Biofortification of cereals to overcome hidden hunger

Review

Bioforti ?cation 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 1and H A R C H A R A N S.D H A L I W A L 3,4

1

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@https://www.360docs.net/doc/228357331.html, With 2?gures

Received February 2,2012/Accepted December 16,2012Communicated by H.Balyan

Abstract

More than 60%of the world population suffers from iron de ?ciency,and over 30%of the global population has zinc de ?ciency.Micronutrient de ?ciency leads to compromised health and economic losses and is pre-valent in populations depending on non-diversi ?ed plant-based diets.Increasing mineral content of staple food crops through bioforti ?cation is the most feasible strategy of combating micronutrient malnutrition.Addi-tionally,it will also enhance the agronomic ef ?ciency of crops on min-eral 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 bioforti ?cation of crop plants.With increased understand-ing of mineral uptake and transport mechanisms in plants,it is becoming ever more possible to engineer bioforti ?ed crop plants with the ultimate goal of overcoming hidden hunger.

Key words:iron —zinc —hidden hunger —bioforti ?cation —uptake —transport —bioavailability

Introduction

The Copenhagen Consensus of 2008listed micronutrient de ?-ciency as the ?fth major global challenge to human health (https://www.360docs.net/doc/228357331.html,).Iron and zinc de ?ciency are the most common and widespread,af ?icting 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 2bil-lion people suffer from iron de ?ciency alone,and the estimates of zinc de ?ciency are also close (Stoltzfus and Dreyfuss 1998,Welch and Graham 2002,Prasad 2003,Gibson 2006,Thacher et al.2006).De ?ciency 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 300enzymatic reactions (King and Keen 1999).

The major reason for micronutrient de ?ciency in the popula-tions of the third world countries is the predominance of non-diversi ?ed 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,?bres and tannins further reduce the bio-availability of these minerals from dietary intakes by preventing their absorp-tion in the intestine (White and Broadley 2005,Pfeiffer and McClafferty 2007).Furthermore,processes like polishing,mill-ing and pearling of cereals make them even poorer in micronu-trients (Welch and Graham 2004,Borg et al.2009).

Strategies for Alleviating Micronutrient Malnutrition

Dietary diversi ?cation,supplementation,forti ?cation and biofor-ti ?cation of crop plants are the approaches proposed for alleviat-ing 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 diversi ?cation and modi ?cation suffers from dif ?culty 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 de ?ciencies.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 sul-phate and zinc acetate.Forti ?cation is the addition of the desired minerals to food stuffs like iodine in salts,iron in ?our,?uorine in toothpaste and zinc in ?ours (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-forti ?ed foods are susceptible to oxida-tion and also alter the taste of the food (Gómez-Galera et al.2010).Similarly,folate-forti ?ed rice loses it while boiling owing to its increased solubility (Brinch-Pederson et al.2007).Further-more,the absorption of oral supplementation also depends on the type of food ingested.These approaches require recurring

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Plant Breeding

doi:10.1111/pbr.12040

?2013Blackwell Verlag

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expenditure,robust distribution system and very careful imple-mentation as overdose may also be harmful(Subbulakshmi and Naik1999,Nantel and Tontisirin2002,Nestel et al.2006). Bioforti?cation refers to increasing genetically the bio-available mineral content of food crops(Bouis2000,Brinch-Pederson et al. 2007).Developing bioforti?ed crops also improves their ef?-ciency of growth in soils with depleted or unavailable mineral composition(Cakmak2008,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(DellaPenna1999,Johns and Eyzaguirre2007, Pfeiffer and McClafferty2007,Tiwari et al.2010).Cereals are the most important source of calories to humans.Rice,wheat and maize provide about23%,17%and10%,respectively,of the calories acquired globally(Khush2003).To effectively target bioforti?cation of cereals,?ve 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 Stephan2003,Colangelo and Guerinot2006).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 non-graminaceous 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 FRO2pro-tein.FRO2is a member of family of eight proteins in Arabidop-sis and is expressed speci?cally in roots.Other members(FRO6, FRO7,FRO8)however,take part in iron transport in shoots (Mukherjee et al.2006,Jeong and Connolly2009,Palmer and Guerinot2009).Solubility of Fe increases by1000-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 Stephan2003).IRT1mediates uptake of Zn,Mn and Cd in addition to Fe(Rogers et al.2000, Curie and Briat2003,Takahashi et al.2011).Strategy-II plants are mostly the members of Poaceae,and they secrete metal che-lators called Phytosiderophores(PS)into the rhizosphere in con-ditions of limiting mineral availability(Mori1999).The metal-PS complex is then taken up by the Yellow Stripe1(YS1)trans-porter,which is an integral membrane protein of roots and shoots(Curie et al.2001).YS1was identi?ed 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 classi?cation 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)and3-epihydroxy-2-hydroxy mugineic acid (epi-HDMA;Mori1999).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 a3”-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 de?cient conditions, ef?ux of DMA in the rhizosphere is carried out by TOM1trans-porter.Overexpression of TOM1led to increased DMA secretion in the rhizosphere,whereas its repession decreased DMA ef?ux (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 OsYSL2using phloem-spe-ci?c OsSUT1promoter and reported an increase of up to4.4 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,Douch-kov et al.2005,Ishimaru et al.2010,Lee et al.2012).

