2 biofortification of cereals to overcome hidden hunger

Plant Breeding 132, 437–445 (2013) © 2013 Blackwell Verlag GmbH doi:10.1111/pbr.12040 Review Biofortification of cereals to overcome hidden hunger N I D H I R A W A T 1, K U M A R I N E E L A M 2, V I J A Y K T I W A R I and H A R C H A R A N S D H A L I W A L 3,4 Wheat Genetic and Genomic Resources Center, Kansas State University, Manhattan, KS 66506, USA; 2School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana 141001, Punjab, India; 3Akal School of Biotechnology, Eternal University, Sirmour, Himachal Pradesh 173101, India; 4Corresponding author, E-mail: hsdhaliwal07@gmail.com With figures Received February 2, 2012/Accepted December 16, 2012 Communicated by H Balyan Abstract More than 60% of the world population suffers from iron deficiency, and over 30% of the global population has zinc deficiency Micronutrient deficiency leads to compromised health and economic losses and is prevalent in populations depending on non-diversified plant-based diets Increasing mineral content of staple food crops through biofortification is the most feasible strategy of combating micronutrient malnutrition Additionally, it will also enhance the agronomic efficiency of crops on mineral poor soils A multipronged strategy towards enhancing mineral content of cereal grains should involve increased uptake of minerals from soil, enhanced partitioning towards grain and improved sequestration in the edible tissues of grains At the same time, it is essential to improve mineral absorption in vivo from cereal-based diets Both conventional and modern breeding approaches and genetic engineering are being employed for biofortification of crop plants With increased understanding of mineral uptake and transport mechanisms in plants, it is becoming ever more possible to engineer biofortified crop plants with the ultimate goal of overcoming hidden hunger Key words: iron — zinc — hidden hunger — biofortification — uptake — transport — bioavailability The Copenhagen Consensus of 2008 listed micronutrient deficiency as the fifth major global challenge to human health (www.copenhagenconsensus.com) Iron and zinc deficiency are the most common and widespread, afflicting more than half of the human population (WHO 2002, White and Broadley 2009) Worst hit are the developing countries of Asia and Africa (Hotz and Brown 2004, Gómez-Galera et al 2010) More than billion people suffer from iron deficiency alone, and the estimates of zinc deficiency are also close (Stoltzfus and Dreyfuss 1998, Welch and Graham 2002, Prasad 2003, Gibson 2006, Thacher et al 2006) Deficiency of iron and zinc, also known as ‘Hidden hunger’, results in poor growth and compromised psychomotor development of children, reduced immunity, fatigue, irritability, weakness, hair loss, wasting of muscles, sterility, morbidity and even death in acute cases (Prasad et al 1961, Haas and Brownlie 2001, Pfeiffer and McClafferty 2007, Wintergerst et al 2007, Stein 2010) Due to its physico-chemical properties, iron takes part in most of the redox reactions in the body and also acts as a cofactor in numerous vital enzymatic reactions (Kim and Geurinot 2007) Likewise, zinc is an essential micronutrient for regulating gene expression and maintaining structural integrity of proteins It acts as a cofactor in more than 300 enzymatic reactions (King and Keen 1999) The major reason for micronutrient deficiency in the populations of the third world countries is the predominance of non-diversified cereal- and plant-based diets, which are poor in micronutrients, as compared to the meat rich diets of people in developed countries (FAO 2004, Grotz and Guerinot 2006, Gómez-Galera et al 2010) Anti-nutritional factors like phytic acid, fibres and tannins further reduce the bio-availability of these minerals from dietary intakes by preventing their absorption in the intestine (White and Broadley 2005, Pfeiffer and McClafferty 2007) Furthermore, processes like polishing, milling and pearling of cereals make them even poorer in micronutrients (Welch and Graham 2004, Borg et al 2009) Strategies for Alleviating Micronutrient Malnutrition Dietary diversification, supplementation, fortification and biofortification of crop plants are the approaches proposed for alleviating micronutrient malnutrition (Zimmerman and Hurrel 2007, Stein 2010) All of the above approaches have their own pros and cons, and a right mix of all the intervention approaches has to be employed to overcome the problem of hidden hunger (Stein 2010) Dietary diversification and modification suffers from difficulty in the change of dietary habits of people and high costs of diets with readily bio-available iron and zinc content (Brinch-Pederson et al 2007, Zimmerman and Hurrel 2007) Supplementation refers to the oral delivery of micronutrients in the forms of tables and syrups, and this strategy has been used in chronic deficiencies For instance, ferrous fumarate, ferrous sulphate and ferrous gluconate are the best absorbed forms of iron Similarly, zinc can be supplied as zinc gluconate, zinc sulphate and zinc acetate Fortification is the addition of the desired minerals to food stuffs like iodine in salts, iron in flour, fluorine in toothpaste and zinc in flours (Rosado 2003, Gómez-Galera et al 2010) The major drawback of these approaches is that these compounds have limited stability in the food stuffs (Allen 2003) For instance, iron-fortified foods are susceptible to oxidation and also alter the taste of the food (Gómez-Galera et al 2010) Similarly, folate-fortified rice loses it while boiling owing to its increased solubility (Brinch-Pederson et al 2007) Furthermore, the absorption of oral supplementation also depends on the type of food ingested These approaches require recurring expenditure, robust distribution system and very careful imple- wileyonlinelibrary.com 438 mentation as overdose may also be harmful (Subbulakshmi and Naik 1999, Nantel and Tontisirin 2002, Nestel et al 2006) Biofortification refers to increasing genetically the bio-available mineral content of food crops (Bouis 2000, Brinch-Pederson et al 2007) Developing biofortified crops also improves their efficiency of growth in soils with depleted or unavailable mineral composition (Cakmak 2008, Borg et al 2009) Conventional breeding and genetic engineering techniques are the two approaches that may be used to biofortify the crops with minerals like iron and zinc (DellaPenna 1999, Johns and Eyzaguirre 2007, Pfeiffer and McClafferty 2007, Tiwari et al 2010) Cereals are the most important source of calories to humans Rice, wheat and maize provide about 23%, 17% and 10%, respectively, of the calories acquired globally (Khush 2003) To effectively target biofortification of cereals, five key steps can be targeted These are (i) enhanced uptake from soil, (ii) increased transport of micronutrients to grains, (iii) increased sequestration of minerals to endosperm rather than husk and aleurone, (iv) reduction in antinutritional factors in grains and (v) increase in promoters of mineral bioavailability in grains Enhancing mineral uptake from soil Plants have developed sophisticated mechanisms for uptake of minerals from soil as they are in forms which are not readily available although abundant (Hell and Stephan 2003, Colangelo and Guerinot 2006) The strategies used by plants to uptake metal ions from soils have been divided into two