Crops With Improved Nutritional Content Though Agricultural Biotechnology

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CROPS WITH IMPROVED NUTRITIONAL CONTENT THOUGH AGRICULTURAL BIOTECHNOLOGY

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Kathleen L. Hefferon Cornell University, Ithaca, NY, United States

1 ­INTRODUCTION Global food and nutrition security is and will continue to be one of our greatest challenges. Approximately 800 million people across the globe are undernourished (meaning that they do not consume an adequate number of calories each day) and over half of the world’s population is malnourished (meaning that they lack access to essential micronutrients such as vitamins and minerals) (Food and Agriculture Organization, 2013; Global Nutrition Report, 2014). Much of food and nutrition insecurity today is located in sub-Saharan Africa and Southeast Asia (Aswath et al., 2016). In late September 2015, the United Nations set out Sustainable Development Goals, intended to address first and foremost, global poverty and hunger. It has been maintained that the number of nutritionally secure foods could be improved by reducing the number of people who live in extreme poverty. This could be achieved through diversification of their diets with the wide range of micronutrients found in fruits and vegetables (Farre et al., 2011; Fitzpatrick et al., 2012). The incorporation of diverse strategies from multiple disciplines will be needed to improve global food and nutrition security. In general, agricultural development in poorly performing regions such as sub-Saharan Africa will require both better plant varieties as well as improved farming methods to bring about the sustainable intensification of crops. Micronutrient deficiencies in staple crops can be addressed through biofortification; either through direct fortification of crops in the form of fertilizers and foliar sprays, by the addition of supplements containing vitamins and minerals to dietary programs, or by direct biofortification of plant varieties (Gomez-Galera et  al., 2010). Agronomic fortification depends on the mineral and crop species, cannot target specific edible plant organs and cannot be a solution for bioactive compounds that require synthesis by the plant, such as vitamins. Supplementation programs can be costly and require an existing infrastructure, thus are not sustainable for the long term. Biofortification of food crops can be advantageous for populations who find it difficult to change their dietary habits or who lack access to nutritional programs (Jeong and Guerinot, 2008). Most staple crops that the world relies upon for calorie intake, such as rice and maize, are low in micronutrients such as vitamin A which are essential to a healthy diet, such as vitamin A, folate, iron (Fe), and zinc (Zn) (Nestel et al., 2006). In many cases, the rural poor have little access to the rich source of micronutrients found in fresh fruits and vegetables. The very poor cannot afford to diversify their diets; a monotonous Plant Micronutrient Use Efficiency. https://doi.org/10.1016/B978-0-12-812104-7.00019-8 Copyright © 2018 Elsevier Inc. All rights reserved.

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diet fails to provide an adequate supply of micronutrients and leads to malnutrition (Bhutta et al., 2013; Hirschi, 2009). Crops can become biofortified through conventional breeding techniques or through the use of biotechnology, such as genetic modification, when the traits needed are not readily available (Waters and Sankaran, 2011; Welch and Graham, 2004; White and Broadley, 2005). The genetic engineering of crops for improved traits has been widely reviewed in the literature and a few examples are listed in Table 1. Briefly, genetic engineering involves the introduction of a novel trait into a crop by through the manipulation of its genetic material. Genetic material can be incorporated into the plant genome either via Agrobacterium-mediated transformation or by biolistic (gene gun) delivery. Transgenic, or genetically modified (GM) crops have been commercially available in the U.S. since 1996 (Basu et al., 2010; Bazuin et al., 2011). A well-known example of a transgenic plant is Golden Rice, which expresses βcarotene and was created philanthropically with the intent of alleviating vitamin A deficiency (VAD) in developing countries (Potrykus, 2010a,b). Cisgenic plants, or plants which express genes from close wild relatives, are also being generated to obtain resistance genes which were lost over years of crop domestication. The Wheat Stem Rust Initiative, for example, is currently generating cisgenic versions of wheat, which possess multiple resistance genes to the fungal pathogen Puccinia graminis f. sp. tritici Ugg99 from wild relatives (Singh et al., 2016). A third technology that falls under the umbrella of genetic engineering is RNA interference, or RNAi technology. In this case, the plant is designed to produce an antisense RNA to a particular gene, whose expression is then blocked via gene silencing. Examples of the use of this technology are genetically modified papaya which are resistant to Papaya ringspot virus. This technology is responsible for saving the Hawaiian papaya industry (Evanega and Lynas, 2015). More recently, a new technology known as ‘gene editing’ has come to the forefront. Gene editing does not require the introduction of new gene sequences, rather, it can direct only one or two nucleotide changes in a plant genome and thus is exempt from the regulations that govern the production of genetically modified organisms (GMOs) (Rani et al., 2016). While no examples of gene edited crops are commercially available at present, much research is being undertaken in this field and many new crop varieties will be realized in years to come using this biotechnological approach (Gilani and Nasim, 2007). It should also be noted that many varieties of crops available today have been generated using mutation breeding. Not considered a form of genetic engineering, mutation breeding involves the introduction of random mutations to plant cuttings using chemical or irradiation mutagen. Explants Table 1  Examples of genetic engineering described in this chapter Technology

