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Biofortification: Introduction, Approaches,
Limitations, and Challenges 1
Ummed Singh, C S Praharaj, S.K. Chaturvedi,
and Abhishek Bohra
Abstract
Micronutrient malnutrition is known to affect more than half of the
world’s population and considered to be among the most serious global
challenges to humankind. Modern plant breeding has been historically
oriented toward achieving high agronomic yields rather than nutritional
quality, and other efforts related to alleviating the problem have been
primarily through industrial fortification or pharmaceutical supplementa-
tion. Micronutrient malnutrition or the hidden hunger is very common
among women and preschool children caused mainly by low dietary
intake of micronutrients, especially Zn and Fe. Biofortification, the pro-
cess of increasing the bioavailable concentrations of essential elements in
edible portions of crop plants through agronomic intervention or genetic
selection, may be the solution to malnutrition or hidden hunger mitigation.
The Consultative Group on International Agricultural Research has been
investigating the genetic potential to increase bioavailable Fe and Zn in
staple food crops such as rice, wheat, maize, common beans, and cassava.
Keywords
Biofortification • Breeding • Genetic engineering • Limitations • Strategy
1.1 Introduction
Biofortification, the process of breeding nutrients
into food crops, provides a comparatively cost-
effective, sustainable, and long-term means of
delivering more micronutrients. This approach
not only will lower the number of severely mal-
nourished people who require treatment by com-
plementary interventions but also will help them
maintain improved nutritional status. Moreover,
U. Singh (*) • C S Praharaj
Division of Crop Production, ICAR-Indian Institute of
Pulses Research, Kanpur, Uttar Pradesh 208 024, India
e-mail: singhummed@yahoo.co.in;cspraharaj@hotmail.
com
S.K. Chaturvedi • A. Bohra
Division of Crop Improvement, ICAR-Indian Institute of
Pulses Research, Kanpur, Uttar Pradesh 208 024, India
e-mail: sushilk.chaturvedi@gmail.com;
abhi.omics@gmail.com
#Springer India 2016
U. Singh et al. (eds.), Biofortification of Food Crops, DOI 10.1007/978-81-322-2716-8_1
3
cspraharaj@gmail.com
biofortification provides a feasible means of
reaching malnourished rural populations who
may have limited access to commercially
marketed fortified foods and supplements.
The biofortification strategy seeks to put the
micronutrient-dense trait in those varieties that
already have preferred agronomic and consump-
tion traits, such as high yield. Marketed surpluses
of these crops may make their way into retail
outlets, reaching consumers in first rural and
then urban areas, in contrast to complementary
interventions, such as fortification and supple-
mentation, that begin in urban centers.
Biofortified staple foods cannot deliver as high
a level of minerals and vitamins per day as
supplements or industrially fortified foods, but
they can help by increasing the daily adequacy
of micronutrient intakes among individuals
throughout the life cycle (Bouis et al. 2011).
1.2 Minerals and Vitamins
Minerals, in the context of the human diet, are
inorganic chemical elements (or more properly
their dissociated ions) that are required for
biological or biochemical processes including
the accumulation of electrolytes. Carbon, hydro-
gen, nitrogen, and oxygen are excluded from the
list as these are found in common organic
molecules. There are 16 essential minerals, but
11 of them are required in such small amounts
and/or are so abundant in food and drinking
water that deficiency arises only in very unusual
circumstances. The remaining five are present in
limiting amounts in many foods, so a monoto-
nous diet can easily result in deficiency. These
minerals are iodine (I), iron (Fe), zinc (Zn), cal-
cium (Ca), and selenium (Se). Deficiency
diseases arise when diets are based predomi-
nantly on staple foods, such as milled cereals,
which have a low bioavailable mineral content
(Christou and Twyman 2004). Mineral defi-
ciency is therefore most prevalent in developing
countries, where there is poor access to fresh
foods, although Ca deficiency is also widespread
in the industrialized world (Galera et al. 2010).
1.2.1 Iodine
Iodine is an essential component of the thyroid
hormones thyroxine and triiodothyronine, which
regulate growth and development and maintain
the basal metabolic rate. However, only 30 % of
the body’s iodine is stored in the thyroid gland,
and the precise role of the 70 % distributed in
other tissues is unknown. It may overlap with the
function of other minerals such as Se or Fe and
Zn (Lyons et al. 2004). Goiter is another impor-
tant symptom of iodine deficiency and results
from the lack of thyroxine inducing the produc-
tion of thyroid-stimulating hormone, which in
turn causes the thyroid gland to swell (Dunn
2003). India is one of the worst affected countries
in the world, with more than 50 million cases of
goiter and more than two million of cretinism.
1.2.2 Vitamin A
Deficiency associated with blindness and
increased risk of disease and death for small
children and pregnant women can be addressed
through supplements, which are now estimated
to reach children at least once a year in
40 countries. The UN Standing Committee on
Nutrition (UN/SCN) estimates that 140 million
children and 7 million pregnant women are VA
deficient, primarily in Africa and South/South-
east Asia. In 1998, WHO, UNICEF, Canadian
International Development Agency, USAID,
and the Micronutrient Initiative launched the
VA Global Initiative. This provides support to
countries in delivering VA supplements.