Barley secretes greatest amounts of MAs in the rhizosphere (Rümheld and Marschner1990,Kanazawa et al.1994).Genetic variability was found in wild germplasm of wheat for amount of MA released in iron suf?cient and iron de?cient growth condi-tions(Neelam et al.2010).Neelam et al.(2012)studied MA released in some wheat-Aegilops addition lines and found

that 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 Stephan2003,Nozoye et al.2011

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group2addition lines released higher MA in the rhizosphere not only in iron de?cient but also in iron suf?cient 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 vari-ability for other genetic traits got eroded.Modern day cultivars of all major crops have limited variability of mineral(Graham et al.2001,Bouis2003).The Consultative Group on Interna-tional Agricultural Research(CGIAR)(https://www.360docs.net/doc/228357331.html,) through its HarvestPlus(https://www.360docs.net/doc/228357331.html,)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 suf-?cient variability for improvement in mineral content(Cakmak et al.2000,Ch a vez et al.2005,Vreugdenhil et al.2005,Rawat et al.2009,White and Broadley2009)which could be used for improvement in modern day varieties.In rice,a fourfold differ-ence 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)eval-uated1814accessions in13trials over6years and reported a range of9.6–63.2mg/kg of grain Fe and12.9–57.6mg/kg of grain Zn.In beans,over1000genotypes of Centro Internacional de Agricultura Tropical(CIAT)core collection were screened and were found to have Fe content in range of34–89mg/kg and Zn in the range of21–54mg/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 culti-vars,wild germplasm,landraces have been done and wild rela-tives 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 trans-fer useful variability for grain iron and zinc content using con-ventional and modern breeding approaches(Stalker1980, Chhuneja et al.2008).Oury et al.(2006)studied G9E interac-tions in wheat cultivars for iron,zinc and magnesium concentra-tions and reported genotypes to have higher effect than environment.

In rice,genetic analysis for grain Fe and Zn was done in pop-ulations 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 chromosomes7,8and9 contributing to19–30%of the variation in Fe content(Gregorio et al.2000).Tiwari et al.(2009)mapped three QTL for grain iron and zinc content on chromosomes2and7in 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 chromosome7A was in the same marker interval as in Tiwari et al.(2009).Chromosomes2and 7of 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 of2S and7U of wheat and found an increase of116%and136%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.turgi-dum 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 fer-ritin,has also been attempted by several workers.Ferritin is an iron storage protein molecule consisting of24subunit shell around a core of up to4500Fe atoms(Thiel and Briat2004). 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)intro-duced soybean ferritin into rice with very strong endosperm-speci?c globulin promoter,and this led to an increase of up to13 times in ferritin protein expression than in Goto et al.(1999). However,the actual increase in iron content was not concomitant, being a mere30%enhancement.Furthermore,the iron content in leaves decreased to as low as10%of the non-transformed plants and was accompanied by chlorosis.Similar observations were made in wheat(Borg et al.2009).This was because the ferritin under endosperm-speci?c 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 bene?cial soil microorgan-isms(Rengel et al.1999,Fageria2009,White and Broadley 2011).The most attractive agronomic strategy of bioforti?cation is foliar application of mineral fertilizers to the plants in readily phytoavailable state.However,iron fertilization has met with limited success in bioforti?cation 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 bet-ter absorption by roots and foliage,but their large scale applica-tion 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,Cakmak2008,White and Broadley2009). 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 satu-rated(Gomez-Becerra et al.2010,White and Broadley2011). Agronomic bioforti?cation has met with limited popularity in cereals because of the recurring expenditure and need for careful time-dependent applications of fertilizers.

Bioforti?cation of cereals3

Sequestration of minerals to endosperm

Most of the mineral content present in the grains is con?ned 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 cereal-based 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 trans-porters facilitating distribution of minerals across the grains (Mori1999,Colangelo and Guerinot2006,Curie et al.2009, Grennan2009,Palmer and Guerinot2009,Puig and Pen~a rrubia 2009,Conn and Gilliham2010).Recently,with the development of techniques like laser capture microdissection,it has been possible to study the expression of different genes across differ-ent 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 trans-port 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 than20-fold. Members of Heavy Metal ATPases(HMA),Zinc-regulated Iron-regulated Protein(ZIP),Cation Diffusion Facilitator(CDF), Natural resistance-associated macrophage proteins(Nramp),Vac-uolar Iron Transporter1(VIT1),CAX(Cation Exchanger),YSL (Yellow Stripe Like),Metallothioneins(MT),NAS(Nicotin-amine 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,ZIP1and CAX family transporters.YSL and ZIP transporters have been proposed to capture zinc-NA complexes from?owing back to the apoplast.The aleurone and embryo expression pro?les 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,NAS9and NAATB are expressed at a higher rate in aleurone than in embryo. Nramp3has been proposed to mediate ef?ux of Zn from the aleurone cells,while others control movement to the aleurone cells where it is stored chie?y in vacuoles.Expression of trans-porter 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 speci?c transporter for Fe movement from transfer cells to the endosperm cavity.