Description

Mutation breeding Transgenesis

Random mutations introduced into genome via chemical or irradiation mutagenesis Introduction of novel traits by delivering DNA from a different organism to the target organism Introduction of trait by delivering DNA from similar, sexually compatible species Introduction of antisense RNA corresponding to a gene from an organism or from an invading pathogen of that organism Targeted nuclease, in conjunction with the cell’s DNA repair machinery, makes small one or a few nucleotide changes within an organism’s genome

Cisgenesis RNA interference (RNAi) Genome editing

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that express new traits are then propagated from these mutagenesis events. According to the Mutant Variety Database, over 17,000 varieties of crops have been developed using mutation breeding. Ruby red grapefruit and single malt scotch are both derived from mutation breeding (Nuijten et al., 2017). The first generation of biotechnology crops focused on agronomic input traits that helped farmers; however, their value was not clear to the general consumer who is disconnected to the demands of farming. Agronomic traits include improved crop yield, and provided resistance against abiotic and biotic stresses such as drought, heat, flooding, and pests (Collinge et al., 2010; Cominelli and Tonelli, 2010; Deikman et  al., 2012). More recently, agricultural biotechnology has expanded to improve human health, including the design of biofortified and functional food crops. The following section describes some of the advances made in generating crops through biotechnology, which are biofortified for vitamins, minerals, and other biological compounds that play an important role in human health.

2 ­BIOFORTIFIED RICE Vitamin A, Fe, and Zn deficiencies are the most widespread of all the human micronutrient deficiencies. They are particularly prevalent for those who live in developing countries where there can be an over-reliance on a monotonous staple crop such as rice. Often sustained supplementation programs in resource-poor regions of the world are difficult to implement due to cost, logistics, and political instability. Today, rice remains the largest source of calories for over one half of the world’s population. The biofortification of rice has been recognized as a potential way to alleviate malnutrition to micronutrients such as vitamin A, Fe, Zn, folate and essential amino acids such as lysine. Rice endosperm is a source of carbohydrate but contains low levels of micronutrients (Beyer, 2010). Rice has long been held as a suitable target for iron biofortification because Fe-deficiency anemia is such a serious problem in developing countries. Rice endosperm also lacks adequate folate levels, and folate deficiency is responsible for birth defects, cardiovascular disease, and some cancers. For example, regions in which folate deficiency is prevalent, such as China’s Shanxi province, exhibit a 38% increased prevalence of neural tube defects in newborns than those found in industrialized countries. Tremendous amounts of research have focused on biofortification of rice with vitamin A, an overview will be provided in the next section. Vitamin A is absolutely essential in the human diet not only for eye health, but also for growth, development, immune function, and reproduction (Tian, 2015). The precursor of vitamin A, B-carotene, cannot be synthesized by humans, and thus must exclusively be derived from a diverse set of dietary sources. Inadequate dietary intake of β-carotene leads to VAD, and pregnant women and young children are particularly vulnerable to this form of malnutrition. As this is written, approximately 100–140 million children have VAD, and 250–500,000 will annually become blind as a result of this deficiency. Half of these again will die within a year of losing their sight (Al-Babili and Beyer, 2005). β-carotene and other carotenoids are compounds synthesized by many fruits and vegetables and can be attributed to the source of colors that can range from yellow to red. After consumption in the form of edible plant tissue, carotenoids pass through the digestive tract, where they are incorporated into lipid micelles and absorbed into intestinal cells. Once absorbed the provitamin A precursors B-carotene undergoes cleavage to form retinol, and free retinol becomes conjugated with long chain fatty acids for storage in the liver. In this way, vitamin A can be secreted into the bloodstream in response to demands from other tissues.