1.2.3 Iron
Iron has numerous important functions in the
human body, reflecting its ability to act as both
an electron donor and acceptor. In this role, it
forms the functional core of the heme complex,
which is found in the oxygen-binding molecules
hemoglobin and myoglobin, and the catalytic
center of cytochromes, which carry out redox
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reactions. Iron is therefore required for oxygen
transport in the body and for energy metabolism,
also contributing to the catalytic activity of a
range of nonheme enzymes such as ribonuclease
reductase (WHO/FAO 1998). The immediate
outcome of Fe deficiency is iron deficiency ane-
mia (IDA), which is thought to affect at least two
billion people worldwide. More than half of these
cases could be addressed by increasing the
amount of Fe in the diet, but as for iodine this
is difficult in developing countries where the
population relies on staples, because cereal
grains contain very low levels of Fe and also
contain antinutritional compounds such as
phytate that inhibit Fe uptake (Zimmermann
et al. 2004).
1.2.4 Zinc
Zinc is an essential functional component of
thousands of proteins. Many contain zinc pros-
thetic groups (e.g., zinc finger, zinc twist) and
approximately 100 enzymes require Zn as a
cofactor. Some olfactory receptors cannot func-
tion without Zn. Many cells in the body also
secrete Zn as a signaling molecule, including
cells in the immune and nervous systems
(WHO/FAO 1998). Nearly two billion people
are at risk of Zn deficiency, predominantly chil-
dren and pregnant women. Signs of severe zinc
deficiency include hair loss, skin lesions,
wasting, and persistent diarrhea. The mineral
appears to be particularly important during
periods of rapid growth, and insufficient intake
during childhood and adolescence can delay
growth, sexual development, and psychomotor
development (WHO/FAO 1998).
1.2.5 Calcium
Calcium is the most abundant mineral in the
human body, accounting for 1–2 % of an adult’s
body mass. Over 99 % of Ca is stored in the teeth
and bones, where it plays an important structural
role (WHO/FAO 1998). However, Ca, like Zn, is
also an enzyme cofactor and an important
signaling molecule (a secondary messenger). It
plays a pivotal role in the blood clotting cascade.
Calcium deficiency has a profound impact on
bone health, resulting in rickets if deficiency
occurs in the young and osteoporosis if it persists
into old age.
1.2.6 Selenium
Selenium is found in two unusual amino acids—
selenocysteine and selenomethionine—which
are the principal functional components of
selenoenzymes. It is an essential cofactor in
approximately 50 enzymes, including those
whose function is to reduce antioxidant enzymes
(such as glutathione peroxidase) and those whose
function is to remove mineral ions from other
proteins (such as thyroid hormone deiodinases)
(Lyons et al. 2004). Se is an antioxidant with
health benefits including the prevention of cancer
and heart disease (WHO/FAO 1998).
1.2.7 Folate
Deficiency associated with increased risk of
maternal death and complications in birth and
also associated with neural tube defects in infants
and with an estimated 200,000 severe birth defects
every year can be addressed through fortification
of wheat products (Haddad et al. 2004).
1.3 Alleviating Hidden Hunger:
Interventions
The term “hidden hunger” has been used to
describe the micronutrient malnutrition inherent
in human diets that are adequate in calories but
lack vitamins and/or mineral elements. The diets
of a large proportion of the world’s population
are deficient in Fe, Zn, Ca, Mg, Cu, Se, or I,
which affects human health and longevity and
therefore national economies. Mineral malnutri-
tion can be addressed by increasing the amount
of fish and animal products in diets, mineral
supplementation, and food fortification and/or
1 Biofortification: Introduction, Approaches, Limitations, and Challenges 5
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increasing the bioavailability of mineral
elements in edible crops. However, strategies to
increase dietary diversification, mineral supple-
mentation, and food fortification have not always
proved successful. For this reason, the bioforti-
fication of crops through the application of min-
eral fertilizers, combined with breeding varieties
with an increased ability to acquire mineral
elements, has been advocated (White and
Broadley 2009).
Food fortification and supplementation are cur-
rently the most cost-effective strategies to address
global mineral malnutrition. The most successful
strategy has been salt iodization (fortification with
iodine) which has reduced the incidence of goiter
and other IDD symptoms markedly where the
scheme has been introduced (Galera et al. 2010).
Most strategies to improve mineral nutrition have
been less successful because of political, socio-
economic, infrastructure-related, and technical
constraints that are apparent in most developing
countries.
1.3.1 Food Fortification
Food fortification is one of the most cost-
effective long-term strategies for mineral nutri-
tion (Horton 2006). Fortification of dairy
products such as bread and milk with different
minerals (and vitamins) has been successful in
industrialized countries However, this strategy is
difficult to implement in developing countries
because it relies on a strong food processing
and distribution infrastructure. Fortification
takes place during food processing and increases
the product price. These factors make fortified
products unaffordable to the most impoverished
people living in remote rural areas. Since many
parts of the world suffer from multiple
deficiencies, strategies must also be developed
to fortify foods simultaneously with several
micronutrients without adverse interactions
among them (Zimmermann et al. 2004). The
addition of a single micronutrient would have
more or less the same cost implications as the
addition of several (Alavi et al. 2008).
