Available online at www.sciencedirect.
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ScienceDirect
Iron biofortification in the 21st century: setting realistic
targets, overcoming obstacles, and new strategies for
healthy nutrition
Marta W Vasconcelos 1, Wilhelm Gruissem2 and
Navreet K Bhullar2
Plant-based foods offer a wide range of nutrients that are
essential for human and animal health. Among these nutrients,
iron stands out as one of the most important micronutrients.
Increasing the iron content in many staple and non-staple plant
foods continues to be a goal of many scientists around the
world. However, the success of such initiatives has sometimes
fallen short of their expected targets. In this review we highlight
the most recent and promising results that have contributed to
increasing the iron content in different crops. We also discuss
methods that to date have been used to reach iron
biofortification goals and new strategies that we believe are
most promising for crop biofortification in the future. Plant
anatomical, physiological and metabolic hurdles still need to be
tackled for making progress on further increasing currently
reached levels of micronutrient improvements. New strategies
need to take into account growing environmental challenges
that may constrain biofortification efforts.
Addresses
1
Universidade Católica Portuguesa, CBQF – Centro de Biotecnologia e
Quı́mica Fina – Laboratório Associado, Escola Superior de
Biotecnologia, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401
Porto, Portugal
2 edible plant parts, is a recommended strategy that is also
Department of Biology, Plant Biotechnology, ETH Zurich, CH-8092
Zurich, Switzerland
Corresponding authors: Vasconcelos, Marta W
(mvasconcelos@porto.ucp.pt) and Bhullar, Navreet K (bhullarn@ethz.ch)
Current Opinion in Biotechnology 2017, 44:8–15
This review comes from a themed issue on Plant biotechnology
Edited by Dominique Van Der Straeten, Hans De Steur and Teresa
B Fitzpatrick
http://dx.doi.org/10.1016/j.copbio.2016.10.001
0958-1669/# 2016 Elsevier Ltd. All rights reserved.
Iron deficiency severely affects human health
Deficiencies of essential micronutrients often have long-
term negative and irreversible impact on human health.
Iron deficiency anemia (IDA) affects around two billion
people globally. Children, pregnant women, and non-
pregnant women in particular are more vulnerable [1].
Current Opinion in Biotechnology 2017, 44:8–15
IDA can decrease cognitive and physical development, as
well as reduce immunity. It often affects work perfor-
mance in all age groups and enhances the risk of maternal
and perinatal mortality [1]. The recommended daily
intake of iron varies between 8–18 mg/day according to
age, body weight and gender, and 30 mg/day are recom-
mended for pregnant women [2].
Dietary diversification, industrial food fortification and
pharmaceutical supplementation are among different in-
tervention strategies aimed at tackling IDA. These inter-
ventions, however, are often constrained by various socio-
economic circumstances, including lack of access to di-
verse diets, insufficient income to afford commercial
fortified food products, lack of agricultural infrastructure,
and long-term governmental policies and food distribu-
tion networks [3,4]. Fortified food products often have
low public acceptance because of color and/or flavor
changes resulting from added micronutrients [5]. Given
these challenges, biofortification of staple crops, that is,
the intrinsic enhancement of nutritional content in the
sustainable and cost effective. The HarvestPlus Chal-
lenge Program of the Consultative Group for Internation-
al Agricultural Research (CGIAR) focuses on the
biofortification of major food crops, including rice, wheat,
maize, beans, sweet potato, cassava and pearl millet [6].
For successful biofortification strategies, knowledge of
the molecular mechanisms controlling iron uptake and
translocation is essential (Figure 1).
Iron biofortification of food crops
To date, conventional breeding and genetic engineering
strategies have the potential of effectively delivering
nutritious iron-rich food to malnourished populations
(Figure 2). To breed high micronutrient-containing vari-
eties, natural genetic variation for the trait of interest
must exist in the germplasm available to breeders. Bean
and Pearl millet varieties with a high iron content have
been developed and were shown to improve the physio-
logical iron status in affected young women in Rwanda
[7] and of school children in India [8]. In several cases,
however, conventional breeding approaches alone cannot
increase the micronutrient content in crops to nutrition-
ally relevant levels (Table 1) because of lacking natural
genetic variation and/or negative correlations between
grain yield and micronutrient content [9]. In rice, for
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 Iron biofortification in the 21st century Vasconcelos, Gruissem and Bhullar 9

Figure 1

Essential parameters for iron biofortification Iron uptake strategies

H+
Storage in the endosperm
ADP ATP Fe2+

Bioavailability ATPase FRO2 IRT1

Ferric chelate
reductase

3+
H+ Fe -chelate Fe2+
Strategy I (Non-Grasses)

PS Fe3+-PS
Translocation within the plant
Intercellular transport
TOM1 YS1
Intracellular transport
Transcription factors regulating iron
homeostasis
PS Fe3+-PS
Strategy II (Grasses)

PS Fe3+-PS
Fe2+

Effective iron uptake by the roots

? ? YS1 IRT1/IRT2
Strategy I: Reduction (non-grasses)
Strategy II: Chelation (grasses)
PS Fe3+-PS Fe2+