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Rice endosperm is white and does not accumulate carotenoids. Since rice is the predominant staple for many VAD populations in the developing world, the design of β-carotene fortified rice, known as Golden Rice, was the first biofortified crop to be considered for its long reaching health benefits (Paine et al., 2005). The first generation of Golden Rice was generated by overexpressing genes from daffodil plants and a specific bacterium that could be used to metabolize β-carotene in rice plants. Rice progeny that were generated in this fashion were found to express 1.6 μg β-carotene/g dry weight in their grain. The most current Golden Rice technology, known as GR2, incorporates genes that are derived from two distinct provitamin A pathways. GR2 rice substitutes the phytoene synthesis gene from maize for the analogous daffodil gene used in GR1 rice. Golden Rice 2 can increase levels of β-carotene to 35 μg/g of dry rice. Servings of 130–200 g of deuterium-labeled GR2 that was grown hydroponically in heavy water and that expressed 0.99–1.53 mg of β-carotene per serving were fed to human volunteers. After thirty-six days post-consumption, blood samples taken exhibited 0.34–0.94 μg of retinol, indicating that β-carotene derived from Golden Rice is effectively converted to vitamin A at a rate of 500–800 μg retinol per 100 g uncooked Golden Rice, or close to the recommended daily allowance for children. In addition to this, the vitamin A value of Golden Rice, nontransformed spinach and pure β-carotene presented in an oil format were provided to children and compared for effectiveness of β-carotene to retinol conversion (Haskell, 2012). The results of this study showed that the β carotene derived from Golden Rice was just as effective as pure β-carotene and in fact more effective than β-carotene from spinach in providing vitamin A to children (Tang et al., 2009, 2012). The above results indicate that Golden Rice could realistically be used to alleviate VAD in rice-consuming populations. Golden Rice could thus be considered the very first genetically engineered crop that has been specifically designed in order to combat malnutrition on a large scale, with the select advantage that it could readily reach remote rural populations that have no access to supplementation programs. A major concern has been over β-carotene degradation in Golden Rice over prolonged periods of storage. Recently, however, Gayen et al. (2015) have demonstrated that by down-regulating the enzyme lipoxygenase (LOX), carotenoid degradation in Golden Rice is greatly reduced, thus reducing post-harvest and economic losses for farmers. Down-regulation of the enzyme was achieved by gene silencing, using RNA interference (RNAi) technology. This study provides new hope to the use of Golden Rice as a means of improving human health with respect to VAD. The development of Fe and Zn biofortified rice is difficult as a result of complex genetic and metabolic networks that control Fe and Zn homeostasis (Grillet et al., 2014; Hotz, 2009). Iron is essential for electron-transfer and as a result is involved in photosynthesis and respiration. Zinc plays a role in the transcription of plant genes and is integral is integral for coordinating the metabolic pathways of proteins, nucleic acids, carbohydrates and lipids. Both Fe and Zn accumulation can be influenced by other nutrients such as phosphorus, sulfur, and nitrogen. As a result of these intricate relationships, under or overexpression of Fe and Zn can impact plant growth and development (Vasconcelos et al., 2003). For example, the constitutive overexpression of the OsNAS genes has improved Fe and Zn concentrations in rice, but has been associated with reductions in plant grain yield in some cases. Plants with lower grain Fe and Zn concentrations exhibited higher grain yield values, and this inverse relationship is most likely due to the source-sink association within the plant whereby micronutrient concentrations in plant tissues become diluted as yield increases, resulting from a discrepancy in the ability of the plant to load micronutrients at the same pace that it is growing (Moreno-Moyano et al., 2016). The research group of Paul et al. (2014) examined the impact of ferritin overexpression on Fe and Zn biofortification in rice. Since the protein ferritin is capable of storing ~4500 bioavailable Fe atoms,