categories (Fig 1) The strategy-I plants include mostly dicots and nongraminaceous monocots These plants secrete protons into the rhizosphere and reduce Fe3+ to more soluble Fe2+ form by the ferric-chelate reductase activity of membrane-bound FRO2 protein FRO2 is a member of family of eight proteins in Arabidopsis and is expressed specifically in roots Other members (FRO6, FRO7, FRO8) however, take part in iron transport in shoots (Mukherjee et al 2006, Jeong and Connolly 2009, Palmer and Guerinot 2009) Solubility of Fe increases by 1000-fold for every unit decrease in the pH (Olsen et al 1981) Iron-regulated transporter (IRT) proteins then transport the readily soluble Fe2+ ions into the plant roots (Hell and Stephan 2003) IRT1 mediates uptake of Zn, Mn and Cd in addition to Fe (Rogers et al 2000, Curie and Briat 2003, Takahashi et al 2011) Strategy-II plants are mostly the members of Poaceae, and they secrete metal chelators called Phytosiderophores (PS) into the rhizosphere in con- N RAWAT, K NEELAM, V K TIWARI et al ditions of limiting mineral availability (Mori 1999) The metalPS complex is then taken up by the Yellow Stripe (YS1) transporter, which is an integral membrane protein of roots and shoots (Curie et al 2001) YS1 was identified and cloned in a maize mutant having interveinal chlorosis and defective in Fe-PS uptake (von Wiren et al 1994, Curie et al 2009) Rice is an exception to the general classification based on strategies of metal uptake because it has been found to express both IRT and YS1 (Ishimaru et al 2006) The phytosiderophores released by Strategy-II plants comprise of mugineic acid family derivatives, namely – mugineic acid (MA), 2′- deoxymugineic acid (DMA), 3-epihydroxymugineic acid (epi-HMA) and 3-epihydroxy-2-hydroxy mugineic acid (epi-HDMA; Mori 1999) S-adenosylmethionine is the precursor for MA synthesis Biosynthesis of MAs begins with trimerization of S-adenosylmethionine to nicotinamine (NA) by the activity of nicotinamine synthase (NAS) NA is then converted to a 3”-keto intermediate by nicotineamine aminotransferase (NAAT), which is then converted to DMA by deoxymugineic acid synthase (DMAS) DMA is subsequently converted to other forms depending upon the species (Bashir et al 2006) All the genes involved in the MA biosynthetic pathway have been cloned and characterized (Okumura et al 1994, Takahashi et al 1999, Nakanishi et al 2000, Higuchi et al 2001, Bashir et al 2006) Once synthesized in the roots under mineral deficient conditions, efflux of DMA in the rhizosphere is carried out by TOM1 transporter Overexpression of TOM1 led to increased DMA secretion in the rhizosphere, whereas its repession decreased DMA efflux (Nozoye et al 2011) Lee et al (2012) reported an increase in iron content in seeds by twofold by overexpressing OsNAS2 Ishimaru et al (2010) overexpressed OsYSL2 using phloem-specific OsSUT1 promoter and reported an increase of up to 4.4 times in iron content of polished rice Thus, metal acquisition from the soil can be increased by increasing the synthesis and release of phytosiderophores together with increased expression of genes encoding YSL proteins (Takahashi et al 2001, Douchkov et al 2005, Ishimaru et al 2010, Lee et al 2012) Barley secretes greatest amounts of MAs in the rhizosphere (Rümheld and Marschner 1990, Kanazawa et al 1994) Genetic variability was found in wild germplasm of wheat for amount of MA released in iron sufficient and iron deficient growth conditions (Neelam et al 2010) Neelam et al (2012) studied MA released in some wheat-Aegilops addition lines and found that group addition lines released higher MA in the rhizosphere not Fig 1: Strategies used by dicot (Strategy-I) and graminaceous (Strategy-II) plants for taking up minerals from soil FRO2- Ferric reductase oxidase, IRT- Iron regulated transporter, TOM1- After the author’s name- Tomoko Nozoye, YSL1- Yellow stripe like Source: Hell and Stephan 2003, Nozoye et al 2011 Biofortification of cereals only in iron deficient but also in iron sufficient growth conditions Interestingly, the higher MA was correlated with high grain iron and zinc concentrations in these addition lines This indicates that better uptake of minerals from the soil in these addition lines may be responsible for enhanced mineral content of grains Enhancing mineral deposition in grains The modern breeding practices have so far targeted improving yield potential of crops as the main objective due to which variability for other genetic traits got eroded Modern day cultivars of all major crops have limited variability of mineral (Graham et al 2001, Bouis 2003) The Consultative Group on International Agricultural Research (CGIAR) (http://www.cgiar.org) through its HarvestPlus (www.harvestplus.org) initiative has been exploring the genetic variability, heritability of mineral traits, stability over different environments, genetic studies and breeding strategies to enhance the mineral content in edible parts of crops – wheat, rice, maize, beans and cassava (CIAT/IFPRI 2002) Wild germplasm of crops has been found to harbour sufficient variability for improvement in mineral content (Cakmak et al 2000, Chavez et al 2005, Vreugdenhil et al 2005, Rawat et al 2009, White and Broadley 2009) which could be used for improvement in modern day varieties In rice, a fourfold difference was found in grain Fe and Zn content in some aromatic lines as compared to popular cultivars (Graham et al 1999, Gregorio et al 2000) In maize, Banziger and Long (2000) evaluated 1814 accessions in 13 trials over years and reported a range of 9.6–63.2 mg/kg of grain Fe and 12.9–57.6 mg/kg of grain Zn In beans, over 1000 genotypes of Centro Internacional de Agricultura Tropical (CIAT) core collection were screened and were found to have Fe content in range of 34–89 mg/kg and Zn in the range of 21–54 mg/kg (Graham et al 1999, Beebe et al 2000) In wheat, many studies exploring variation in the grain iron and zinc content in the old and modern wheat cultivars, wild germplasm, landraces have been done and wild relatives were found to contain three-fourfold higher grain iron and zinc content than the popular cultivars (Cakmak et al 2000, Gregorio et al 2000, Chhuneja et al 2006, Rawat et al 2009) Wild relatives have been used to transfer genes for biotic and abiotic stress tolerance and yield and quality improvement in cultivated varieties, and likewise, these can also be used to transfer useful variability for grain iron and zinc content using conventional and modern breeding approaches (Stalker 1980, Chhuneja et al 2008) Oury et al (2006) studied G E interactions in wheat cultivars for iron, zinc and magnesium concentrations and reported genotypes to have higher effect than environment In rice, genetic analysis for grain Fe and Zn was done in populations of aromatic-high-Fe and popular-low-Fe varieties The analysis of variance indicated that although environment affects the mineral content, the genetic component was more profound in determining the Fe, Zn status of the grains Three groups of high Fe genes were mapped to rice chromosomes 7, and contributing to 19–30% of the variation in Fe content (Gregorio et al 2000) Tiwari et al (2009) mapped three QTL for grain iron and zinc content on chromosomes and in a diploid wheat RIL population Peleg et al (2009) also reported three major QTL for grain iron and zinc concentrations in a Triticum durum–T dicoccoides RIL population