Zinc fortification has been implemented in the
industrial world but rarely in developing
countries. One exception is Zn-fortified wheat
and maize flours in Mexico, which are used to
make bread and tortillas, the two principal
staples (IZINCG 2007). Organizations such as
the Zinc Task Force (ZTF) and the International
Zinc Nutrition Consultative Group (IZiNCG) are
fighting Zn malnutrition by promoting diverse
strategies to eliminate it. As Zn and Fe
deficiencies tend to go hand in hand, it has been
suggested that double fortification would be
effective with little additional cost, particularly
if Fe fortification were already in place.
1.3.2 Industrial Fortification
The marketed supply of a widely consumed sta-
ple food can be fortified by adding
micronutrients at the processing stage, and his-
torically this is how micronutrient deficiencies
have been addressed in the developed world.
Concentration in the food industry also tends to
strengthen compliance and quality assurance.
Consumption of wheat flour products is growing
around the world, even where wheat is not a
traditional food staple, opening new fortification
opportunities at the milling stage. Public support
for traditional fortification has recently been
enhanced by new promotion and coordination
efforts: Micronutrient Initiative (based in
Canada), Flour Fortification Initiative (based in
Emory University), Mid Day Meal Scheme
(India), and the Global Alliance for Improved
Nutrition (GAIN, based in Geneva). Other
important global actors include the Network for
Sustained Elimination of Iodine Deficiency and
the International Zinc Nutrition Consultative
Group. In addition, efforts are underway to set
regional standards for fortification. The Flour
Fortification Initiative provides an assessment
of global progress, and they report that 26 % of
the global wheat market is fortified, benefiting
1.8 billion people. Most wheat fortification
efforts in the developing world are still prelimi-
nary or on a pilot scale; they are primarily in
the Western Hemisphere, with little sustained
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activity in Asia and Africa, where most of the
micronutrient deficient populations live. Certain
kinds of fortification may be impractical for
some important food staples (e.g., VA fortifica-
tion of milled rice) or may introduce off colors or
flavors (e.g., VA fortification of white maize).
Industrial fortification will only apply to
marketed supplies and therefore may not reach
those among the poor who obtain food outside of
commercialized channels. Given these
limitations, it is clear that industrial fortification
of food cannot provide a complete solution to the
problem of micronutrient deficiencies in the
medium term. It is in this context that a role
emerges for biofortification as a complementary
strategy.
1.3.3 Promotion of Dietary
Diversification
Education is an important element in ensuring
that improvements in income result in better
maternal and child health. However, dietary
diversification is constrained by resource avail-
ability for poor households and seasonal avail-
ability of fruits and vegetables. Promotion of
home gardens is often touted, but the poor have
a high opportunity cost for their labor and often
limited land. Increased production of fruits and
vegetables for household use reduces resources
available for other income-earning or food pro-
duction activities. This type of effort is also rela-
tively expensive and difficult to sustain on any
large scale.
1.3.4 Food Supplementation
Supplementation is the best short-term interven-
tion to improve nutritional health, involving the
distribution of pills or mineral solutions for imme-
diate consumption. This helps to alleviate acute
mineral shortages but is unsustainable for large
populations and should be replaced with fortifica-
tion at the earliest opportunity (Shrimpton and
Schultink 2002). In industrialized countries, with
few mineral malnutrition problems, supplementa-
tion is focused on a small subset of the population
with specific deficiencies resulting from medical
conditions. In developing countries, where acute
and chronic deficiencies are common, supplemen-
tation is highly recommended to complement the
diet (fortified or otherwise) of the entire popula-
tion (Nantel and Tontisirin 2002).
Periodic provision of supplements (often in the
form of tablets) can address deficiencies of
micronutrients that are stored in the body, such
as vitamin A and iron. Supplementation can be
cheap compared to the large public health benefit.
The total annual cost of iron tablet supplementa-
tion in India to reach 27 million women and
128 million children at risk is only $5.2 million.
Yet even small budgets can be difficult to sustain
year after year when they are dedicated to the
welfare of politically weak or socially marginal
beneficiaries. Furthermore, while certain
populations are easy to reach through existing
institutions (e.g., schoolchildren through schools),
it is often difficult to accomplish full coverage of
those most at risk—poor women and very young
children. Thus, supplementation has often been
most effective when delivered together with
other maternal and child health interventions.
The distribution of vitamin A supplements has
been one of the most cost effective and success-
ful acute intervention programs in the developing
world (Shrimpton and Schultink 2002), but this is
a rarity. Like fortification, successful supplemen-
tation strategies require a robust infrastructure
and a government determined to improve the
nutritional health of its population (Shrimpton
and Schultink 2002). Even more than fortifica-
tion, supplementation requires compliance mon-
itoring because more people will neglect to take
regular supplements at prescribed intervals than
fortified staple foods. Mineral supplements are
prescribed for acute deficiency diseases in
industrialized countries as well as serving a
niche health market. Zn supplements rank very
highly according to the Copenhagen Consensus
report cost–benefit analysis.