Ramesh et al.(2004)overexpressed Zn transporter AtZIP1of Arabidopsis in barley under a ubiquitin promoter.The trans-genic lines produced smaller seeds with high Zn concentration. Manipulating the expression of genes regulating CAX transport-ers has been proposed as an approach to increase Zn concentra-tions 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 dif?cult by the antinutritional factors like phytic acid,tannins,lignans etc.present in cereals(Welch and House1984).Phytic acid(1,2,3,4,5,6)-hexakisphosphate is a chief storage form of phosphorus in seeds and accounts for about1%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(Bouis2000).Monogastric animals and humans do not have speci?c enzymes(Phytases)for break-ing 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,only5%of Fe and25%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

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present in legume and cereal-based diets is bioavailable(Pfeiffer and McClafferty2007).

Numerous attempts have been made to reduce phytic acid con-tent of the cereal grains.Transgenic crops with microbial phyta-ses is one such approach.Brinch-Pederson et al.(2000) introduced Aspergillus niger phyA gene in wheat under the maize ubiquitin-1promoter,which resulted in signi?cantly 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)reported95%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-speci?c rice glutelin-1promoter.Chen et al.(2008) transferred A.niger phyA2gene to maize under maize embryo-speci?c globulin-1promoter,resulting in a50-fold increase over the non-transgenic maize.Transgenic approach to increase phy-tase,however,suffers from the bottleneck of phytase being non-tolerant 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-speci?c over-expression of which can be used to generate barley lines with reduced phytic acid.

Signi?cant genetic variation has been reported for grain phy-tate 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 Fossati1997)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 path-way of phytic acid(Stephens and Irvine1990,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 Hatzack1998)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-speci?c Ole18promoter.The transgenic rice had68%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 conven-tional varieties in monogastric animals and humans(Mendoza et al.1998,Mendoza2002).Salunke et al.(2012)studied the bioavailability of zinc in a diploid wheat(T.monococcum)lpa mutant using Caco2cell lines and found that it actually had higher bioavailability than wild type.

The agronomic performance of the lpa mutants is variable (Bregitzer and Raboy2006,Guttieri et al.2006,Raboy2007). Shi et al.(2007)developed maize and soybean lines with embryo-speci?c gene silencing of a multidrug resistance-associ-ated 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 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(Rober-froid2007,White and Broadley2009).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 carote-noids has been suggested to play a role in improving iron absorption in vitro and in human studies,although the exact mechanism is unknown(Garcia-Casal2006).Inulin is a polysaccharide formed of b(2-1)linked fructose monomers.Hav-ing a prebiotic effect,inulin promotes the growth of useful lacto-bacilli and bi?dobacteria,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).

Signi?cant genetic variation has been reported for b-carotene in wheat,rice(Welch and Graham2005,Howitt and Pogson2006, 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 Naik1970).Signi?cant breakthroughs have been made in genetic engineering attempts to increase b-carotene.Ye et al.(2000)produced golden rice with increased b-carotene con-tent by introducing the phytoene synthase(psy)gene from daffodil and a bacterial phytoene desaturase(crtI)gene from Erwinia uredovora under the control of endosperm-speci?c glutelin(Gt1) and the constitutive CaMV(cauli?ower mosaic virus)35S pro-moter,respectively.Paine et al.(2005)developed golden rice-2 with23-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-speci?c‘super gamma-zein promoter’leading to a34-fold increase in carotenoid(mainly)b-carotene level.

Chen et al.(2003)reported a2–4-fold increase in the ascorbic acid levels in maize and tobacco by overexpressing a wheat dehy-droascorbate 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,by169-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 Lee2000).Five QTL for fructan accumulation were mapped on wheat chromosomes 2B,3B,5A,6D and7A by Huynh et al.(2008).The QTL on 6D and7A contributed to the largest phenotypic variance of 17%and27%,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 ara-binoxylans also increased inulin content(4.2mg/kg)by42-fold compared with the wild type variety(0.1mg/kg).However, more investigations are needed to propose SSIIa manipulation as a generalized strategy for increasing inulin content.

Bioforti?cation of cereals5

Conclusions and Future Directions

Bioforti?cation of crops is a feasible and most economical approach for overcoming‘hidden hunger’.Increasing the con-centration of minerals in edible portions of cereals involves better uptake from soil and improved translocation to grains from leaves and?nally 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,chela-tors 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 com-pounds and/or enhancing concentration of mineral absorption promoters.To effectively combat hidden hunger through bio-forti?cation,even after the development of bioforti?ed varie-ties,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 over-coming hidden hunger.

Con?ict of Interests

Authors declare no con?icts of interest.

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