where the QTL common for zinc and iron on chromosome 7A was in the same marker interval as in Tiwari et al (2009) Chromosomes and 439 of wild wheats – Aegilops kotschyi, Ae peregrina and Ae longissima have also been found to carry genes for high grain iron and zinc content (Tiwari et al 2010, Neelam et al 2011, Rawat et al 2011) Tiwari et al (2010) produced addition and substitution lines of 2S and 7U of wheat and found an increase of 116% and 136% in grain iron and zinc content, respectively, over cultivated varieties Uauy et al (2006) cloned a high grain protein (Gpc-B1) locus from a wild wheat T turgidum ssp dicoccoides and reported that it promotes senescence and simultaneously increases mobilization of iron and zinc from leaves to grains Genetic engineering to increase the mineral content in grains, by overexpressing metal-storage proteins like lactoferrin and ferritin, has also been attempted by several workers Ferritin is an iron storage protein molecule consisting of 24 subunit shell around a core of up to 4500 Fe atoms (Thiel and Briat 2004) Introduction of soybean ferritin gene into rice and wheat using maize ubiquitin promoter led to increase in iron content of leaves, but decrease in grain iron content Drakakaki et al (2000) The reason for this observation is that leaves act as a stronger sink for the iron than the seeds Goto et al (1999) transformed rice with soybean ferritin under rice storage protein glutelin promoter (GluB-1) and reported a threefold increase in grain iron content as compared to non-transformed lines Qu et al (2005) introduced soybean ferritin into rice with very strong endospermspecific globulin promoter, and this led to an increase of up to 13 times in ferritin protein expression than in Goto et al (1999) However, the actual increase in iron content was not concomitant, being a mere 30% enhancement Furthermore, the iron content in leaves decreased to as low as 10% of the nontransformed plants and was accompanied by chlorosis Similar observations were made in wheat (Borg et al 2009) This was because the ferritin under endosperm-specific promoter led to increased translocation to seeds from leaves without a simultaneous increase in iron uptake from soil by roots Attempts are being made to improve the concurrent uptake by the plants from soil in addition to improving ferritin expression in endosperm (Borg et al 2009) Agronomic strategies to enhance mineral content of cereal crops involve application of micronutrient fertilizers to the plants in readily phytoavailable state, correcting soil alkalinity, adopting crop rotation practices or introducing beneficial soil microorganisms (Rengel et al 1999, Fageria 2009, White and Broadley 2011) The most attractive agronomic strategy of biofortification is foliar application of mineral fertilizers to the plants in readily phytoavailable state However, iron fertilization has met with limited success in biofortification because the applied Fe2+ gets rapidly oxidized to Fe3+ state, which is not absorbed by the plants Foliar spray of ferrous compounds resulted in reduction in chlorosis and a little increase in yield in sorghum and some other crops (Zhang et al 2008) Chelated forms of Fe show better absorption by roots and foliage, but their large scale application is economically not feasible (Brinch-Pederson et al 2007) Zn fertilization in the form of ZnSO4, ZnO and synthetic Zn-chelates results in increase in fruits and seeds of crops (Rengel et al 1999, Cakmak 2008, White and Broadley 2009) Grain Zn concentration has been found to be correlated with grain protein concentration, and thus higher N-fertilization increases Zn concentration to an extent, but after that it is saturated (Gomez-Becerra et al 2010, White and Broadley 2011) Agronomic biofortification has met with limited popularity in cereals because of the recurring expenditure and need for careful time-dependent applications of fertilizers 440 Sequestration of minerals to endosperm Most of the mineral content present in the grains is confined to the aleurone layer and embryo, which is removed during milling and processing in major crops like wheat and rice A useful approach towards increasing the iron and zinc status of cerealbased diets would be to increase the sequestration of minerals to the endosperm portion of the grains Metal transporters involved in uptake from soil and long distance transport from roots to shoot have been studied in detail, but little is known about transporters facilitating distribution of minerals across the grains (Mori 1999, Colangelo and Guerinot 2006, Curie et al 2009, Grennan 2009, Palmer and Guerinot 2009, Puig and Pen~arrubia 2009, Conn and Gilliham 2010) Recently, with the development of techniques like laser capture microdissection, it has been possible to study the expression of different genes across different issues of the grains (Borg et al 2009, Tauris et al 2009, Schiebold et al 2011) In a beautiful study, Tauris et al (2009) examined zinc transport across different tissues of the developing barley grain They isolated transfer cells, aleurone layer, endosperm and embryo of developing barley grain using laser capture microdissection and studied the expression of various zinc transporters by microarray (Fig 2) Transfer cells are the interface between phloem of the maternal tissue and the endosperm tissue and have numerous wall ingrowths increasing the surface area by more than 20-fold Members of Heavy Metal ATPases (HMA), Zinc-regulated Iron-regulated Protein (ZIP), Cation Diffusion Facilitator (CDF), Natural resistance-associated macrophage proteins (Nramp), Vacuolar Iron Transporter (VIT1), CAX (Cation Exchanger), YSL (Yellow Stripe Like), Metallothioneins (MT), NAS (Nicotinamine Synthase) and NAAT (Nicotinamine amino transferase) are highly expressed in transfer cells It has been proposed that Zn combines with NA (Nicotinamine) or MA (Mugineic acid) and then is taken up from the phloem and stored in vacuoles by CDF, VIT1, ZIP1 and CAX family transporters YSL and ZIP transporters have been proposed to capture zinc-NA complexes from flowing back to the apoplast The aleurone and embryo N RAWAT, K NEELAM, V K TIWARI et al expression profiles have a lot of resemblance HMA8, ZIP and CDF are expressed in both the tissues at about the same level, although Nramp3, ZIF1, CAX1a, VIT1_2, NAS9 and NAATB are expressed at a higher rate in aleurone than in embryo Nramp3 has been proposed to mediate efflux of Zn from the aleurone cells, while others control movement to the aleurone cells where it is stored chiefly in vacuoles Expression of transporter genes in the endosperm tissue is limited Borg et al (2009) proposed a similar but preliminary roadmap for transport of iron across developing barley grain (Fig 2) There are several similarities in the two pathways More detailed studies are required to further delineate specific transporter for Fe movement from transfer cells to the endosperm cavity Ramesh et al (2004) overexpressed Zn transporter AtZIP1 of Arabidopsis in barley under a ubiquitin promoter The transgenic lines produced smaller seeds with high Zn concentration Manipulating the expression of genes regulating CAX transporters has been proposed as an approach to increase Zn concentrations in the edible tissues of transgenic plants (Shigaki et al 2005) Reducing the antinutritional factors in cereals To achieve effective availability of minerals from