Combination of strategy I and II in Rice

Current Opinion in Biotechnology

The parameters affecting the success of iron biofortification strategies are represented. Effective iron uptake by the roots is one of the primary
requisites. Iron acquisition from the soil is generally categorized into two distinct strategies: a reduction-based strategy (Strategy I) and a
chelation-based strategy (Strategy II) [45]. In Strategy I, ATPases are released into the soil to acidify the rhizosphere in order to increase the
solubility of Fe3+. NADPH-dependent ferric chelate reductases then reduce Fe3+ to Fe2+ and ZINC-REGULATED TRANSPORTER/IRON-
REGULATED TRANSPORTER-RELATED PROTEIN (ZIP) family transporters, including IRT1, transfer Fe2+ across the epidermal plasma membrane
[46]. Iron uptake in grass species mainly involves the chelation strategy (Strategy II), which comprises of four main steps involving (1) biosynthesis
of mugineic acid family phytosiderophores, (2) release of phytosiderophores (PS) to the rhizosphere, (3) iron solubilization and chelation to PS and
(4) Fe(III)-PS transport to the plant. Rice is a unique grass species which can use both strategies for iron uptake, that is, uptake of iron in the form
of Fe(III)-PS complexes, as well as in the form of Fe(II). However, rice is not capable of reducing Fe(III) to Fe(II) form, as other non-grass species
can do (Strategy I). FRO2: FERRIC REDUCTASE OXIDASE 2; TOM1: TRANSPORTER OF MUGINEIC ACID 1; YS1: YELLOW STRIPE 1; IRT1:
IRON-REGULATED TRANSPORTER 1.

example, long-term breeding efforts to achieve the
recommended iron content of 14 mg/gDW in polished
grains have failed [10], but this target has now been
achieved using genetic engineering strategies
[11,12,13].
Different processes — root uptake, transport, remobiliza-
tion, storage and enhanced iron bioavailability — can be
modulated in order to produce plant foods with higher iron
content [14]. In cereals, including rice and wheat, engi-
neering of iron transport and storage in the endosperm has
been an important goal. To achieve this, plants have been
transformed with FERRITIN expressed under the control
of endosperm-specific promoters, resulting in considerable
increases in iron content in polished rice grains and in
wheat flour [15–18] (Table 2). However, expressing FER-
RITIN alone could not achieve the recommended target
iron levels in either rice or wheat. In parallel, rice trans-
formed with genes related to phytosiderophore synthesis
(Figure 2) or encoding iron transporters has also met with
varied degrees of success in increasing iron in rice grains
[11,19–21]. More recently, iron biofortification of rice has

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10 Plant biotechnology

Figure 2

Natural variation
Mutants with in germplasm -
high Fe content in crossing of high
desired tissue Fe content variety
to popular variety

Conventional
breeding

Initial: Final:
TALENs, RNAi, Field and
30 μg/g Variety Farmer 59 μg/g
CRISPR/Cas9, bioavailability
release fields
iron in the
Smart Breeding assessment∗ iron in the
grain grain

Genetic
engineering

Facilitate efficient Alter inter- and intra-

Fe transport cellular Fe storage
and transport

Enhance Fe Increase Fe
uptake (strategy I storage in target
or II) tissue

Current Opinion in Biotechnology

Methods for iron biofortification in crop plants. Schematic representation of the current (solid circles) and emerging (dashed circle) methods being
used in iron biofortification programs. Key steps (blue arrow) are shown that are necessary for a biofortified wheat crop to be available to the
farmer. * In the case of GM crops, a prior step of regulatory and product assessment is required for field trials and human bioavailability studies.

achieved noteworthy success by expressing combinations requirement (EAR) recommended by HarvestPlus, and
of genes, including FERRITIN, NICOTIANAMINE these iron levels have been verified in two field locations
SYNTHASE (NAS), and/or other gene(s) encoding iron [13]. However, in the rice line with the highest grain iron
transporter(s) [12,13,22,23]. Rice lines expressing the level the transgene construct was inserted as an inverted
genes for bean FERRITIN and rice NAS2 have polished repeat [13]. It is currently unknown if the duplicated and
grain iron levels reaching 30% of the estimated average inverted transgene locus can be stably maintained in
breeding lines. Cassava lines expressing the Chlamydomo-
nas reinhardtii IRON ASSIMILATORY 1 (FEA1) gene in
Table 1
the starchy storage roots [24] and those expressing the
Baseline concentration and recommended targets of iron levels Arabidopsis VACUOLAR IRON TRANSPORTER 1 (VIT1)
in staple crops in order to achieve 30% of the estimated average
gene [25] accumulated significantly higher iron levels in
daily dietary requirement for iron on a dry weight (DW) or fresh
weight (FW) basis the storage roots. Despite these encouraging successes,
however, there is still a long way to go before these
Crop Target tissue Concentration Recommended
biofortified crops will reach the needy populations.
in popular iron levels
varieties (mg/g)
Wheat Whole grains 30 59 mg/g DW
From the lab to the field and to the plate
Rice Polished grains 2 15 mg/g DW The total iron content in any given crop is not always a
Beans Whole beans 50 107 mg/g DW good indicator of its useful nutritional quality because the
Pearl millet Whole grains 47 88 mg/g DW human body will not absorb all of the iron, that is, not all
Maize Whole kernels 30 60 mg/g DW iron is bioavailable (Figure 3). In addition to ensuring high
Sweet potato Tuber 6 85 mg/g FW
Cassava Roots 4 45 mg/g FW
iron bioavailability, it is also necessary to consider post-
harvest storage effects and especially the role of the pre-
Modified from [6]. harvest environment. Unlike other nutrients, in particular

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 Iron biofortification in the 21st century Vasconcelos, Gruissem and Bhullar 11