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1.3.5 Biofortification
Conventional interventions have a limited
impact, so biofortification has been proposed as
an alternative long-term approach for improving
mineral nutrition (Zhu et al. 2007). Bioforti-
fication focuses on enhancing the mineral
nutritional qualities of crops at source, which
encompasses processes that increase both min-
eral levels and their bioavailability in the edible
part of staple crops. The former can be achieved
by agronomic intervention, plant breeding, or
genetic engineering, whereas only plant breeding
and genetic engineering can influence mineral
bioavailability. Plant breeding and genetic engi-
neering are often compared because, in contrast
to agronomic interventions, both involve chang-
ing the genotype of a target crop. The two pro-
cesses are similar in aim, albeit different in
scope. Both attempt to create plant lines carrying
genes that favor the most efficient accumulation
of bioavailable minerals—plant breeding
achieves this by crossing the best performing
plants and selecting those with favorable traits
over many generations—whereas genetic engi-
neering accesses genes from any source and
introduces them directly into the crop. Plant
breeding is limited to genes that can be sourced
from sexually compatible plants, whereas genetic
engineering has no taxonomic constraints and
even artificial genes can be used.
The main advantage of genetic engineering and
plant breeding approaches for mineral enhance-
ment is that investment is only required at the
research and development stage, and thereafter
the nutritionally enhanced crops are entirely sus-
tainable. Furthermore, as stated above, mineral-
rich plants tend to be more vigorous and more
tolerant of biotic stress, which means yields are
likely to improve in line with mineral content
(Frossard et al. 2000; Nestel et al. 2006). Unlike
conventional intervention strategies, genetic engi-
neering and plant breeding are both economically
and environmentally sustainable (Stein
et al. 2008). Although there are no commercial
nutritionally enhanced plants derived from either
method at the current time, this approach has the
greatest long-term cost-effectiveness overall and
is likely to have an important impact over the next
few decades. Biofortification is also likely to be
more accessible than conventional interventions
in the long term because it removes hurdles such
as the reliance on infrastructure and compliance.
Moreover, plants assimilate minerals into organic
forms that are naturally bioavailable and contrib-
ute to the natural taste and texture of the food.
Economic studies have shown many potential
health benefits of biofortification strategies, espe-
cially in combination with conventional strategies
(Buois 2002; Stein et al. 2008).
1.3.5.1 Plant Breeding
Plant breeding programs focus on improving the
level and bioavailability of minerals in staple
crops using their natural genetic variation
(Welch and Graham 2005). Breeding approaches
include the discovery of genetic variation affect-
ing heritable mineral traits, checking their stabil-
ity under different conditions, and the feasibility
of breeding for increasing mineral content in
edible tissues without affecting yields or other
quality traits. Breeding for increased mineral
levels has several advantages over conventional
interventions (e.g., sustainability); no high-
mineral varieties produced by this method have
been introduced onto the market thus far. This
reflects long development times, particularly if
the mineral trait needs to be introgressed from a
wild relative. Breeders utilize molecular biology
techniques such as quantitative trait locus (QTL)
maps and marker-assisted selection (MAS) to
accelerate the identification of high-mineral
varieties, but they have to take into account
differences in soil properties (e.g., pH, organic
composition) that may interfere with mineral
uptake and accumulation. For example, the min-
eral pool available to plant roots may be
extremely low in dry, alkaline soils with a low
organic content (Cakmak 2008).
1.3.5.2 Conventional Plant Breeding
This allows crop scientists to make significant
improvement in the nutritional, eating quality,
and agronomic traits of major subsistence food
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crops. Conventional breeding is limited, how-
ever, because it can only use the genetic
variability already available and observable in
the crop being improved or occasionally in the
wild varieties that can cross with the crop. Fur-
thermore, conventional breeders usually have to
trade away yield and sometimes grain quality to
obtain higher levels of nutrition. One example is
quality protein maize (QPM), which has taken
decades of conventional plant breeding work to
develop into varieties acceptable to farmers.
However, multiple gains are at times possible,
as with iron and zinc in rice and wheat, where the
characteristics that lead to more iron and zinc in
the plant can also lead, by some accounts, to
higher yield. Other biofortified crops, such as
the orange-fleshed sweet potatoes (OFSP) pro-
moted through the HarvestPlus program in
Africa, have been successfully selected and
developed for both nutrient and (at least rainy
season) yield traits (Unnevehr et al. 2007).
1.3.5.3 Mutation Breeding
Mutation breeding has been used extensively in
developed and developing countries to develop
grain varieties with improved grain quality and in
some cases higher yield and other traits. This
technique makes use of the greater genetic
variability that can be created by inducing
mutations with chemical treatments or irradia-
tion. The FAO/International Atomic Energy
Agency (IAEA) website contains more than
2500 varieties that have been developed through
mutation breeding (Mutant varieties database).