cereal-based diets, it is essential to make it easily absorbable in the intestines, a task which is made difficult by the antinutritional factors like phytic acid, tannins, lignans etc present in cereals (Welch and House 1984) Phytic acid (1,2,3,4,5,6)-hexakisphosphate is a chief storage form of phosphorus in seeds and accounts for about 1% of seed weight (Lott et al 2000) In ionic form, it has dense negative charge around it due to which it strongly binds to metal cations in the seeds and makes a stable phytate-metal complex Phytic acid is considered to be the most important anti-nutritional factor in food (Bouis 2000) Monogastric animals and humans not have specific enzymes (Phytases) for breaking down this phytate-metal complex in their guts and so the minerals present in their diets are not available for absorption in them (Raboy et al 2000) In fact, only 5% of Fe and 25% of Zn Fig 2: Generalized roadmap for iron and zinc sequestration in a developing grain Fe- iron, Zn- zinc, NA- nicotinamine, MA- mugineic acid, P- phytate, VIT- Vacuolar Iron Transporter, CAX- Cation Exchanger, Nramp- Natural resistance-associated macrophage proteins, HMA- Heavy metal ATPase, YSL -Yellow Stripe Like, ZIP- Zinc-regulated Iron-regulated Protein Source: Borg et al 2009, Tauris et al 2009 Biofortification of cereals present in legume and cereal-based diets is bioavailable (Pfeiffer and McClafferty 2007) Numerous attempts have been made to reduce phytic acid content of the cereal grains Transgenic crops with microbial phytases is one such approach Brinch-Pederson et al (2000) introduced Aspergillus niger phyA gene in wheat under the maize ubiquitin-1 promoter, which resulted in significantly increased seed phytase activity Phytases from Escherichia coli, Selenimonas ruminatum, A fumigatus and Schwanniomyces occidentalis have been used subsequently in cereals (Lucca et al 2001, Hong et al 2004, Hamada et al 2005) Drakakaki et al (2005) reported 95% reduction in phytic acid and concomitant increase in bioavailability of iron by co-expressing recombinant soybean ferritin and A niger phytase in maize under endosperm-specific rice glutelin-1 promoter Chen et al (2008) transferred A niger phyA2 gene to maize under maize embryospecific globulin-1 promoter, resulting in a 50-fold increase over the non-transgenic maize Transgenic approach to increase phytase, however, suffers from the bottleneck of phytase being nontolerant to heat As such, phytases in transgenic cereal grains tend to lose their activity while cooking and processing Dai et al (2011) have cloned barley phytase, embryo-specific overexpression of which can be used to generate barley lines with reduced phytic acid Significant genetic variation has been reported for grain phytate concentration in rice (Glahn et al 2002), wheat (Raboy et al 1991, Welch et al 2005), barley (Dai et al 2007), pearl millet (Abdalla et al 1998), oat (Lolas et al 1976), triticale (Feil and Fossati 1997) and Sorghum (Reddy et al 2005); but attempts to use genetic variability for lowering phytic acid in crops are limited An alternative strategy to lower phytic acid content of seeds has been to manipulate the biosynthetic pathway of phytic acid (Stephens and Irvine 1990, Brinch-Pedersen et al 2002) Low phytic acid (lpa) mutants have been reported in maize (Raboy et al 2000), rice (Larson et al 2000), soybean (Wilcox et al 2000), barley (Rasmussen and Hatzack 1998) and wheat (Guttieri et al 2004) These lpa mutants have low phytate and high inorganic phosphorus content in their seeds Kuwano et al (2008) used antisense RNA for RINO1gene, which controls myo-inositol phosphate synthase activity, under aleurone-specific Ole18 promoter The transgenic rice had 68% lower phytic acid levels than the wild type, which was even lower than the mutant lpa rice Greater mineral absorption has been reported from lpa mutants in corn, rice, barley and soybean than in the conventional varieties in monogastric animals and humans (Mendoza et al 1998, Mendoza 2002) Salunke et al (2012) studied the bioavailability of zinc in a diploid wheat (T monococcum) lpa mutant using Caco2 cell lines and found that it actually had higher bioavailability than wild type The agronomic performance of the lpa mutants is variable (Bregitzer and Raboy 2006, Guttieri et al 2006, Raboy 2007) Shi et al (2007) developed maize and soybean lines with embryo-specific gene silencing of a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter These lines had normal seed dry weight and germination rate showing that this may be an agronomically feasible strategy to produce commercial lpa lines of crops QTL mapping for phytate content has been done in rice (Stangoulis et al 2007), soybean (Walker et al 2006) and bean (Cichy et al 2009), and the loci affecting phytic acid have been found to be different than grain micronutrient content, suggesting that it would be possible to enhance the micronutrient content simultaneously 441 along with decreasing the phytic acid content (White and Broadley 2009) Enhancing the promoters of mineral bioavailability As opposed to antinutritional compounds like phytic acid and polyphenols, there are compounds like ascorbic acid, b-carotene and inulin that enhance the absorption of minerals in gut (Roberfroid 2007, White and Broadley 2009) Ascorbic acid reduces Fe3+ ions to Fe2+ ions which being more soluble are readily absorbed in the gut (Ballot et al 1987) Similarly, b-carotene also increases the solubility of Fe, making it more bioavailable (Garcia-Casal et al 1998) The provitamin-A activity of carotenoids has been suggested to play a role in improving iron absorption in vitro and in human studies, although the exact mechanism is unknown (Garcia-Casal 2006) Inulin is a polysaccharide formed of b(2-1) linked fructose monomers Having a prebiotic effect, inulin promotes the growth of useful lactobacilli and bifidobacteria, which produce short-chain fatty acids (SCFA) These SCFA lower the pH of the gut and increase the solubility of minerals, making them more bioavailable (Abrams et al 2007, Jenkins et al 2011) Significant genetic variation has been reported for b-carotene in wheat, rice (Welch and Graham 2005, Howitt and Pogson 2006, Abdel-Aal et al 2007), maize (Ortiz-Monasterio et al 2007, Harjes et al 2008), sorghum (Reddy et al 2005) and pearl millet (Kapoor and Naik 1970) Significant breakthroughs have been made in genetic engineering attempts to increase b-carotene Ye et al (2000) produced golden rice with increased b-carotene content by introducing the phytoene synthase (psy) gene from daffodil and a bacterial phytoene desaturase (crtI) gene from Erwinia uredovora under the control of endosperm-specific glutelin (Gt1) and the constitutive CaMV (cauliflower mosaic virus) 35S promoter, respectively Paine et al (2005) developed golden rice-2 with 23-fold higher total carotenoids compared with the original golden rice (Ye et al 2000) by introducing the psy gene from maize which led to increased carotenoid accumulation, which was limiting in the original golden rice In maize, Aluru et al (2008) overexpressed bacterial genes crtB and crtI, under the control of endosperm-specific ‘super gamma-zein promoter’ leading to a 34fold increase in carotenoid (mainly) b-carotene level Chen et al (2003) reported a 2–4-fold increase in the ascorbic acid levels in maize and tobacco by overexpressing a wheat dehydroascorbate reductase (DHAR), an