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overexpression of the ferritin gene in transgenic seeds should increase Fe accumulation. The increased need for Fe is transmitted to the roots of the plants via Fe transporters, and these proteins are also capable of translocating Zn. This group therefore incorporated the soybean ferritin 1 gene into a highyielding local indica rice cultivar known as Swarna, and milled rice grains exhibited 2.6-fold overexpression of ferritin, as well as a 2.54-fold increase in Fe, and 1.54-fold increase in Zn accumulation, respectively, with few morphological differences from the parental wild type Swarna plants. Masuda et al. (2013) also demonstrated that Fe biofortified rice expressing the soybean ferritin gene as well as the barley IDS3 MA synthase gene were tolerant to Fe deficient calcareous soils and still displayed an increase in Fe content, although not as significant as that found on commercially supplied soil. The results of this study show that these biofortified plants will function well and improve iron bioavailability on iron-poor soils. Authors Tan et al. (2015) examined the effect of the high-efficiency metal transporter MxIRT1 on rice Fe and Zn micronutrient content. Blancquaert et  al. (2014, 2015) assessed the stability of folate in two biofortified rice lines and found that folate concentrations were reduced to half of their original levels after four months of storage. To address this problem, the authors used endosperm-specific overexpression of genes from another plant species, Arabidopsis thaliana, which encode enzymes responsible for catalyzing folate polyglutamylation, and placed them under control of the rice glutelin B1 promoter. The authors also overexpressed a plant-optimized, soluble version of FBP (derived from bovine milk) under control of the rice glutelin B4 promoter. Transgenic folate biofortified rice produced in this fashion did not show any visible phenotypic differences compared with WT plants but resulted in higher and more stable concentrations of folate in stored grains compared to the earlier levels obtained. Three 100 g servings per day of this folate biofortified rice, taking into account folate loss after boiling and long term storage, could satisfy the daily folate requirements of the average adult. Kiekens et al. (2015) examined the bioavailability of folates present in genetically engineered biofortified rice during a 12-week feeding trial using rats as a model organism. The authors analyzed folate in the blood and homocysteine levels and found that folates released in the GI tract becomes incorporated into red blood cells and alleviate both anemia and hyperhomocysteinemia, suggesting that folate rice which is locally grown may serve as a fortification technique. The essential amino acid lysine is also missing from rice and is a source of malnutrition. The authors sought to generate transgenic rice biofortified with protein-bound lysine. To address food safety and allergenicity, Wong et al. (2015) first surveyed the GenBank database to identify rice endogenous proteins that were rich in lysine (>10 mol%). The choice of generating a “cisgenic” plant was deliberately made to lessen ethical concerns regarding GMOs. Two histone genes, RLRH1 and RLRH2, were chosen as candidate transgenes to be expressed in rice seed because they have high lysine content and low allergen potential. The genes were placed under careful regulation so that they were expressed at similar levels of other amino acids in rice seed in order to avoid triggering any physiological abnormalities. The authors were able to improve lysine content by up to 35% in transgenic rice lines as compared to wild type rice used as a control.

3 ­BIOFORTIFIED MAIZE AND CASSAVA Maize differs from rice as it is capable of producing β-carotene. β-carotene content, however, can vary greatly between maize varieties. Maize can either undergo conventional breeding or be genetically

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engineered to increase levels of β-carotene (Moretti et  al., 2014; Tanumihardjo et  al., 2010, 2017). β-carotene content, for example, has been increased up to 59 ug β-carotene/g dry weight in white endosperm varieties of maize through the use of transgenic technologies. Consumption of transgenic maize biofortified with β-carotene in the form of a porridge by North American women resulted in a conversion of β-carotene to retinol of 6.48 ± 3.51 μg β-carotene to 1 μg retinol (Li et  al., 2010). Similarly, healthy Zimbabwean men fed with biofortified maize and supplemented with fat demonstrated a conversion of 3.2 ± 1.5 μg β-carotene to 1 μg retinol (Muzhingi et al., 2011; Gomes et al., 2013; Mugode et al., 2014). Moreno et al. (2016) showed that chickens fed transgenic biofortified maize laid eggs that were deleted in vitamin A, whereas levels were increased within their livers. On the other hand, other carotenoids such as lutein and zeaxanthin, increased in concentration in the eggs, indicating that carotenoid transport and deposition can follow select and differential pathways (Twyman et al., 2016). The BioCassava Plus project specifically targets cassava, the nutritionally deficient staple of 250 million sub-Saharan Africans (De Moura et al., 2013; Sayre et al., 2011). Transgenic cassava expressing high levels of β-carotene have been produced and fed to healthy volunteers in the form of a porridge. Blood samples taken from these volunteers demonstrated that biofortified cassava increases β-carotene and retinyl palmitate TRL plasma concentrations (4.5 μg β-carotene to 1 μg retinol conversion). The results of this study suggest that biofortified cassava could be used to prevent VAD (La Frano et al., 2013, 2014). Programs such as these could therefore generate cassava crops with more lasting nutritional benefits (Sayre et al., 2011). Cassava roots also express a low protein: energy ratio, and less than 10%–20% of the required amounts of Fe, Zn, vitamin A and vitamin E. By reducing levels of the toxin cyanogen in roots, iron root uptake and protein accumulation in cassava could be enhanced (Bouis et al., 2011). Recently, Telengech et al. (2015), found that β-carotene biofortification was also found in the stems and leaves of cassava plants, not only the tubers, despite the fact that a tuber-specific (patatin) promoter was used in the construction of these transgenic plants (Leyva-Guerrero et al., 2012).