Figure 3

Phytic acid

Tannic acid

Fe
?
Inulin

Vitamin C

Fe3+

Fe3+

Fe3+
Fe3+
Fe3+ Fe3+
Fe3+
Fe3+ HO
HO O Fe3+
HO
HO OH
O O HO O
H OH
O
Fe 3+ Fe3+
HO OH OH n
HO
OH O
O
Fe3+
OH OH
Current Opinion in Biotechnology

Main factors affecting iron bioavailability. Nutritional iron is usually divided into heme iron (found in meat products, and whose absorption is
generally unaffected by other food components), and non-heme iron (from plant sources, and whose absorption is strongly dependent on other
components of the diet). Most staple foods have high concentrations of anti-nutritional compounds [59] such as phytic acid (PA), which
complexes iron and negatively influences its bioavailability in beans [60], rice [61] and other crops. Breeders have focused on lowering the PA
concentration of high-iron crops. Flour from low PA beans, for example, has recently been used for the production of nutritionally enhanced
biscuits [62]. Two low phytic acid (lpa) mutants have been developed for rice, and map-based cloning and complementation identified the
underlying lpa gene to be the SULFATE TRANSPORTER 3;3 (SULTR3;3) [61]. Tannic acid (TA) is a complex polyphenol molecule that can block
non-heme iron absorption, but strategies to reduce TA levels have not been a widely explored approach. With regards to compounds that
promote iron uptake, two of the most promising candidates are inulin and vitamin C. Inulin showed a positive influence on iron bioavailability in
pigs [63], anemic rats [64], and in iron-deficient broiler chickens [65] but not in humans [66]. Increasing inulin in crops has not been a high priority,
but vitamin C biofortification has been successful, for example in tomato using transgenic approaches [67] or in potato by exploiting natural
variation [68]. However, vitamin C synthesis and accumulation is a highly regulated multigenic trait, and levels vary depending on the
developmental stage of the plant, agronomic practices, environmental conditions [69] or fruit maturity [33]. This is a major obstacle for any
biofortification strategy to increase vitamin C to predictable levels. Lastly, techniques for iron bioavailability studies must be chosen carefully, as
discussions regarding the best methodologies still exist [70].

vitamins that are highly labile in high temperature or light,
iron levels in the grain are stable after harvest. In most
biofortification proof-of-concept studies plants are grown
in strictly controlled environmental conditions (often in-
cluding well-defined temperature settings, humidity and
soil substrates). It is well known that soil type influences
the amount of iron available to the plant for absorption
[26]. It is very important to test biofortified lines in the field
during several growing seasons to ensure that the increased
iron levels achieved by breeding or genetic engineering are
reproducible and robust. For example, a maize recombi-
nant inbred population (RIL) grown to maturity in the
field in four environments (two locations  two years)
showed significant genotype  location  year effects
for grain iron content [27]. Many such studies have been
performed using conventionally bred biofortified material
[28]. Fewer field studies using genetically modified mate-
rial have been reported [13], notably because of the
current regulatory hurdles and destruction of field sites.
In both cases, polished rice grains retained the required
iron levels regardless of the field conditions in which
plants were grown. Transgenic iron-biofortified cassava
plants grown to a 6-month stage in Puerto Rico showed
no phenotypic differences to non-transformed cassava
plants in greenhouse or field conditions [28]. Repeated
field testing of high-iron maize in different years and
locations in the United States revealed three modest
QTLs for iron content, even when considering high trait
heritability and location-independent enhancement of
seed iron [29].

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12 Plant biotechnology

Table 2

Biotechnological strategies for iron biofortification in staple crops utilizing genes targeting: (i) storage; (ii) efficient uptake and
translocation; (iii) regulation of deficiency responses; (iv) inter-cellular and intra-cellular storage and transport; and (v) combination
strategies

Target Gene(s) Crop/cultivar used Iron increases compared Reference

for transformation to controla
Storage GmFERRITIN Rice/Japonica cv. Kitaake Up to 3-fold (T1 whole seeds) [15]
GmFERRITIN Rice/Indica cv. IR68144 Up to 3.7-fold (T2 polished seeds) [16]
OsFERRITIN 2 Rice/Indica cv. Pusa-Sugandhi II Up to 2.09-fold (T3 polished seeds) [47]
GmFERRITIN H1 Rice/Indica cv. IR64 Up to 3.4-fold (T3 polished seeds) [17]
TaFERRITIN 1-A Wheat/Bobwhite 50–85% increase (wheat flour) [18]
GmFERRITIN Maize/HiII (A188  B73) 0.2 fold (whole kernels) [48]
Gm FERRITIN Maize/HiII (A188  B73) and Jubilee 20–70% increase (maize flour) [49]
GmFERRITIN Lettuce (cv. Green Leaf) Up to 1.7 fold (leaves) [50]
Uptake and HvNAS1 Rice/Japonica cv. Tsukinohikari Up to 2.3-fold (T2 polished seeds) [20]
translocation OsNAS1 Rice/Japonica cv. Nipponbare 2.2-fold (T1 polished seeds) [11]
OsNAS2 Rice/Japonica cv. Nipponbare Up to 4.2-fold (T1 polished seeds) [11]
OsNAS3 Rice/Japonica cv. Nipponbare 2.2-fold (T1 polished seeds) [11]
OsNAS2 Wheat/Bobwhite Up to 2.1-fold (T4 whole grains); [51]
2.5-fold (flour)
OsYSL2 Rice/Japonica cv. Tsukinohikari Up to 4.4-fold (polished seeds) [52]
HvIDS3 genome Rice/Japonica cv. Tsukinohikari 1.4-fold (polished seeds) [53]
fragment
CrFEA1 Cassava/TMS60444 >3-fold (12 month old storage roots) [24]
Deficiency responses OsIRO2 Rice/Japonica cv. Tsukinohikari >2-fold (whole seeds) [54]
OsFRO2 Soybean 10% (grain) [55]
Inter-cellular and OsVIT1 or Rice/Japonica cv. Zhonghua11 1.5-fold (brown rice grains) [56]
intra-cellular storage OsVIT2 functional and cv. Dongjin genetic
and transport disruption (mutants) background, respectively
OsVIT2 T-DNA Rice >1.5-fold (polished seeds) [57]
insertion line
AtVIT1 Cassava/TME 204 3–4 fold (storage roots) [25]
Combination strategies PvFERRITIN, AtNAS1, Rice/Japonica cv. Taipei 309 Up to 6.3-fold (T4 polished seeds) [58]
and AfPHYTASE
GmFERRITIN H2  2, Rice/Japonica cv. Tsukinohikari Up to 6-fold (T2 polished seeds) and [23]
HvNAS1, OsYSL2  2 up to 4.4-fold (T3 polished seeds)
GmFERRITIN H2  2, Rice/Japonica cv. Paw San Yin Up to 3.4-fold (T2 polished seeds) [22]
HvNAS1, OsYSL2  2
GmFERRITIN H2  2, Rice/Japonica cv. Tsukinohikari Up to 4-fold (T3 polished seeds) [22]
HvNAS1, HvNAAT-A,-B,
and IDS3 genome fragment
AtIRT1, PvFERRITIN, Rice/Japonica cv. Taipei 309 and Up to 4.24-fold (T3 polished seeds) b [12]
AtNAS1, and AfPHYTASE NFP rice [58]
GmFERRITIN and OsNAS2 Rice/IR64 Up to 6-fold (T3 polished seeds) [13]