enzyme regenerating ascorbic acid from dehydroascorbate Naqvi et al (2009) manipulated three separate metabolic pathways in maize to simultaneously increase the levels of b-carotene, ascorbate and folate, by 169-fold, 6-fold and twice, respectively, over non-transformed control Genetic variation has been studied for inulin concentration in wheat, maize, barley and rye (Shelton and Lee 2000) Five QTL for fructan accumulation were mapped on wheat chromosomes 2B, 3B, 5A, 6D and 7A by Huynh et al (2008) The QTL on 6D and 7A contributed to the largest phenotypic variance of 17% and 27%, respectively A barley mutant (M292) was reported to lower plasma cholesterol and enhanced short-chain fatty acids in the guts of rats and pigs (Bird et al 2004a,b) Clarke et al (2008) reported that a mutation in Starch synthase (SSIIa) gene in M292-enhanced free sugars, b-glucans and arabinoxylans also increased inulin content (4.2 mg/kg) by 42-fold compared with the wild type variety (0.1 mg/kg) However, more investigations are needed to propose SSIIa manipulation as a generalized strategy for increasing inulin content 442 Conclusions and Future Directions Biofortification of crops is a feasible and most economical approach for overcoming ‘hidden hunger’ Increasing the concentration of minerals in edible portions of cereals involves better uptake from soil and improved translocation to grains from leaves and finally enhanced sequestration to endosperm Genetic diversity can be utilized to enhance micronutrient composition through conventional and modern breeding approaches At the same time genetic engineering approaches can progress based on increased understanding of metabolic pathways and expression patterns of metal transporters, chelators and associated compounds The most promising work plan to successfully alleviate micronutrient malnutrition will be to increase mineral content in the crops and simultaneously enhance their bioavailability by reducing antinutritional compounds and/or enhancing concentration of mineral absorption promoters To effectively combat hidden hunger through biofortification, even after the development of biofortified varieties, it will be essential to address various socio-economical and socio-political challenges to popularize their cultivation by farmers and ultimately their consumption by the end users A multi-tier coordinated strategy will play a pivotal role in overcoming hidden hunger Conflict of Interests Authors declare no conflicts of interest References Abdalla, A A., A H E Tinay, B E Mohamed, and A H Abdalla, 1998: Proximate composition, starch, phytate and mineral contents of 10 pearl millet genotypes Food Chem 63, 243—246 Abdel-Aal, El.-S., J C Young, I Rabalski, P Hucl, and J FregeauReid, 2007: Identification and quantification of seed carotenoids in selected wheat species J Agric Food Chem 55, 787—794 Abrams, S A., K M Hawthorne, O Aliu, P D Hicks, C Chen, and I J Griffin, 2007: An Inulin-type fructan enhances calcium absorption primarily via an effect on colonic absorption in humans J Nutr 137, 2208—2212 Allen, L H., 2003: Interventions for micronutrient deficiency control in developing countries: past, present and future J Nutr 133, 3877S— 3878S Aluru, M., Y Xu, R Guo, Z Wang, S Li, W White, K Wang, and S Rodermel, 2008: Generation of transgenic maize with enhanced provitamin A content J Exp Bot 59, 3551—3562 Ballot, D., R D Baynes, T H Bothwell, M Gillooly, B J Macfarlane, A P Macphail, G Lyons, D P Dermon, W R Bezwoda, J D Torrance, J D Torrance, J E Bothwell, and F Mayet, 1987: The effects of fruit juices and fruits on the absorption of iron from a rice meal Br J Nutr 57, 331—343 Banziger, M., and J Long, 2000: The potential for increasing the iron and zinc density of maize through plant-breeding Food Nutr Bull 21, 397—400 Bashir, K., H Inoue, S Nagasaka, M Takahashi, H Nakanishi, S Mori, and N K Nishizawa, 2006: Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants J Biol Chem 281, 32395—32402 Beebe, S., A Gonzalez, and J Rengifo, 2000: Research on trace minerals in the common bean Food Nutr Bull 21, 387—391 Bird, A R., C Flory, D A Davies, S Usher, and D L Topping, 2004a: A novel barley cultivar (Himalaya 292) with a specific gene mutation in starch synthase IIa raises large bowel starch and shortchain fatty acids in rats J Nutr 134, 831—835 Bird, A R., M Jackson, R A King, D A Davies, S Usher, and D L Topping, 2004b: A novel high-amylose barley cultivar (Hordeum vulg- N RAWAT, K NEELAM, V K TIWARI et al are var Himalaya 292) lowers plasma cholesterol and alters indices of large-bowel fermentation in pigs Br J Nutr 92, 607—615 Borg, S., H Brinch-Pedersen, B Tauris, and P B Holm, 2009: Iron transport, deposition and bioavailability in the wheat and barley grain Plant Soil 325, 15—24 Bouis, H E., 2000: Special issue on improving human nutrition through agriculture Food Nutr Bull 21, 351—576 Bouis, H E., 2003: Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proc Nutr Soc 62, 403—411 Bregitzer, P., and V Raboy, 2006: Effects of four independent low-phytate mutations on barley agronomic performance Crop Sci 46, 1318—1322 Brinch-Pedersen, H., L D Sørensen, and P B Holm, 2002: Engineering crop plants: getting a handle on phosphate Trends Plant Sci 7, 118—125 Brinch-Pederson, H., A Olesen, S K Rasmussen, and P B Holm, 2000: Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase Mol Breed 6, 195—206 Brinch-Pederson, H., S Borg, B Tauris, and P B Holm, 2007: Molecular genetic approaches to increasing mineral availability and vitamin content of cereals J Cereal Sci 46, 308—326 Cakmak, I., 2008: Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant Soil 302, 1—17 Cakmak, I., H Ozkan, H J Braun, R M Welch, and V Romheld, 2000: Zinc and iron concentrations in seeds of wild, primitive, and modern wheats Food Nutr Bull 21, 401—403 Chavez, A L., T Sanchez, G Jaramillo, J M Bedoya, J Echeverry, E A Bola~nos, H Ceballos, and C A Iglesias, 2005: Variation of quality traits in cassava roots evaluated in landraces and improved clones Euphytica 143, 125—133 Chen, Z., T E Young, J Ling, S C Chang, and D R Gallie, 2003: Increasing vitamin C content of plants through enhanced ascorbate recycling Proc Natl Acad Sci USA 100, 3525—3530 Chen, R., G Xue, P Chen, B Yao, W Yang, Q Ma, Y Fan, Z Zhao, M C Tarczynski, and J Shi, 2008: Transgenic maize plants expressing a fungal phytase gene Transgenic Res 17, 633—643 Chhuneja, P., H S Dhaliwal, N S Bains, and K Singh, 2006: Aegilops kotschyi and Aegilops tauschii as sources for higher levels of grain iron and zinc Plant Breeding 125, 529—531 Chhuneja, P., S Kaur, R K Goel, M Aghaee-Sarbarzeh, M Prashar, and H S Dhaliwal, 2008: Transfer of leaf rust and stripe rust resistance from Aegilops umbellulata Zhuk to bread wheat (Triticum aestivum L.) Genet Res Crop Evol 55, 849—849 CIAT/IFPRI, 2002: Biofortified Crops for Improved Human Nutrition A Challenge Program Proposal Consultative Group on International Agricultural Research, Washington Available at: http://www.cgiar.org/ pdf/biofortification.pdf (last accessed on Feburary 26, 2012) Cichy, K A., G V Caldas, S S Snapp, and M W Blair, 2009: QTL analysis of seed iron, zinc, and phosphorus levels in an Andean bean population Crop Sci 49, 1742—1750 Clarke, B., R Liang, M K Morell, A R Bird, C L D Jenkins, and Z Li, 2008: Gene expression in a starch synthase IIa mutant of barley: changes in the level of gene