4 ­BIOFORTIFIED POTATOES The carotenoid astaxanthin, synthesized from the precursor B-carotene, is produced by some bacteria, a few green algae, and several fungi and plants Astaxanthins can accumulate through dietary intake in salmon, trout, krill, shrimp, crayfish, crustaceans, and the feathers of some birds, resulting in a characteristic red/pink color. Since astaxanthin is only available from a few natural sources, the metabolic engineering of plants to generate this compound would be beneficial. This requires the introduction and expression of ketolase genes from other sources, such as algae or bacteria, in plants. To this end, Campbell et al. (2015), generated astaxanthin in potato tubers. The authors chose a variety of potato that already produced high levels of astaxanthin precursor compounds, and transformed them with codon-optimized Brevundimonas SD212 crtZ and crtW genes under the CaMV 35S promoter. The authors were able to produce astaxanthin in potato at levels that approach the daily recommended dose (55 g of potato provides 5 mg of astraxanthin).

5 ­BIOFORTIFIED BANANA Bananas, grown widely throughout the tropics, are the world’s most important fruit crops. They are eaten raw and are often cooked as a major staple in many African countries. While the vitamin A

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content is high in many types of bananas that are cooked, the Cavendish banana, which is used as a dessert banana by much of the world, and the cooking banana, East African highland banana (EAHB), consumed in Uganda, has low levels of this micronutrient. In countries like Uganda, for example, vitamin A prevalence for children under the age of five and women of childbearing age has increased. It is difficult to conventionally breed these types of bananas due to low fertility, however, a genetically engineering Cavendish banana to increase vitamin A content has been successfully achieved. Research on the Cavendish banana can be used as a model system for the EAHB. Transgenic lines expressing both phytoene synthase (from the fruit of the Fe’I banana that expresses high levels of Vitamin A) under the control of the banana ubiquitone promoter (Ubi) were able to produce greater levels of vitamin A than were set as a target (20 μg/g dry weight). These transgenic lines were characterized by a “golden bunch” phenotype. It is interesting to note that the vitamin A content of these transgenic lines increased over successive vegetative generations (Paul et al., 2017).

6 ­BIOFORTIFIED SORGHUM AND MILLET In Sub-Saharan Africa, sorghum and millet are often the staples of a typical diet. Sorghum is the cereal of over 300 million African people. Sorghum is a hardy plant and grows well in marginal land. Sorghum has a high homology to maize, yet has low levels of lysine, iron, zinc, and vitamin A, with vitamin A deficiency being the greatest cause of mortality in children under the age of five for this region. Both conventional and (traditional) breeding and transgenic approaches to biofortify sorghum are underway, with the Africa Biofortified Sorghum (ABS) Project dedicated to enhance bioavailable content of provitamin A carotenoids, Zn, and Fe. The Biosorghum project sought out to increase iron and zinc availability by 50%, to increase provitamin A levels to up to 20 mg/kg, to increase lysine content by 80%–100%, to increase tryptophan and threonine by 20%, and to improve protein digestibility from its current to approx. 60%–80% (Henley et al., 2010). Authors Lipkie et al (2013) examined the bioaccessibility of vitamin A in sorghum based cooked porridge using an in vitro digestion model. One transgenic line that was studied had 9–15 times greater vitamin A content than the germplasm background in the raw sorghum meal and 4–8 times greater after in vitro digestion (which simulated oral, gastric, and intestinal conditions by consecutively adding amylase, pepsin and lipase/bile). The authors found that carotenoids were less bioaccessible from transgenic sorghum events than from the wild type controls. Furthermore, the vitamin A bioaccessibility in transgenic crops was not improved despite the enhanced content in the meal itself. It is possible that the digestibility of proteins in sorghum after cooking is poor, and carotenoids such as vitamin A are likely bound to kafirin proteins in sorghum. The authors discovered that provitamin A bioaccessibility become enhanced 3- to 5-fold by processing 10% of a lipid source, such as sunflower or peanut oil, into the porridge. Lysine is an amino acid that is essential for growth, bone calcification, and the immune system. Increasing lysine availability has been developed both by chemical mutagenesis breeding and by genetic engineering. The Africa Biofortified Sorghum project, led by Africa Harvest Biotechnology Foundation International generated a transgenic, nutritionally enhanced sorghum with improved lysine and wet-cooked protein digestibility. When included in a daily diet, these improvements should enable young children to meet sufficient protein and energy requirements. Taylor and Taylor (2011) examined whether this genetically engineered variety of sorghum would retain these improvements when ­prepared in the form of typical African meals, such as flatbread, couscous, injera, and cookies.