Of these, 1568 are in Asia, 695 in Europe, and
165 in the United States. Most of the European
and US mutants are flowers, but most in Asia are
basic food crops such as wheat, rice, maize, and
soybeans. According to their website,
FAO/IAEA include biofortification as one of
the objectives of their mutagenesis program, but
there do not seem to be any applicable results yet.
Varieties produced using mutagenesis can be
grown and certified as organic crops in the
United States, whereas transgenic crops devel-
oped using recombinant DNA (rDNA) technol-
ogy cannot.
1.3.5.4 Molecular Breeding
Also called marker-assisted breeding, this is a
powerful tool of modern biotechnology that
encounters little cultural or regulatory resistance
and has been embraced so far even by organic
growers because it relies on biological breeding
processes rather than engineered gene insertions
to change the DNA of plants. This technique is
expanding rapidly with the development of geno-
mics, which is the study of the location and
function of genes, and with the rapid decline in
costs of screening plant tissue. Once scientists
have identified the location of a gene for a desir-
able trait, they build a probe that attaches itself
only to a DNA fragment, a so-called marker,
unique to that gene. They then can use this
marker as a way to monitor and speed up their
efforts to move this trait into relatives of the plant
using conventional breeding. For example, since
the marker can be detected in the tissue of new
seedlings, the presence or absence of the desired
trait can be determined without having to wait for
a plant to mature, often reducing by years the
length of a typical crop development process. If
molecular breeding reduces the number of
generations required to develop a pure line vari-
ety by three generations, this can save 3 years of
research time. The use of molecular breeding has
increased dramatically both by private seed
companies and government plant breeders in
developed countries, and it is gradually spread-
ing to developing countries (Pray 2006). Using
this technique, plant breeders also can stack into
one variety several different genes that code for
different traits, for example, QPM, disease resis-
tance, and drought tolerance in maize (Pray
2006). This technique has also been used to find
recessive traits in plants that cannot be located by
conventional breeding or other techniques.
1.3.5.5 Genetic Engineering
Genetic engineering is the latest weapon in the
armory against mineral deficiency and uses
advanced biotechnology techniques to introduce
genes directly into breeding varieties. The genes
can come from any source (including animals
and microbes) and are designed to achieve one
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or more of the following goals (Zhu et al. 2007):
(a) improve the efficiency with which minerals are
mobilized in the soil, (b) reduce the level of
antinutritional compounds, and (c) increase the
level of nutritional enhancer compounds such as
inulin. Genetic engineering, or rDNA, is a tech-
nique that offers still greater speed and reach
because it moves specific genes with desired traits
from a source organism—one which does not
have to be a related organism—directly into the
living DNA of a target organism. The transgenic
trait is added without normal biological reproduc-
tion, but once in the plant it becomes inheritable
through normal reproduction. Scientists first
developed this technique in the laboratory in
1973 and have been using it to transform agricul-
tural crop plants since the 1980s. Once a useful
gene has been identified (which can require a
major research project and many years), it is
attached to both marker and promoter genes and
then inserted into a plant, usually using a nonvia-
ble virus called Agrobacterium as a carrier. GE
produces plants that are known as transgenics or
less precisely as GMOs. GE has great reach
because it can add valuable characteristics that
are not currently found in the seeds of individual
plant species. GE was necessary for the develop-
ment of golden rice, which contains the precursor
to VA from a daffodil plant. This was a trait
missing from rice plants, and it could not be
introduced conventionally since daffodils cannot
be crossed with rice plants. In addition, GE can
take much less time to incorporate desired traits
into a crop plant than either traditional or molecu-
lar breeding. The choice of which technology to
use when biofortifying crops comes down to a
calculation by breeders of how to get the best
results most quickly, given their budget constraint.
Conventional plant breeding requires less invest-
ment in labs or highly trained human resources
(molecular biologists) than either marker-assisted
selection or genetic engineering, and it faces lower
and less costly regulatory hurdles. However, if
there are no genes for the VA precursors in the
genome of a crop (as one example), no amount of
conventional plant breeding can put them there,
and scientists must turn to GE. Molecular breeding
and GE also have advantages over traditional
breeding because they make it easier to develop
crops with multiple desired nutritional traits, main-
tain agronomic viability of biofortified crops,
adapt agriculture improvements arising in the
United States for obscure crops in developing
countries, etc.
1.3.5.6 Tissue Cultures
Modern tissue culture techniques can allow
scientists to reproduce plants from a single cell.
These techniques are now used extensively to pro-
duce disease-free planting material of clonally
propagated crops such as bananas. When tissue
culture is combined with embryo rescue
techniques, plant breeders can use the genes from
wild and weedy relatives of a crop, which would
normally not cross with the cultivated crop. This
allows breeders to increase genetic variability of
the cultivated crop and then bring in valuable traits
of the wild and weedy relatives. These techniques
have allowed scientists to cross Asian and African
rice varieties and develop Nerica rice varieties
with agronomic traits, such as higher yield and
resistance to water stresses, that have met with
growing success in Africa. Tissue culture is an
important tool for propagation of roots and tubers,
such as potatoes and cassava, and both of these
crops are part of current biofortification research.