transcription and grain composition Funct Integr Genomics 8, 211—221 Colangelo, E P., and M L Guerinot, 2006: Put the metal to the petal: metal uptake and transport throughout plants Curr Opin Plant Biol 9, 322—330 Conn, S., and M Gilliham, 2010: Comparative physiology of elemental distributions in plants Annal Bot 105, 1081—1102 Curie, C., and J F Briat, 2003: Iron transport and signaling in plants Annu Rev Plant Biol 54, 183—206 Curie, C., Z Panaviene, C Loulergue, S L Dellaporta, J F Briat, and E L Walker, 2001: Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake Nature 409, 346—349 Curie, C., G Cassin, D Couch, F Divol, K Higuchi, M L Jean, J Misson, A Schikora, P Czernic, and S Mari, 2009: Metal movement within the plant: contribution of nicotinamine and yellow stripe 1-like transporters Annal Bot 103, 1—11 Biofortification of cereals Dai, F., J M Wang, S H Zhang, Z Z Xu, and G P Zhang, 2007: Genotypic and environmental variation in phytic acid content and its relation to protein content and malt quality in barley Food Chem 105, 606—611 Dai, F., L Qiu, L Ye, D Wu, M Zhou, and G Zhang, 2011: Identification of a Phytase Gene in Barley (Hordeum vulgare L.) PLoS ONE 6, e18829 DellaPenna, D., 1999: Nutritional genomics: manipulating plant micronutrients to improve human health Science 285, 375—379 Douchkov, D., C Gryczka, U W Stephan, R Hell, and H Baumlein, 2005: Ectopic expression of nicotinamine synthase genes results in improved iron accumulation and increase nickel tolerance in transgenic tobacco Plant, Cell Environ., 28, 365—374 Drakakaki, G., P Christou, and E Stoger, 2000: Constitutive expression of soybean ferritin cDNA in transgenic wheat and rice results in increased iron levels in vegetative tissues but not in seeds Transgen Res 9, 445—452 Drakakaki, G., S Marcel, R P Glahn, E K Lund, S Pariagh, R Fischer, P Christou, and E Stoger, 2005: Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron Plant Mol Biol 59, 869—880 Fageria, N K., 2009: The Use of Nutrients in Crop Plants CRCPress, Boca Raton, FL FAO, 2004: Cereals and other starch-based staples: are consumption patterns changing? Feil, B., and D Fossati, 1997: Phytic acid in triticale grains as affected by cultivar and environment Crop Sci 37, 916—921 Garcia-Casal, M N., 2006: Carotenoids increase iron absorption from cereal-based food in the human Nutr Res 26, 340—344 Garcia-Casal, M N., M Layrisse, L Solano, M A Baron, F Arguello, D Llovera, J Ramirez, I Leets, and E Tropper, 1998: Vitamin A and beta-carotene can improve non-heme iron absorption from rice, wheat and corn by humans J Nutr 128, 646—650 Gibson, R S., 2006: Zinc: the missing link in combating micronutrient malnutrition in developing countries Proc Nutr Soc 65, 51—60 Glahn, R P., S Q Chen, R M Welch, and G B Gregorio, 2002: Comparison of iron bioavailability from 15 rice genotypes: Studies using an in vitro digestion/caco-2 cell culture model J Agric Food Chem 50, 3586—3591 Gómez-Galera, S., E Rojas, D Sudhakar, C Zhu, A M Pelacho, T Capell, and P Christou, 2010: Critical evaluation of strategies for mineral fortification of staple food crops Transgenic Res 19, 165— 180 Gómez-Becerra, H F., H Erdem, A Yazici, Y Tutus, B Torun, L Ozturk, and I Cakmak, 2010: Grain concentrations of protein and mineral nutrients in a large collection of spelt wheat grown under different environments J Cereal Sci 52, 342—349 Goto, F., T Yoshihara, N Shigemoto, S Toki, and F Takaiwa, 1999: Iron fortification of rice seed by the soybean ferritin gene Nat Biotechnol 17, 282—286 Graham, R., D Senadhira, S Beebe, C Iglesias, and I Monasterio, 1999: Breeding for micronutrient density in edible portions of staple food crops: conventional approaches Field Crop Res 60, 57—80 Graham, R D., R M Welch, and H E Bouis, 2001: Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: principles, perspectives and knowledge gaps Advance Agron 70, 77—142 Gregorio, G B., D Senadhira, T Htut, and R D Graham, 2000: Breeding for trace mineral density in rice Food Nutr Bull 21, 382—386 Grennan, A K., 2009: Identification of genes involved in metal transport in plants Plant Physiol 149, 1623—1624 Grotz, N., and M L Guerinot, 2006: Molecular aspects of Cu, Fe and Zn homeostasis in plants Biochim Biophys Acta 1763, 595—608 Guttieri, M., D Bowen, J A Dorsch, V Raboy, and E Souza, 2004: Identification and characterization of a low phytic acid wheat Crop Sci 44, 418—424 Guttieri, M J., K M Peterson, and E J Souza, 2006: Agronomic performance of low phytic acid wheat Crop Sci 46, 2623—2629 443 Haas, J D., and T IV Brownlie, 2001: Iron deficiency and reduced work capacity: a critical review of the research to determine a causal relationship J Nutr 131, 691S—696S Hamada, A., K Yamaguchi, N Ohnishi, M Harada, S Nikumaru, and H Honda, 2005: High-level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene Plant Biotechnol J 3, 43—55 Harjes, C E., T R Rocheford, L Bai, T P Brutnell, C B Kandianis, S G Sowinski, A E Stapleton, R Vallabhaneni, M Williams, E T Wurtzel, J B Yan, and E S Buckler, 2008: Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification Science 319, 330—333 Hell, R., and U W Stephan, 2003: Iron uptake, trafficking and homeostasis in plants Planta 216, 541—551 Higuchi, K., S Watanabe, M Takahashi, S Kawasaki, N Hiromi, N K Nishizawa, and S Mori, 2001: Nicotinamine synthase gene expression differs in barley and rice under Fe-deficient conditions Plant J 25, 159—167 Hong, C Y., K J Cheng, T H Tseng, C S Wang, L F Liu, and S M Yu, 2004: Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds Transgenic Res 13, 29—30 Hotz, C., and K H Brown, 2004: Assessment of the risk of zinc deficiency in populations and options for its control Food Nutr Bull 25, S91—S204 Howitt, C A., and B J Pogson, 2006: Carotenoid accumulation and function in seeds and nongreen tissues Plant, Cell Environ 29, 435— 445 Huynh, B.-L., H Wallwork, J C R Stangoulis, R D Graham, K L Willsmore, S Olson, and D E Mather, 2008: Quantitative trait loci for grain fructan concentration in wheat (Triticum aestivum L.) Theor Appl Genet 117, 701—709 Ishimaru, Y., M Suzuki, T Tsukamoto, K Suzuki, M Nakazono, T Kobayashi, Y Wada, S Watanabe, S Matsuhashi, M Takahashi, H Nakanishi, S Mori, and N K Nishizawa, 2006: Rice plants take up iron as an Fe3-phytosiderophore and as Fe2 Plant J 45, 335—346 Ishimaru, Y., H Masuda, K Bashir, H Inoue, T Tsukamoto, M Takahashi, H Nakanishi, N Aoki, T Hirose, R Ohsugi, and N K Nishizawa, 2010: Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese Plant J 62, 379—390 Jenkins, C L D., D Lewis, R Bushell, D P Belobrajdic, and A R Bird, 2011: Chain length of cereal fructans isolated from wheat stem and barley grain modulates in vitro fermentation J Cereal Sci 53, 188—191 Jeong, J., and E L Connolly, 2009: Iron uptake mechanisms in plants: functions of the FRO family of ferric reductases Plant Sci 176, 709—714 Johns, T., and P B Eyzaguirre, 2007: Biofortification, biodiversity and diet: a search for complementary applications against poverty and malnutrition Food Policy 32, 1—24 Kanazawa, K., K Higuchi, N K Nishizawa, S Fushiya, M Chino, and S Mori, 1994: Nicotianamine aminotransferase activities are correlated to the phytosiderophore secretions under Fe-deficient conditions in Gramineae J Exp Bot 45, 1903—1906 Kapoor, H C., and M S Naik, 1970: Effects of soil and spray applications of urea and storage on the b-carotene content of yellow endosperm sorghum and pearl millet grains Indian J Agric Sci 40, 942—947 Khush, G S., 2003: Productivity improvements in rice Nutr Rev 61, S114—S116 Kim, S A., and M L Geurinot, 2007: Mining iron: iron uptake and transport in plants FEBS Letter 581, 2273—2280 King, J C., and C L Keen, 1999: Zinc In: M E Shills, J A S Olsem, M Shike, and A C Ross (eds), Modern Nutrition in Health and Disease, 9th edn, 223—239 Williams and Wilkins, Baltimore Kuwano, M., T Mimura, F Takaiwa, and K T Yoshida, 2008: Generation of stable ‘low phytic acid transgenic rice through antisense 444 repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter Plant Biotechnol J 7, 96—105 Larson, S R., J N Rutger, K A Young, and V Raboy, 2000: Isolation and genetic mapping of a non-lethal rice (Oryza sativa L.) low phytic acid mutation Crop Sci 40, 1397—1405 Lee, S., Y.