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Couscous and cookies, which underwent the harshest heat treatments, lost the greatest lysine content, while fermented sorghum maintained the highest. Millets provide up to 75% total calorie intake in Asia and Africa, next to cereal grains. Hardy in nature, millets are drought and disease resistant. Millets contain high amounts of proteins and micronutrients; however, they also carry an abundance of anti-nutrient factors such as phytates and tannins that reduce nutrient bioavailability (Vinoth and Ravindhran, 2017). Efforts must be made to reduce the action of anti-nutrients in millets in order to increase the bioavailablility of minerals such as Fe, Zn, and calcium (Sharma et al., 2017). Genetic engineering technologies including RNA interference and gene editing could address these challenges.

7 ­BIOTECHNOLOGY TO ACHIEVE ADDITIONAL HEALTH BENEFITS There is an increased interest of developed countries in nutritionally enhanced plants which containing phytochemicals besides β-carotene. One reason for such focus stems from evidence that phytochemicals play a prominant role in battling cardiovascular disease, cancer, hypertension, and diabetes; all considered to be leading causes of death in industrialized countries. These “nutritionally enhanced” plants can be generated using technologies such as RNAi and cisgenesis, as well as transgenesis. Manipulation of metabolic networks to produce more of a desired phytochemical (or less of an undesirable one) can take place by overexpressing or down-regulating transcription factors (TFs). Carotenoids represent a large group of colored pigments that are synthesized by plants as well as algae and bacteria, and can protect plants from photo-oxidative stress (Zhai et al., 2016). Dietary carotenoids found in leafy greens, seeds, fruit, and vegetables have been found to protect humans in a number of ways, although the mechanisms of action remain unclear. For example, lycopene, a carotenoid found in tomato, has a protective effect against prostate cancer, and lutein and zeaxanthin provide protection against age-related macular degeneration. Astaxanthin is also increasingly associated with a wide range of health benefits, including cancer prevention and immune function. The next section describes some of the research that has been conducted on the use of biotechnology to enhance carotenoid production in food crops.

8 ­NUTRITIONALLY ENHANCED TOMATO Tomatoes can act as phytochemical food vehicles for carotenoids due to their high consumption across the world, low cost, and ease of growth (Zhai et al., 2016). During ripening, the red carotenoid lycopene accumulates to give tomatoes their red color. Other carotenoids such as β-carotene, lutein, and zeathanthin are produced in tomato fruit (D’Ambrosio et al., 2016). The carotenoid pathway is complex and several engineering steps are necessary to control the production of these and other metabolites. Several transgenic lines in tomato have been developed to increase carotenoid expression, for example, the Red Setter line overexpresses Lycopene β-cyclase (Lcy-b) which in turn increases β-carotene production. Another transgenic line of tomato known as high Delta overexpresses Lyc-e to increase lutein content. Ralley et al. (2016), have demonstrated that transgenic Solanum lycopersicum plants expressing an additional copy of the lycopene β-cyclase gene (LCYB) from Nicotiana tabacum, under the control of the Arabidopsis polyubiquitin promoter (UBQ3), increased β-carotene content some 10-fold in ripening tomato fruit. Tomato fruit increased in orange color; no other carotenoid levels were changed.