AfPHYTASE, Aspergillus fumigatus PHYTASE; AtIRT1, Arabidopsis thaliana IRON-REGULATED TRANSPORTER 1; AtNAS1, Arabidopsis thaliana
NICOTIANAMINE SYNTHASE 1; GmFERRITIN H1 and H2, Glycine max FERRITIN H1 and H2; HvIDS3, Hordeum vulgare iron deficiency specific
clone 3; HvNAAT-A,-B, Hordeum vulgare NICOTIANAMINE AMINOTRANSFERASE-A, -B; HvNAS1, Hordeum vulgare NICOTIANAMINE SYNTHASE
1; MxIRT1, Malus xiaojinensis IRON-REGULATED TRANSPORTER 1; OsFERRITIN2, Oryza sativa FERRITIN 2; OsIRO2, Oryza sativa IRON
DEFICIENCY-INDUCIBLE BASIC HELIX-LOOP-HELIX (bHLH) TRANSCRIPTION FACTOR 2; OsIRT1, Oryza sativa IRON-REGULATED TRANS-
PORTER 1; OsNAS1, 2 and 3, Oryza sativa NICOTIANAMINE SYNTHASE 1, 2 and 3; CrFEA 1, Chlamydomonas reinhardtii IRON ASSIMILATORY
GENE; OsVIT1 and 2, Oryza sativa VACUOLAR IRON TRANSPORTER 1 and 2; AtVIT1, Arabidopsis thaliana VACUOLAR IRON TRANSPORTER 1;
OsYSL2 and 15, Oryza sativa YELLOW STRIPE1-LIKE 2 and 15; PvFERRITIN, Phaseolus vulgaris FERRITIN.
a
In the case of transgenic plants, the target tissue and generation of tested plant material written in parentheses, if available.
b
Fold change values are provided in comparison to the non-transgenic rice lines and not to the transgenic lines used for super-transformation.

Modern technologies to design future
biofortification strategies
Recently, new technologies have emerged that can pro-
vide significant advances in iron biofortification programs.
These include oligo-directed mutagenesis, reverse breed-
ing, RNA-directed DNA methylation and sequence-spe-
cific nuclease technology or ‘genome editing’, commonly
performed using zinc finger nucleases, meganucleases,
TALENs and CRISPR-Cas9 [30]. These technologies
have benefited from the high speed and low cost of next
generation sequencing methods that facilitate non-tar-
geted gene and allele discovery [14]. In addition, it is now
current practice to combine different molecular breeding
and genomics methods such as marker-assisted selection,