-S Kim, U S Jeon, Y.-K Kim, J K Schjoerring, and G An, 2012: Activation of rice nicotinamine synthase (OsNAS2) enhances iron availability for biofortification Mol Cell 3, 33 Lolas, G M., N Palamidis, and P Markakis, 1976: The phytic acid–total phosphorus relationship in barley, oats, soybeans, and wheat Cereal Chem 53, 867—871 Lott, J N A., I Ockenden, V Raboy, and G D Batten, 2000: Phytic acid and phosphorus in crop seeds and fruits: a global estimate Seed Sci Res 10, 11—33 Lucca, P., R Hurrell, and I Potrykus, 2001: Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains Theor Appl Genet 102, 392—397 Mendoza, C., 2002: Effect of genetically modified low phytic acid plants on mineral absorption Int J Food Sci Technol 37, 759—767 Mendoza, C., F E Viteri, B L€onnerdal, K A Young, V Raboy, and K H Brown, 1998: Effect of genetically modified, low-phytic acid maize on absorption of iron from tortillas Am J Clin Nutr 68, 1123—1128 Mori, S., 1999: Iron acquisition by plants Curr Opin Plant Biol 2, 250—253 Mukherjee, I., N H Campbell, J S Ash, and E L Connolly, 2006: Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper Planta 223, 1178—1190 Nakanishi, H., H Yamaguchi, T Sasakuma, N K Nishizawa, and S Mori, 2000: Two dioygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores Plant Mol Biol 44, 199—207 Nantel, G., and K Tontisirin, 2002: Policy and sustainability issues J Nutr 132, S839—S844 Naqvi, S., C Zhu, G Farre, K Ramessar, L Bassie, J Breitenbach, D P Conesa, G Ros, G Sandmann, T Capell, and P Christou, 2009: Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways Proc Natl Acad Sci USA 106, 7762—7767 Neelam, K., V K Tiwari, N Rawat, S K Tripathi, G S Randhawa, and H S Dhaliwal, 2010: Identification of Aegilops species with higher production of phytosiderophore and iron and zinc uptake under micronutrient-sufficient and -deficient conditions Plant Genet Resour Charac Utiliz., 8, 132—141 Neelam, K., N Rawat, V K Tiwari, S Malik, S K Tripathi, G S Randhawa, and H S Dhaliwal, 2011: Molecular and cytological characterization of high grain iron and zinc Wheat- Aegilops peregrina derivatives Mol Breed 28, 623—634 Neelam, K., N Rawat, V K Tiwari, S K Tripathi, G S Randhawa, and H S Dhaliwal, 2012: Evaluation and identification of wheat- Aegilops addition lines controlling high grain iron and zinc content and mugineic acids production Cer Res Commun 40, 53— 61 Nestel, P., H E Bouis, J V Meenakshi, and W Pfeiffer, 2006: Biofortification of staple food crops J Nutr 136, 1064—1067 Nozoye, T., S Nagasaka, T Kobayashi, S Takahashi, Y Sato, Y Sato, N Uozumi, H Nakanishi, and N K Nishizawa, 2011: Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants J Biochem 286, 5446—5454 Okumura, N., N K Nishizawa, Y Umehara, T Ohata, H Nakanishi, T Yamaguchi, M Chino, and S Mori, 1994: A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare Plant Mol Biol 25, 705—719 Olsen, R A., R B Clark, and J H Bennet, 1981: The enhancement of soil fertility by plant roots Am Sci 69, 378—384 Ortiz-Monasterio, J I., N Palacios-Rojas, E Meng, K Pixley, R Trethowan, and R J Pe~ na, 2007: Enhancing the mineral and vitamin content N RAWAT, K NEELAM, V K TIWARI et al of wheat and maize through plant breeding J Cereal Sci 46, 293— 307 Oury, F X., F Leenhardt, C Remesy, E Chanliaud, B Duperrier, F Balfourier, and G Charmet, 2006: Genetic variability and stability of grain magnesium, zinc and iron concentrations in bread wheat Eur J Agron 25, 177—185 Paine, J A., C A Shipton, S Chaggar, R M Howells, M J Kennedy, G Vernon, S Y Wright, E Hinchliffe, J L Adams, A L Silverstone, and R Drake, 2005: Improving the nutritional value of Golden Rice through increased pro-vitamin A content Nat Biotechnol 23, 482—487 Palmer, C M., and M L Guerinot, 2009: Facing the challenges of Cu, Fe and Zn homeostasis in plants Nat Chem Biol 5, 333— 340 Peleg, Z., I Cakmak, L Ozturk, A Yazici, Y Jun, H Budak, A B Korol, T Fahima, and Y Saranga, 2009: Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat x wild emmer wheat RIL population Theor Appl Genet 119, 353—369 Pfeiffer, W H., and B McClafferty, 2007: HarvestPlus: breeding crops for better nutrition Crop Sci 47, S88—S105 Prasad, A S., 2003: Zinc deficiency, Has been known of for 40 years but ignored by global health organisations Br Med J 326, 409—410 Prasad, A S., J A Halsted, and M Nadimi, 1961: Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia Am J Med 31, 532—546 Puig, S., and L Pen~arrubia, 2009: Placing metal micronutrients in context: transport and distribution in plants Curr Opin Plant Biol 12, 299—306 Qu, L Q., T Yoshihara, A Ooyama, F Goto, and F Takaiwa, 2005: Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds Planta 222, 225—233 Raboy, V., 2007: Seed phosphorus and the development of low-phytate crops In: Turner B.L., A E Richardson, and E J Mullaney (eds), Inositol Phosphates: Linking Agriculture and the Environment, 111— 132 CABI, Wallingford, UK Raboy, V., M M Noaman, G A Taylor, and S G Pickett, 1991: Grain phytic acid and protein are highly correlated in winter wheat Crop Sci 31, 631—635 Raboy, V., P F Gerbasi, K A Young, S D Stoneberg, S G Pickett, A T Bauman, P P N Murthy, W F Sheridan, and D S Ertl, 2000: Origin and seed phenotype of maize low phytic acid 1–1 and low phytic acid 2–1 Plant Physiol 12, 355—368 Ramesh, S A., S Choimes, and D P Schachtman, 2004: Over-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases shortterm zinc uptake after zinc deprivation and seed zinc content Plant Mol Biol 54, 373—385 Rasmussen, S K., and F Hatzack, 1998: Identification of two low-phytate barley (Hordeum vulgare L.) grain mutants by TLC and genetic analysis Hereditas 129, 107—112 Rawat, N., V K Tiwari, N Singh, G S Randhawa, K Singh, P Chhuneja, and H S Dhaliwal, 2009: Evaluation and utilization of Aegilops and wild Triticum species for enhancing iron and zinc content in wheat Genet Resour Crop Evol 56, 53—64 Rawat, N., V K Tiwari, K Neelam, G S Randhawa, B Friebe, B S Gill, and H S Dhaliwal, 2011: Development and molecular characterization of wheat-Aegilops kotschyi addition and substitution lines with high grain protein, iron and zinc Genome 54, 943—953 Reddy, B V S., S Ramesh, and T Longvah, 2005: Prospects of breeding for micronutrients and b-carotene-dense sorghums Int Sorghum Millets Newslett 46, 10—14 Rengel, Z., G D Batten, and D E Crowley, 1999: Agronomic approaches for improving the micronutrient density in edible portions of field crops Field Crops Res 60, 27—40 Rümheld, V., and H Marschner, 1990: Genotypical differences among graminaceous species in release of