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Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) exhibited enhanced levels of Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) and a significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. These transgenic fruits were also fortified with important nutraceuticals including lycopene, ascorbate and antioxidants (Pandey et al., 2015). Another metabolic engineering strategy involves the introduction of two transcription factors from a snapdragon that are involved in anthocyanin production (Butelli et al., 2008). This led to high levels of expression of these flavonoids in tomato fruit, which as a result become dark purple in color. Found in blueberries and cranberries, anthocyanin is an antioxidant with clearly demonstrable health benefits (Gonzali et al., 2009). Tomato fruit that were metabolically engineered to produce at least seven different anthocyanins and were able to extend the life spans of cancer-susceptible mice by up to thirty percent (Kiferle et al., 2015; Su et al., 2016). These tomatoes may also fight cardiovascular disease and possess anti-inflammatory properties, and are currently being prepared for commercialization. In the future, tomatoes expressing anthocyanin may make their way into food products such as juice, ketchup and pizza sauce. Lim et al. (2014) also metabolically engineered flavonoids to improve the nutritional value of tomatoes. By genetically engineering the onion chalcone isomerase (CHI) gene into tomato, total flavonol content was significantly increased. Coexpression of the Delila (Del) and Rosea1 (Ros1) genes from the snapdragon Antirrhinum majus produced an anthocyanin-rich tomato that was purple in color. A consumer panel reported marginal but significantly higher preference for the flavor and overall liking of CHI tomatoes over Del/Ros1 and wild-type tomatoes. Several of the panel stated afterwards that they would purchase transgenic food if they believed that it would promote their health. In a farther step forward, Juárez et al. (2012) generated a monoclonal antibody toward rotavirus in transgenic purple tomatoes high in anthocyanin content. Extracts of purple tomatoes were shown to neutralize rotavirus infection in an in vitro assay, as well as produce nutraceutically valuable anthocyanins. Transgenic tomatoes that express the gene encoding grape (Vitis vinifera L.) stilbene synthase under the control of a fruit specific promoter have also been developed. Transgenic tomato plants accumulated resveratrol concentrations during tomato ripening of up to 53 microg/g fresh weight (D’Introno et al., 2009; Giovinazzo et al., 2005). Resveratrol is a bioactive compound with a number of beneficial health properties found in red grapes and wine, but not many other common food sources. Producing resveratrol in commonly produced and well-distributed fruit such as tomato would improve its accessibility to a wider population. Flavonols, found in tomato, have a proven role for bone health. Transgenic tomato fruit expressing AtMYB12, an Arabidopsis transcription factor, led to increased flavonol biosynthesis and significant increases in bone growth for pre-pubertal mice fed tomatoes containing the transgene for six weeks (Choudhary et al., 2016) The increases in bone growth and density (both tibial and femoral) were the result of an increase in the number and size of hypertrophic chondrocytes. These tomatoes could enable individuals to better achieve peak bone mass during adolescence.

9 ­NUTRITIONALLY ENHANCED OIL CROPS Oil crops have also been developed to supply the world’s population with sufficient nutrients. Omega 3 fatty acid is one such oil that is available in seafood and is highly beneficial for the human diet, in terms of brain function and cardiovascular health. A daily intake of up to 500 mg omega-3 fatty

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a­ cids (LC-PUFA) is recommended, and this amounts to an annual requirement of 1.25 million metric tones for a population of 7 billion. The annual global supply of Ω-3 LC-PUFA cannot meet this level of requirement due in part to overfishing, and as a result, transgenic “designer oilseed” plants have been metabolically engineered to synthesize omega-3 fatty acids derived from fish and other types of marine life (Ruiz-López et al., 2012a,b, 2014). The metabolic pathway to produce this fatty acid has been reconstituted in plants such as false flax (Camelina sativa), a relative to canola, and several other beneficial fatty acids have also been produced in plant seed oils, including γ-linolenic, stearidonic and arachidonic acids (Haslam et  al., 2013). As another example, Betancor et  al. (2015) developed transgenic lines of Camelina sativa to reproduce fish oil using a metabolic pathway derived from algal genes. Camelina oil containing n-3 LC-PUFA was used to replace fish oil in farmed salmon feeds, with no detrimental effects on fish performance, metabolic responses or the nutritional quality. Other types of designer oils are under development. Liu et al. (2015) have developed oilseed crops with higher levels of acetyl glyceride, with altered properties such as viscosity, freezing point, and calorific value. In another example, Augustine and Bisht (2015) developed transgenic lines and were able to alter the metabolic pathways of Brassica juncea to enrich glucoraphanin content, and improve the cancer fighting value of Brassica based oil and vegetable products. Not only did the increased glucosinolate concentration prevent cancer, but it also defended the plant against the stem rot pathogen, Sclerotinia sclerotiorum.