1.3.6 Microbiological Interventions
1.3.6.1 Plant Growth Promoting
Rhizobacteria (PGPR)
These include beneficial bacteria that colonize
plant roots and enhance plant growth by a wide
variety of mechanisms. The use of PGPR is
steadily increasing in agriculture, as it offers an
attractive way to reduce the use of chemical
fertilizers, pesticides, and related agrochemicals
(Rana et al. 2012). Interventions using PGPR or
other biological agents are limited. Secretion of
phytosiderophores by microorganisms and plants
in restricted spatial and temporal windows
represents an efficient strategy for uptake of
iron and other micronutrients by plants from the
rhizosphere. Analysis of the complex
interactions between soils, plants, and microbes
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in relation with micronutrient dynamics
represents a unique opportunity to enhance our
knowledge of the rhizosphere ecology. Such
progress can provide information and tools
enabling us to develop strategies to improve
plant nutrition and health with decrease in the
application of chemical inputs. Microorganisms
are known to differ significantly in competing
with higher plants for micronutrients (Steven
1991). Among bacteria, a lot of attention has
been dedicated to the siderophore-mediated iron
uptake by fluorescent pseudomonads. A number
of other mechanisms are also involved in the
sequestration and transformation by
microorganisms in soil such as production of
acids, alkalis, etc. PGPR constitute a significant
part of the protective flora that benefit plants by
enhancing root function, suppressing disease,
and accelerating growth and development
(Glick 1995). Microorganisms differ in compet-
ing with higher plants for micronutrients. Species
of Azotobacter differed in their competitiveness
with wheat plants in extracting Fe and Zn
(Shivay et al. 2010). Biofortification of crops
through application of PGPR can be therefore
considered as a possible supplementary measure,
which along with breeding varieties can lead to
increased micronutrient concentrations in wheat
crop, besides improving yield and soil fertility.
1.3.6.2 AM Fungi
Most plants, including all major grain crops and
almost all vegetables and fruits, are associated with
mycorrhizal fungi that improve the uptake of essen-
tial mineral elements from soils and, therefore,
enhance plant growth and productivity.These sym-
biotic fungi, therefore, change, directly or indi-
rectly, the mineral nutrition of plant products that
are also essential for humans. However, the role of
mycorrhizas on element biofortification may be
piloted through agricultural practices. Mycorrhizas
can potentially offer a more effective and sustain-
able element biofortification to curb global human
malnutrition. Approximately 90 % of land plants
form mycorrhizas (literally “fungus roots”): they
exist everywhere, from tiny home gardens to large
ecosystems (Smith and Read 1997). Six types of
mycorrhizas (arbuscular, arbutoid, ecto-, ericoid,
monotropoid, and orchid) are categorized by their
distinct morphological characteristics (Wang and
Qiu 2006). Of them, arbuscular mycorrhiza
(AM) is the most common and predominant type.
Arbuscules, specific “little-tree-shaped” fungal
structures inside root cortical cells, serve as the
main sites of nutrient exchange between the plant
and the fungus. AM also has external hyphae that
provide an extensive surface area or network for
nutrient uptake from soils. Thus, AM is the most
important mycorrhiza in agriculture and closely
relates to human nutrition. Obligately depending
on plant photosynthates as energy sources, the
extensive AM mycelial systems (the vegetative
parts of the fungus) effectively explore soil
substrates and acquire soil inorganic nutrients,
including major macronutrients N, P, and K and
micronutrients Cu, Fe, and Zn (Caris et al. 1998),
with some capacity for acquiring organic N and P
(Koide and Kabir 2000). These soil-derived
nutrients are not only essential for AM develop-
ment but are also partly transferred to the host
plant. It is believed that many plants that usually
form this symbiotic relationship would be unable to
survive without the mycorrhiza. Mycorrhizal
mycelia and their exudates also constitute a large
carbon source (20–30 % total soil microbial bio-
mass) for the functioning of other belowground
microorganisms that ameliorate soil nutrient avail-
ability through decomposition of organic
compounds and weathering of inorganic materials.
So, not only do the activities of AM fungi have
multiple functions that enhance plant performance
but they also play crucial roles in the development
of soil properties and the health of the entire
ecosystem.
1.3.7 Agronomic Intervention
Farmers have applied mineral fertilizers to soil
for hundreds of years in order to improve the
health of their plants, but within certain limits
the same strategy can also be used to increase
mineral accumulation within cereal grains for
nutritional purposes (Rengel et al. 1999). This
strategy only works if the mineral deficiency in
the grain reflects the absence of that mineral in
1 Biofortification: Introduction, Approaches, Limitations, and Challenges 11
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the soil and if the mineral fertilizer contains
minerals that are rapidly and easily mobilizable.