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 Iron biofortification in the 21st century Vasconcelos, Gruissem and Bhullar 13
association genetics, high-throughput phenotyping
and genotyping for targeting specific traits [31]. RNAi-
mediated gene silencing has also progressed, for example
in developing low-phytate rice by reducing the expres-
sion of the INOSITOL 1,3,4,5,6-PENTAKISPHOSPHATE
2-KINASE GENE (IPK1) [32]. These methods can soon
be integrated in breeding and genetic engineering pro-
grams aimed at complex micronutrient biofortification
strategies, for example that combine increased iron con-
centrations with increased promotive compounds or de-
creased levels of antinutritional factors. By combining
bioinformatics approaches, omics resources and transcrip-
tome collections, scientists have now unraveled the ascor-
bic metabolic network in tomato [33], which can facilitate
the deployment of novel vitamin C biofortification strat-
egies for different crops, thereby augmenting iron bio-
availability. However, vitamin C biofortification remains
a challenge due to its complex genetic regulation. Ge-
nome-wide association studies (GWAS) have also
revealed genes involved in plant tolerance to iron toxicity
[34] and deficiency [35]. Similarly, a GWAS study in
sorghum successfully identified novel quantitative trait
loci for polyphenols, some of which co-localize with
homologues of flavonoid pathway genes from other plants
[36]. Polyphenols are important anti-nutritional factors for
iron bioavailability, and thus these QTLs may lead to
novel candidate genes for decreasing polyphenol concen-
trations in the grain to improve iron bioavailability.
Today, genetically modified (GM) crops still face difficult
regulatory hurdles and a lack of consumer acceptance,
especially in Europe. New gene editing methods based
on TALENs and CRISPR/Cas9, for example, could prove
very useful for future biofortification programs because
improved nutrient traits can be obtained faster and more
efficiently [37]. Gene editing methods can be used to
change one or a few nucleotides in the DNA, replace
existing alleles with new ones, or insert new gene(s) at
predetermined sites in the genome. New and improved
crop varieties generated by gene editing may not be
subject to regulation commonly applied to GM crops if
gene-edited traits are indistinguishable from the same
traits derived from conventional breeding approaches
[38,39]. Assuming that allelic variants have been identi-
fied for proteins that are more efficient at reducing,
absorbing, transporting or accumulating iron, CRISPR/
Cas9 can be used for targeted and precise gene editing to
improve gene function in a more supportive regulatory
framework.
Exome sequencing of iron homeostasis-related genes in
germplasm with contrasting efficiencies in iron uptake
and accumulation would provide the best allelic variants
for relevant genes to use in biofortification strategies (see
Figure 1). Until now, most CRISPR/Cas9 applications
were tested in model crops, but CRISPR/Cas9-mediated
genome editing is now quickly advancing in all major
www.sciencedirect.com
crops. Research is underway to improve crops for higher
yield, stress tolerance, disease and pest resistance, nutri-
ent use efficiency, and increased nutritional value
[37,38,40]. Finally, when considering current achieve-
ments of increasing iron content in staple crops for human
health, we must also be aware of the potential impact of
climate change and loss of genetic diversity. Rising tem-
peratures, unpredictable precipitation and droughts as
well as increasing atmospheric CO2, which are projected
in climate change scenarios for the 21st century [41],
could affect soil fertility and in turn plant iron absorption.
Genetic diversity, which has revealed higher reductase
activity in bean [42] or higher seed iron concentrations in
chickpea [43] and rice [44], must be protected and better
utilized because it will allow breeders and scientists to
respond to future challenges.
Conflict of interest
There is no conflict of interest relating to this article.
Acknowledgements
This work was supported by National Funds from FCT through projects
PEst-OE/EQB/LA0016/2013 and PTDC/AGR-PRO/3972/2014 to MWV,
and by ETH Zurich funds to NKB and WG, who also gratefully
acknowledge support from a private donor. We thank Meng Wang for the
wheat plant drawing used in the figures and we thank Kumar Vasudevan for
the rice plant drawing. We apologize to all colleagues whose relevant work
we could not cite because of space limitations
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Stevens GA, Finucane MM, De-Regil LM, Paciorek CJ,
Flaxman SR, Branca F, Peña-Rosas JP, Bhutta ZA, Ezzati M:
Global, regional, and national trends in haemoglobin
concentration and prevalence of total and severe anaemia in
children and pregnant and non-pregnant women for 1995–
2011: a systematic analysis of population-representative data.
Lancet Glob Health 2013, 1:16-25.
2. National Institutes of Health: Iron: Dietary Supplement Fact
Sheet. 2016 [no volume].
3. Nantel G, Tontisirin K: Forging effective strategies to combat
iron deficiency prevention and control of iron deficiency:
policy and strategy issues 1, 2. J Nutr 2002, 132:839S-844S.
4. Vijayaraghavan K: Control of micronutrient deficiencies in
India: obstacles and strategies. Nutr Rev 2002, 60:S73-S76.
5. Hurrell R: How to ensure adequate iron absorption from iron-
fortified food. Nutr Rev 2002, 60:7-15.
6. Bouis HE, Hotz C, McClafferty B, Meenakshi JV, Pfeiffer WH:
Biofortification: a new tool to reduce micronutrient
malnutrition. Food Nutr Bull 2011, 32:202-215.
7. Haas JD, Luna SV, Lung MG, Wenger MJ, Murray-kolb LE,
 Beebe S, Gahutu J, Egli IM: Consuming iron biofortified beans
increases iron status in Rwandan women after 128 days in a
randomized controlled feeding trial. J Nutr 2016 http://
dx.doi.org/10.3945/jn.115.224741.
In a cohort of 195 Rwandan women subgroups were fed either Fe-
biofortified beans, or standard unfortified beans 2 times/d for 128 days.
The consumption of iron-biofortified beans significantly improved iron
status in Rwandan women.
Current Opinion in Biotechnology 2017, 44:8–15
14 Plant biotechnology
8. Finkelstein JL, Mehta S, Udipi SA, Ghugre PS, Luna SV,
 Wenger MJ, Murray-Kolb LE, Przybyszewski EM, Haas JD: A
randomized trial of iron-biofortified pearl millet in school
children in India. J Nutr 2015, 145:1576-1581.
Iron-biofortified pearl millet significantly improved iron status in children
compared to control plants. Feeding biofortified pearl millet may be an
effective strategy for other age groups as well.
9. Amiri R, Bahraminejad S, Sasani S, Jalali-Honarmand S, Fakhri R:
Bread wheat genetic variation for grain’s protein, iron and zinc
concentrations as uptake by their genetic ability. Eur J Agron
2015, 67:20-26.
10. Bhullar NK, Gruissem W: Nutritional enhancement of rice for
human health: the contribution of biotechnology. Biotechnol
Adv 2013, 31:50-57.
11. Johnson AAT, Kyriacou B, Callahan DL, Carruthers L, Stangoulis J,
Lombi E, Tester M: Constitutive overexpression of the OsNAS
gene family reveals single-gene strategies for effective iron-
and zinc-biofortification of rice endosperm. PLoS ONE 2011, 6.
12. Boonyaves K, Gruissem W, Bhullar NK: NOD promoter-
 controlled AtIRT1 expression functions synergistically with
NAS and FERRITIN genes to increase iron in rice grains. Plant
Mol Biol 2016, 90:207-215.
High-iron rice lines expressing AtNAS1 and FERRITIN were super-trans-
formed with the AtIRT1 gene expressed under control of a NOD promoter.
The synergistic action of AtIRT1, AtNAS1 and PvFERRITIN further
increases iron in both polished and unpolished rice grains.
13. Trijatmiko KR, Dueñas C, Tsakirpaloglou N, Torrizo L, Arines FM,
 Adeva C, Balindong J, Oliva N, Sapasap MV, Borrero J et al.:
Biofortified indica rice attains iron and zinc nutrition dietary
targets in the field. Sci Rep 2016, 6:19792.
Successfully attained Fe/Zn levels in transgenic polished rice grains to
fulfill 30% of EAR in the human diet in a well-characterized GM event of a
widely consumed indica rice cultivar without a yield penalty.
14. Carvalho SMP, Vasconcelos MW: Producing more with less:
strategies and novel technologies for plant-based food
biofortification. Food Res Int 2013, 54:961-971.
15. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F: Iron
fortification of rice seed by the soybean ferritin gene. Nat
Biotechnol 1999, 17:282-286.
16. Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L,
Krishnan S, Oliveira M, Goto F, Datta SK: Enhanced iron and zinc
accumulation in transgenic rice with the ferritin gene. Plant Sci
2003, 164:371-378.
17. Oliva N, Chadha-Mohanty P, Poletti S, Abrigo E, Atienza G,
Torrizo L, Garcia R, Dueñas C, Poncio MA, Balindong J et al.:
Large-scale production and evaluation of marker-free indica
rice IR64 expressing phytoferritin genes. Mol Breed 2014,
33:23-37.
18. Borg S, Brinch-Pedersen H, Tauris B, Madsen LH, Darbani B,
Noeparvar S, Holm PB: Wheat ferritins: improving the iron
content of the wheat grain. J Cereal Sci 2012, 56:204-213.
19. Lee S, An G: Over-expression of OsIRT1 leads to increased
iron and zinc accumulations in rice. Plant Cell Environ 2009,
32:408-416.
20. Masuda H, Usuda K, Kobayashi T, Ishimaru Y, Kakei Y,
Takahashi M, Higuchi K, Nakanishi H, Mori S, Nishizawa NK:
Overexpression of the barley nicotianamine synthase gene
HvNAS1 increases iron and zinc concentrations in rice grains.
Rice 2009, 2:155-166.
21. Lee S, Kim YS, Jeon US, Kim YK, Schjoerring JK, An G: Activation
of rice nicotianamine synthase 2 (OsNAS2) enhances iron
availability for biofortification. Mol Cells 2012, 33:269-275.
22. Aung MS, Masuda H, Kobayashi T, Nakanishi H, Yamakawa T,
Nishizawa NK: Iron biofortification of myanmar rice. Front Plant
Sci 2013, 4:158.
23. Masuda H, Ishimaru Y, Aung MS, Kobayashi T, Kakei Y,
Takahashi M, Higuchi K, Nakanishi H, Nishizawa NK: Iron
biofortification in rice by the introduction of multiple genes
involved in iron nutrition. Sci Rep 2012, 2:543.
Current Opinion in Biotechnology 2017, 44:8–15
24. Ihemere U: Iron biofortification and homeostasis in transgenic
cassava roots expressing the algal iron assimilatory gene,
FEA1. Front Plant Sci 2012, 3:1-22.
25. Narayanan N, Beyene G, Chauhan RD, Gaitán-Solis E, Grusak MA,
 Taylor N, Anderson P: Overexpression of Arabidopsis VIT1