10 ­CONCLUSIONS AND FUTURE PERSPECTIVES Overcoming micronutrient deficiency will remain one of our greatest challenges for many years to come (Mayer et al., 2008; McGloughlin, 2010). Agricultural biotechnology represents one tool by which to address micronutrient deficiency in resource-poor countries, where staple crops such as rice have low levels of micronutrients including Vitamin A, Fe, Zn and folate (Zhu, 2007). The advent of ‘designer crops’ with enhanced nutritional benefits, including carotenoids and omega-3 fatty acids, will also find a place in the dietary patterns of the industrialized world (Ricroch and HénardDamave, 2016). A principal hurdle to overcome with respect to achieving these goals will be how crops produced by biotechnology are addressed in the future (Pérez-Massot et al., 2013; Potrykus, 2010a,b). Evidence exists that negative viewpoints could be changing. In 2010, De Steur et al. theoretically investigated the potential health impact and willingness of the populace to accept transgenic folate-biofortified rice in China and found general acceptance. The implementation of this modified crop could have a positive effect on the health and economic burden of folate deficiency in Asia. However, regulatory constraints where marketing approvals are pending could bottleneck what progress is being made with respect to delivering biofortified crops to the rural poor in developing countries and this will remain a formidable challenge (Sperotto et al., 2012). While the political environment has been discouraging, to say the least, for university and public institute scientists with respect to the de-regulation of transgenic plants, this may not necessarily be the case for newer technologies, such as RNAi and genome editing. RNA interference has already proven its importance in altering metabolic pathways to improve the storage time of Golden Rice by eliminating undesirable metabolites, as well as the nutritional content of tomato by enhancing secondary metabolite synthesis (Saurabh et al., 2014; Tiwari et al., 2014; Koch and Kogel, 2014).

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Moreover, CRISPR-Cas9 and other genome editing strategies lack stringent regulatory structures and will be vital for the future development of biofortified crops. Inexpensive, rapid, and easy to use, genome editing could pave the way for the future of agriculture as we now know it. Recently for example, Pan et al. (2016) used the CRISPR/Cas9 system to target two genes responsible for altering the color of tomato fruit. Similarly, Čermák et al. (2015), examined the use of CRISPR/Cas9 delivered by a geminivirus vector to overexpress anthocyanin in tomato (Solanum lycopersicum). These two examples represent a plethora of accomplishments that can be realized using gene editing technologies, most likely without the regulatory barriers or stigma that has been associated with genetically modified (GM) crops. The future holds great promise for agricultural biotechnology, and more specifically, for crop biofortification as a whole (Gartland et al., 2013).

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­FURTHER READING De Steur, H., Blancquaert, D., Strobbe, S., Lambert, W., Gellynck, X., Van Der Straeten, D., 2015. Status and market potential of transgenic biofortified crops. Nat. Biotechnol. 33 (1), 25–29. Farré, G., Bai, C., Twyman, R.M., Capell, T., Christou, P., Zhu, C., 2011. Nutritious crops producing multiple carotenoids—a metabolic balancing act. Trends Plant Sci. 16 (10), 532–540. Food and Agriculture Organization of the United Nations FAO GM Foods Platform, 2014. Available online: http:// www.fao.org/food/food-safety-quality/gm-foods-platform/en/ (accessed 15.08.14). International Food Policy Research Institute, 2014. Global Hunger Index ISAAA Brief 46-2013: Executive Summary Global Status of Commercialized Biotech/GM. Lucca, P., Hurrel, R., Potrykus, I., 2002. Genetic engineering approaches to improve the bioavailability and the level of iron in the rice grains. Theor. Appl. Genet. 102, 392–397. Pandey, A., Misra, P., Khan, M.P., Swarnkar, G., Tewari, M.C., Bhambhani, S., Trivedi, R., Chattopadhyay, N., Trivedi, P.K., 2014. Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity. Plant Biotechnol. J. 12 (1), 69–80. Pillay, K., Siwela, M., Derera, J., Veldman, F.J., 2014. Provitamin A carotenoids in biofortified maize and their retention during processing and preparation of South African maize foods. J. Food Sci. Technol. 51 (4), 634–644. Reguera, M., Peleg, Z., Blumwald, E., 2012. Targeting metabolic pathways for genetic engineering abiotic stresstolerance in crops. Biochim. Biophys. Acta 1819 (2), 186–194. Sanahuja, G., Farré, G., Berman, J., Zorrilla-López, U., Twyman, R.M., Capell, T., Christou, P., Zhu, C., 2013. A question of balance: achieving appropriate nutrient levels in biofortified staple crops. Nutr. Res. Rev. 26 (2), 235–245. Wilson, S.A., Roberts, S.C., 2014. Metabolic engineering approaches for production of biochemicals in food and medicinal plants. Curr. Opin. Biotechnol. 26, 174–182.