Also, even if plants can absorb minerals effi-
ciently from the soil, they may store the mineral
in leaves but not fruits or seeds, or they may
accumulate the mineral in a form that is not
bioavailable, thus having no impact on nutrition
(Frossard et al. 2000). Like supplements and
fortification, agronomic intervention is probably
best applied in niche situations or in combination
with other strategies (Cakmak 2008). One draw-
back of agronomic intervention is the cost and
impact of the fertilizers. Fertilizer use is likely to
increase the cost of food, thus reducing its avail-
ability to the most impoverished people. The
expensive fertilizers must be applied regularly,
with no direct yield incentive to farmers in devel-
oping countries, so the intervention would likely
be omitted to save costs even though seeds pro-
duced under rich mineral conditions germinate
more vigorously than those in poor soils
(Cakmak 2008). There is also concern about the
impact of increased fertilizer use on the environ-
ment (Graham 2003).
Agronomic strategies to increase the
concentrations of mineral elements in edible
tissues generally rely on the application of min-
eral fertilizers and/or improvement of the solubi-
lization and mobilization of mineral elements in
the soil (White and Broadley 2009). When crops
are grown where mineral elements become
immediately unavailable in the soil, targeted
application of soluble inorganic fertilizers to
roots or to leaves is practiced. In situations
where mineral elements are not readily
translocated to edible tissues, foliar applications
of soluble inorganic fertilizers are made. It has
been observed that the human population of the
world has exceeded the carrying capacity of
low-input agriculture, and modern inorganic
fertilizers are necessary to obtain the crop yields
required to prevent starvation (Graham
et al. 2007). Essential plant nutrients are mainly
applied to soil and plant foliage for achieving
maximum economic yields. Soil application
method is more common and most effective for
nutrients, which are required in higher amounts.
However, under certain circumstances, foliar fer-
tilization is more economic and effective. Foliar
symptoms, soil and plant tissue tests, and crop
growth responses are principal nutrient disorder
diagnostic techniques. Soil applications of
fertilizers are mainly done on the basis of soil
tests, whereas foliar nutrient applications are
mainly done on the basis of visual foliar
symptoms or plant tissue tests. Hence, correct
diagnosis of nutrient deficiency is fundamental
for successful foliar fertilization. In addition,
there are some more requirements for successful
foliar fertilization. Foliar fertilization requires
higher leaf area index for absorbing applied
nutrient solution in sufficient amount; it may be
necessary to have more than one application
depending on severity of nutrient deficiency.
1.3.7.1 Parboiled Rice
Fe fortification in parboiled rice is a rapid and
cost-effective solution to Fe deficiency anemia in
economically disadvantaged populations with
rice as the major staple food and poor access to
animal proteins. It focused initially on the feasi-
bility of this innovative approach, by examining
the effectiveness of Fe fortification and retention,
the solubility of Fe in the grain in response to
fortification treatments, and the likely pathway of
Fe movement into the endosperm. NaFeEDTA
has been recently approved as an ingredient to be
used in supervised food fortification programs
(Hurrell 2003) and is the most promising Fe
fortification compound for food additives and is
used intensively to prevent oxidation and color
changes in food and promote its bioavailability in
the human diet. The effectiveness of enhancing
Fe density in white rice through the Fe-fortified
parboiling process is far greater than that
achieved from conventional and transgenic rice
breeding. For example, Fe concentration in
milled rice of IR68144-2B-2-2-3, an improved
rice cultivar by conventional breeding, is
7–13 mg Fe kg
−1
(Graham et al. 1999) and
37 mg Fe kg
−1
in rice containing transferred
soybean ferritin gene (Vasconcelos et al. 2003).
In contrast, Fe concentration in the milled rice
grains Fe fortified in the parboiling had 70–144
and 30–110 mg Fe kg
−1
in 60 and 120 s milled
grains, respectively.
Comparatively, a substantial loss of Fe
sprayed on raw rice surface occurs if the rice is
12 U. Singh et al.
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rinsed before cooking. Although a polymer coat-
ing technique has been advocated for painting Fe
on the rice grain surface to minimize Fe loss from
washing and/or cooking, these techniques are
expensive and not practical in rice mills of devel-
oping countries. In comparison, parboiled rice is
a rapid and cost-effective vehicle to deliver Fe
nutrition benefits through the already established
parboiling infrastructure, market network, and
consumers’ acceptance in Asia (particularly in
the subcontinent) and Africa, where the high
risk of Fe malnutrition-induced anemia is present
(Graham et al. 1999). Most of the fortified Fe in
the milled rice grains remained in the dilute acid-
soluble pool, which is considered as potentially
bioavailable in the human diet in all cultivars.
One of the significant advantages of parboiled
rice is that parboiling resulted in a significant
inward movement of the fortified Fe into the
endosperm, countering milling-induced Fe loss
in raw rice grains due to restricted distribution of
Fe in the aleurone and embryo (the bran fraction)
of brown rice (Bhattacharya 2004). Parboiling
itself may cause inward migration of some min-
eral nutrients present in the surface layers of rice
grain, resulting in a higher retention rate of these
nutrients when being milled to produce white
rice (Ali and Bhattacharya 1980; Palipane and
Swarnasiri 1985), such as P, Ca, Fe, Mn, Mo, and
Cr in milled parboiled rice.