increases accumulation of iron in cassava roots and stems.
Plant Sci 2015, 240:170-181.
AtVIT1 transgenic cassava plants showed 2–4 times higher values of iron
content when compared to wild-type plants. Vacuolar iron sequestration
is a useful transgenic strategy to biofortify crops with iron and to help
eliminate micronutrient malnutrition in at-risk human populations.
26. Santos CS, Carvalho SMP, Leite A, Moniz T, Roriz M,
Rangel AOSS, Rangel M, Vasconcelos MW: Effect of tris(3-
hydroxy-4-pyridinonate) iron(III) complexes on iron uptake
and storage in soybean (Glycine max L.). Plant Physiol Biochem
2016, 106:91-100.
27. Gu R, Chen F, Liu B, Wang X, Liu J, Li P, Pan Q, Pace J,
 Soomro AA, Lübberstedt T et al.: Comprehensive phenotypic
analysis and quantitative trait locus identification for grain
mineral concentration, content, and yield in maize (Zea mays
L.). Theor Appl Genet 2015, 128:1777-1789.
A maize RIL was field grown to maturity across four environments (two
locations  two years) and QTL analysis revealed 28, 25, and 12 QTLs for
mineral concentration, content and yield, respectively. All QTLs were
assigned into 12 distinct QTL clusters.
28. Sayre R, Beeching JR, Cahoon EB, Egesi C, Fauquet C, Fellman J,
Fregene M, Gruissem W, Mallowa S, Manary M et al.: The
BioCassava plus program: biofortification of cassava for sub-
Saharan Africa. Annu Rev Plant Biol 2011, 62:251-272.
29. Lung’aho MG, Mwaniki AM, Szalma SJ, Hart JJ, Rutzke MA,
Kochian LV, Glahn RP, Hoekenga OA: Genetic and physiological
analysis of iron biofortification in Maize Kernels. PLoS ONE
2011, 6:1-10.
30. Schaart JG, van de Wiel CCM, Lotz LAP, Smulders MJM:
Opportunities for products of new plant breeding techniques.
Trends Plant Sci 2015, xx:1-12.
31. Ricroch AE, Hénard-Damave M-C: Next biotech plants: new
traits, crops, developers and technologies for addressing
global challenges. Crit Rev Biotechnol 2015, 8551:1-16.
32. Ali N, Paul S, Gayen D, Sarkar SN, Datta K, Datta SK:
Development of low phytate rice by RNAi mediated seed-
specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-
kinase gene (IPK1). PLoS ONE 2013, 8:1-12.
33. Ruggieri V, Bostan H, Barone A, Frusciante L, Chiusano ML:
Integrated bioinformatics to decipher the ascorbic acid
metabolic network in tomato. Plant Mol Biol 2016, 91:1-16.
34. Matthus E, Wu LB, Ueda Y, Höller S, Becker M, Frei M: Loci,
genes, and mechanisms associated with tolerance to ferrous
iron toxicity in rice (Oryza sativa L.). Theor Appl Genet 2015,
128:2085-2098.
35. Li W, Lan P: Genome-wide analysis of overlapping genes
regulated by iron deficiency and phosphate starvation reveals
new interactions in Arabidopsis roots. BMC Res Notes 2015,
8:555.
36. Rhodes DH, Hoffmann L Jr, Rooney WL, Ramu P, Morris GP,
Kresovich S: Genome-wide association study of grain
polyphenol concentrations in global sorghum [Sorghum
bicolor (L.) Moench] germplasm. J Agric Food Chem 2014,
62:10916-10927.
37. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J-L:
Simultaneous editing of three homoeoalleles in hexaploid
bread wheat confers heritable resistance to powdery mildew.
Nat Biotechnol 2014. Advance on: 1–6.
38. Abdallah N, Prakash C, McHughen A: Genome editing for crop
improvement: challenges and opportunities. Statew Agric L
Use Baseline 2015 2015, 1:183-205.
39. Huang S, Weigel D, Beachy R, Li J: A proposed regulatory
 framework for genome-edited crops. Nat Genet 2016, 48:109-
111.
Graphical comparison of the three main breeding methodologies. The
authors propose a regulatory framework for gene edited crops and
www.sciencedirect.com
 Iron biofortification in the 21st century Vasconcelos, Gruissem and Bhullar 15
recommend five main steps as the primary guiding principles when
considering the generation and regulation of such materials.
40. Li T, Liu B, Spalding MH, Weeks DP, Yang B: High-efficiency
TALEN-based gene editing produces disease-resistant rice.
Nat Biotechnol 2012, 30:390-392.
41. Myers SS, Zanobetti A, Kloog I, Huybers P, Leakey ADB,
Bloom AJ, Carlisle E, Dietterich LH, Fitzgerald G, Hasegawa T
et al.: Increasing CO2 threatens human nutrition. Nature 2014,
510:139-142.
42. Blair MW, Knewtson SJ, Astudillo C, Li C-M, Fernandez AC,
Grusak MA: Variation and inheritance of iron reductase activity
in the roots of common bean (Phaseolus vulgaris L.) and
association with seed iron accumulation QTL. BMC Plant Biol
2010, 10:215.
43. Diapari Anoop, Bett Kirstin, Deokar Amit, Warkentin Thomas D,
Tar’an Bunyamin M: Genetic diversity and association mapping
of iron and zinc concentrations in chickpea (Cicer arietinum
L.). Genome 2014, 57:459-468.
44. Huang Y, Sun C, Min J, Chen Y, Tong C, Bao J: Association
mapping of quantitative trait loci for mineral element contents
in whole grain rice (Oryza sativa L.). J Agric Food Chem 2015
http://dx.doi.org/10.1021/acs.jafc.5b04932.
45. Marschner H, Römheld V: Strategies of plants for acquisition of
iron. Plant Soil 1994, 165:261-274.
46. Eng B, Guerinot M, Eide D, Saier M: Sequence analyses and
phylogenetic characterization of the ZIP family of metal ion
transport proteins. J Membr Biol 1998, 166:1-7.
47. Paul S, Ali N, Gayen D, Datta SK, Datta K: Molecular breeding of