phytosiderophores and uptake of iron phytosiderophores Plant Soil 123, 147—153 Roberfroid, M B., 2007: Inulin-type fructans: functional food ingredients J Nutr 137, S2493—S2502 Biofortification of cereals Rogers, E E., D J Eide, and M L Geurinot, 2000: Altered selectivity in an Arabidopsis metal transporter Proc Natl Acad Sci USA 97, 12356—12360 Rosado, J L., 2003: Zinc and copper: proposed fortification levels and recommended zinc compounds J Nutr 133, 2985S—2989S Salunke, R., N Rawat, K Neelam, V K Tiwari, G S Randhawa, H S Dhaliwal, and P Roy, 2012: Determination of bioavailable-zinc from biofortified wheat using a coupled in vitro digestion/Caco-2 reportergene based assay J Food Comp Anal 25, 149—159 Schiebold, S., H Tschiersch, L Borisjuk, N Heinzel, R Radchuk, and H Rolletschek, 2011: A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus Plant Method 7, 19 Shelton, D R., and W J Lee, 2000: Cereal Carbohydrates In: K Kulp, and J G Ponte (eds), Handbook of Cereal Science and Technology, 2nd edn, 385À414 Marcel Dekker, NY, USA Shi, J., H Wang, K Schellin, B Li, M Faller, J M Stoop, R B Meeley, D S Ertl, J P Ranch, and K Glassman, 2007: Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds Nat Biotechnol 25, 930—937 Shigaki, T., B J Barkla, M C Miranda-Vergara, J Zhao, O Pantoja, and K D Hirschi, 2005: Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1 J Biol Chem 280, 30136—30142 Stalker, H T., 1980: Utilization of wild species for crop improvement Adv Agron 33, 111—147 Stangoulis, J C R., B L Huynh, R M Welch, E Y Choi, and R D Graham, 2007: Quantitative trait loci for phytate in rice grain and their relationship with grain micronutrient content Euphytica 154, 289— 294 Stein, A J., 2010: Global impacts of human malnutrition Plant Soil 335, 133—154 Stephens, L R., and R F Irvine, 1990: Stepwise phosphorylation of myo-inositol leading to myo-inositol hexakisphosphate in Dictyostelium Nature 346, 580—583 Stoltzfus, R J., and M L Dreyfuss, 1998: Guidelines for the Use of Iron Supplements to Prevent and Treat Iron Deficiency Anemia ILSI Press, Washington, DC Subbulakshmi, G., and M Naik, 1999: Food fortification in developing countries- current status and strategies J Food Sci Technol., 365, 371—395 Takahashi, M., H Yamaguchi, H Nakanishi, T Shioiri, N K Nishizawa, and S Mori, 1999: Cloning two genes for nicotinamine aminotransferase, a critical enzyme in iron acquisition (strategy II) in graminaceous plants Plant Physiol 121, 947—956 Takahashi, M., H Nakanishi, S Kawasaki, N K Nishizawa, and S Mori, 2001: Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes Nat Biotechnol 19, 466—469 Takahashi, R., Y Ishimaru, T Senoura, H Shimo, S Ishikawa, T Arao, and N K Nakanishi, 2011: The OsNRAMP1 iron transporter is involved in Cd accumulation in rice J Exp Bot 62, 4843—4850 Tauris, B., S Borg, P L Gregersen, and P B Holm, 2009: A roadmap for zinc trafficking in the developing barley grain based on laser capture microdissection and gene expression profiling J Exp Bot 60, 1333—1347 Thacher, T D., P R Fischer, M A Strand, and J M Pettifor, 2006: Nutritional rickets around the world: causes and future directions Ann Trop Paediatr 26, 1—16 Thiel, E C., and J F Briat, 2004: Plant Ferritin and Non-Haeme Iron Nutrition in Humans: HarvestPlus Technical Monograph International food policy research institute and International center for tropical agriculture, Washington, D.C 445 Tiwari, V K., N Rawat, P Chhuneja, K Neelam, R Aggarwal, G S Randhawa, H S Dhaliwal, and K Singh, 2009: Mapping of quantitative trait loci for grain iron and zinc concentration in diploid A genome wheat J Hered 100, 771—776 Tiwari, V K., N Rawat, K Neelam, S Kumar, G S Randhawa, and H S Dhaliwal, 2010: Substitution of 2S and 7U chromosomes of Aegilops kotschyi in wheat enhances grain iron and zinc concentration Theor Appl Genet 121, 259—269 Uauy, C., A Distelfeld, T Fahima, A Blech, and J Dubcovsky, 2006: A NAC gene regulating senescence improves grain protein, zinc and iron content in wheat Science 314, 1298—1301 Vreugdenhil, D., M G M Aarts, and M Koorneef, 2005: Exploring natural genetic variation to improve plant nutrient content In: M R Broadley, and P J White (eds), Plant Nutritional Genomics, 201— 219 Blackwell, Oxford, UK Walker, D R., A M Scaboo, V R Pantalone, J R Wilcox, and H R Boerma, 2006: Genetic mapping of loci associated with seed phytic acid content in CX1834-1-2 soybean Crop Sci 46, 390—397 Welch, R M., and R D Graham, 2002: Breeding crops for enhanced micronutrient content Plant Soil 245, 205—214 Welch, R M., and R D Graham, 2004: Breeding for micronutrients in staple food crops from a human nutrition perspective J Exp Bot 55, 353—364 Welch, R M., and R D Graham, 2005: Agriculture: the real nexus for enhancing bioavailable micronutrients in food crops J Trace Element Med Biol 18, 299—307 Welch, R M., and W A House, 1984: Factors affecting the bioavailability of mineral nutrients in plant foods In: R M.Welch, and W H Gabelman (eds), Crops as Sources of Nutrients for Humans, 37—54 American Society of Agronomy, WI Welch, R M., W A House, I Ortiz-Monasterio, and Z Cheng, 2005: Potential for improving bioavailable zinc in wheat grain (Triticum species) through plant breeding J Agric Food Chem 53, 2176—2180 White, P J., and M R Broadley, 2005: Biofortifying crops with essential mineral elements Trends Plant Sci 10, 586—593 White, P J., and M R Broadley, 2009: Biofortification of crops with seven mineral elements often lacking in human diets – iron, zinc, copper, calcium, magnesium, selenium and iodine New Phytol 182, 49— 84 White, P J., and M R Broadley, 2011: Physiological limits to zinc biofortification of edible crops Front Plant Sci 2, 1—11 Wilcox, J., G Premachandra, K Young, and V Raboy, 2000: Isolation of high seed inorganic P, low-phytate soybean mutants Crop Sci 40, 1601—1605 Wintergerst, E S., S Maggini, and D H Hornig, 2007: Contribution of selected vitamins and trace elements to immune function Ann Nutr Metab 51, 301—323 von Wiren, N., H Marschner, and V Rӧmheld, 1994: Iron inefficiency in maize mutant ysl (Zea mays L cv Yellow-Stripe) is caused by a defect in uptake of iron phytosiderophores Plant Physiol 141, 1446— 1458 World Health Organization, 2002: World health report Available at: http://www.who.int/whr/2002/ (last accessed on February 16, 2012) Ye, X., S Al-Babili, A Kloti, J Zhang, P Lucca, P Beyer, P Beyer, and I Potrykus, 2000: Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm Science 287, 303—305 Zhang, F., M Fan, X Gao, C Zou, and Y Zuo, 2008: Soil and crop management for improving iron and zinc nutrition of crops In: Banuelos G.S., and Lin ZQ (eds), Development and Uses of Biofortified Agricultural Products, 71—93 CRC Press, FL, USA Zimmerman, M B., and R F Hurrel, 2007: Nutritional iron deficiency Lancet 370, 511—519  ... Ferritin is an iron storage protein molecule consisting of 24 subunit shell around a core of up to 4500 Fe atoms (Thiel and Briat 2004) Introduction of soybean ferritin gene into rice and wheat... mineral absorption promoters To effectively combat hidden hunger through biofortification, even after the development of biofortified varieties, it will be essential to address various socio-economical... genetic component was more profound in determining the Fe, Zn status of the grains Three groups of high Fe genes were mapped to rice chromosomes 7, and contributing to 19–30% of the variation in Fe