combination with a fungal phytase contained up to threefold more bioavailable Fe as determined by the in vitro digestion/Caco-2 system (Drakakaki et al. 2005). Expression of ferritin, an iron-storage protein, in seeds causes a threefold to fourfold increase in iron levels (Goto et al. 2000, 1999; Vasconcelos et al. 2003). Although polishing of rice causes a decrease in mineral levels, ferritin-enhanced rice still has increased iron levels in the transgenic polished rice. Rats fed with a diet containing the transgenic rice demonstrate that the iron in the rice had bioavailability equal to that found in diets containing FeSO4 at equal concentrations (Murray-Kolb et al. 2002).

A potentially promising alternative approach was suggested to indirectly enhance Fe and Zn bioavailability through increasing the concentration of nondigestible carbohydrates, thereby promoting beneficial microbiota that stimulates Fe and Zn absorption by human gut cells (Murgia et al. 2012; Shahzad et al. 2014). The main focus has been on inulin, a polymer consisting of beta-2-1-linked fructose units. Supplemented inulin had enhanced bioavailability of Fe in maize and soybean meals (Yasuda et al. 2006). Variation in concentrations among crop plants and engineering strategies has to be explored more extensively for this prebiotic (Rawat et al. 2013). A combined approach in rice and maize has been developed that involves the expression of iron-storage proteins and fungal phytase (Bashir et al. 2013). This combined approach for mineral biofortification should provide maximal levels of bioavailable iron.

7 Conclusions

In conclusion, with the increasing global population and nutritional insecurity mainly in developing nations, research priorities should be redefined and reoriented to a more meaningful approach to solve the problem. Combined approach of genetic engineering, breeding, and agronomic intervention could help figure out above nutritional problem. Future research should be diverted to analyze the interaction of augmented nutrients with other nutrients in vivo. It is also advised that the impact of genetic engineering on the agronomic performance of crops and biotic and abiotic stresses should also be assessed. In addition, the focus should be on studies involving field crop trials and human beings as experimental subjects to analyze the effectiveness of agronomic or genetic biofortification.