Osfer2 gene to increase iron nutrition in rice grain. GM Crops
Food 2012, 3:310-316.
48. Kanobe MN, Rodermel SR, Bailey T, Scott MP: Changes in
endogenous gene transcript and protein levels in maize plants
expressing the soybean ferritin transgene. Front Plant Sci 2013,
4:196.
49. Drakakaki G, Marcel S, Glahn RP, Lund EK, Pariagh S, Fischer R,
Christou P, Stoger E: 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 2005, 59:869-880.
50. Goto F, Yoshihara T, Saiki H: Iron accumulation and enhanced
growth in transgenic lettuce plants expressing the iron-
binding protein ferritin. TAG Theor Appl Genet 2000, 100:658-
664.
51. Singh S, Gruissem W, Bhullar N: Rice NICOTIANAMINE
SYNTHASE 2 expression improves dietary iron and zinc levels
in wheat. Theor Appl Genet 2016 http://dx.doi.org/10.1007/
s00122-016-2808-x. (in press).
52. Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T,
Takahashi M, Nakanishi H, Aoki N, Hirose T, Ohsugi R et al.: Rice
metal-nicotianamine transporter, OsYSL2, is required for the
long-distance transport of iron and manganese. Plant J 2010,
62:379-390.
53. Masuda H, Suzuki M, Morikawa KC, Kobayashi T, Nakanishi H,
Takahashi M, Saigusa M, Mori S, Nishizawa NK: Increase in Iron
and zinc concentrations in rice grains via the introduction of
barley genes involved in phytosiderophore synthesis. Rice
2008, 1:100-108.
54. Ogo Y, Itai RN, Kobayashi T, Aung MS, Nakanishi H, Nishizawa NK:
OsIRO2 is responsible for iron utilization in rice and
improves growth and yield in calcareous soil. Plant Mol Biol
2011, 75:593-605.
55. Vasconcelos MW, Clemente TE, Grusak MA: Evaluation of
constitutive iron reductase (AtFRO2) expression on mineral
www.sciencedirect.com
accumulation and distribution in soybean (Glycine max L.).
Front Plant Sci 2014, 5:112.
56. Zhang Y, Xu YH, Yi HY, Gong JM: Vacuolar membrane
transporters OsVIT1 and OsVIT2 modulate iron translocation
between flag leaves and seeds in rice. Plant J 2012, 72:400-410.
57. Bashir K, Takahashi R, Akhtar S, Ishimaru Y, Nakanishi H,
Nishizawa NK: The knockdown of OsVIT2 and MIT affects iron
localization in rice seed. Rice 2012, 6:31.
58. Wirth J, Poletti S, Aeschlimann B, Yakandawala N, Drosse B,
Osorio S, Tohge T, Fernie AR, Günther D, Gruissem W et al.: Rice
endosperm iron biofortification by targeted and synergistic
action of nicotianamine synthase and ferritin. Plant Biotechnol
J 2009, 7:631-644.
59. Vasconcelos M, Grusak MA: Status and future developments
involving plant iron in animal and human nutrition. In Iron
Nutrition in Plants and Rhizospheric Microorganisms. Edited by
Barton LL, Abadı́a J. New York, USA: Springer, Life Sciences;
2006:1-22.
60. Petry N, Egli I, Gahutu JB, Tugirimana PL, Boy E, Hurrell R: Phytic
acid concentration influences iron bioavailability from
biofortified beans in Rwandese women with low iron status 1,
2. J Nutr 2014, 144:1681-1687.
61. Zhao H, Frank T, Tan Y, Zhou C, Jabnoune M, Arpat AB, Cui H,
Huang J, He Z, Poirier Y et al.: Disruption of OsSULTR3;3
reduces phytate and phosphorus concentrations and alters
the metabolite profile in rice grains. New Phytol 2016 http://
dx.doi.org/10.1111/nph.13969.
62. Sparvoli F, Laureati M, Pilu R, Pagliarini E, Toschi I, Giuberti G,
Fortunati P, Daminati MG, Cominelli E, Bollini R: Exploitation of
common bean flours with low antinutrient content for making
nutritionally enhanced biscuits. Front Plant Sci 2016, 7:1-14.
63. Samolińska W, Grela ER: Comparative effects of inulin with
different polymerization degrees on growth performance,
blood trace minerals, and erythrocyte indices in growing-
finishing pigs. Biol Trace Elem Res 2016 http://dx.doi.org/
10.1007/s12011-016-0796-y.
64. Lobo AR, Gaievski EHS, De Carli E, Alvares EP, Colli C: Fructo-
oligosaccharides and iron bioavailability in anaemic rats: the
effects on iron species distribution, ferroportin-1 expression,
crypt bifurcation and crypt cell proliferation in the caecum.
Br J Nutr 2014, 112:1286-1295.
65. Tako E, Glahn RP: Intra-amniotic administration and dietary
inulin affect the iron status and intestinal functionality of iron-
deficient broiler chickens. Poult Sci 2012, 91:1361-1370.
66. Petry N, Egli I, Chassard C, Lacroix C, Hurrell R: Inulin modifies
the bifidobacteria population, fecal lactate concentration, and
fecal pH but does not influence iron absorption in women with
low iron status. Am J Clin Nutr 2012, 96:325-331.
67. Bulley S, Wright M, Rommens C, Yan H, Rassam M, Lin-Wang K,
Andre C, Brewster D, Karunairetnam S, Allan AC et al.: Enhancing
ascorbate in fruits and tubers through over-expression of the
l-galactose pathway gene GDP-l-galactose phosphorylase.
Plant Biotechnol J 2012, 10:390-397.
68. Love SL, Salaiz T, Mosley AR, Shafii B, Price WJ, Thornton RE:
Ascorbic acid concentration and stability in North American
potato germplasm. Acta Hortic 2003, 619:87-93.
69. Locato V, Cimini S, De Gara L: Strategies to increase vitamin C
in plants: from plant defense perspective to food
biofortification. Front Plant Sci 2013, 4:152.
70. Glahn RP: Iron biofortification and revisiting the accuracy of
extrinsic labeling in studies of iron absorption. J Nutr 2014,
145:1025-1032.
Current Opinion in Biotechnology 2017, 44:8–15