1.3.8 Biofortification of Feed
for Livestock
For many decades plant breeding primarily
focused on yield, and little attention was given to
the nutritional value of cereal residues (bran and
straw) that were not used for human consumption.
However, bran and straw are among the most
important feed for ruminant livestock in many
parts of the world, where sorghum and millet are
important staple cereals in addition to rice and
wheat. In sorghum and pearl millet, various crop
management interventions (Reddy et al. 2003) and
plant breeding (Zerbini and Thomas 2003)
strategies were shown to influence yield and
improve quality of the straw for animal feed.
These efforts are now being complemented with
the introduction of molecular markers (QTL
mapping and MAS) as tools to increase breeding
efficiency (Hash et al. 2003). Progress has also
been reported for a completely different approach
to improving cereal nutrient availability. In trans-
genic animals like the Enviropig, phytase is pro-
duced in the salivary glands and the active enzyme
is secreted into the saliva (Forsberg et al. 2003).
These pigs show improved phosphorus uptake.
Such a transgenic pig might also show improved
iron uptake in the intestine, owing to lower content
of the antinutritional phytate.
1.4 Hindrances or Limiting Factors
1.4.1 Antinutrients
Phytate and tannins are the limiting factors in the
absorption of Fe, Zn, and Ca by the gut (Mendoza
2002). Phytate occurs widely in plant tissues but is
concentrated in seeds or grain. There is consider-
able intraspecific variation in phytate concentra-
tion in edible portions (Glahn et al. 2002; Coelho
et al. 2005) that is independent of variation in Fe
and Zn concentrations. In addition, several low
phytic acid (lpa) mutants have been produced by
non-transgenic techniques in rice, maize, wheat,
barley, and soybean (Banziger and Long 2000).
Fortuitously, plants with lpa mutations often show
raised levels of seed Fe, Zn, and Mg (or similar
levels to those found in wild type), although they
do have reduced concentrations of seed
Ca. Tannin concentration in edible tissues also
varies greatly between varieties (Lin et al. 2005).
Hence, breeding for reduced concentrations of
these antinutrients appears feasible.
Phytic acid, as well as other metabolites pro-
duced by plants, such as PP, is considered an
“antinutrient” because by chelating iron, it can
reduce its absorption in the human gut (Jin
et al. 2009). In plants, however, phytic acid
fulfills essential biological functions (Murgia
et al. 2012). Phytate, a mixed cation salt of
D-myo-inositol, hexakisphosphate, commonly
known as PA, InsP6, or IP6, constitutes up to
1–8 % of mature seed dry weight and accounts
1 Biofortification: Introduction, Approaches, Limitations, and Challenges 13
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for up to 90 % of phosphorus content in cereal
grains, legumes, nuts, and oil seeds. Phytate
represents an important metal cation reserve
(magnesium, potassium, calcium, manganese,
barium, and iron) in seeds, either in the aleurone
cell layer or in the seed embryo, depending on the
plant species. Degradation of phytate occurs dur-
ing seed germination, by means of phytases, a
class of phosphatases capable of releasing at
least one phosphate from phytic acid (IP6)
(Bohn et al. 2008). The consequent release of
phosphorus and mineral nutrients supports growth
and development of the seedling. Besides its well-
known role in mineral storage in seeds, IP6 also
acts in the leaves in the signaling cascade trig-
gered by drought/osmotic stress leading to stoma-
tal closure. In guard cells ABA produces rapid
changes in IP6 which trigger release of Ca2+
from endomembrane stores and inhibition of K+
inward rectifying channels. Some low IP6 cereals
are less tolerant to stress and possess undesirable
agronomical traits, such as reduced seed yield and
lower seed viability, as observed in bread wheat
(Triticum aestivum), rice (Oryza sativa), and bar-
ley (Hordeum vulgare) (Zhao et al. 2008),
suggesting that IP6 is indeed involved in essential
biological functions in the whole plant. More
recently, a key role for IP6 in the maintenance of
basal resistance against a wide range of pathogens
has been demonstrated transgenic potatoes with
compromised synthesis of IP6 (through IPS anti-
sense RNA) are less resistant to virus infection.
Disruption of IP6 biosynthesis in Arabidopsis is
also associated with increased susceptibility to
viruses and to bacterial and fungal pathogens
(Murphy et al. 2008).
1.4.2 Promoters
Some organic compounds stimulate absorption of
essential mineral elements by humans (Table 1.1).
These include ascorbate (vitamin C), b-carotene
(provitamin A), protein cysteine, and various
organic and amino acids. There is considerable
intraspecific variation in both ascorbate and
b-carotene concentrations in fruit and vegetables
(Frossard et al. 2000). For example, ascorbate
concentration in cassava varied by 250-fold in
leaves and 40-fold in roots among the
530 accessions of the CIAT core collection,
whereas b-carotene concentration varied by 3.7-
fold in leaves and 10-fold in roots. Similarly, ascor-
bate concentration varied almost 20-fold among
Dioscorea alata accessions. There is also apprecia-
ble intraspecific variation in amino acid
concentrations in edible tissues (Guzman-
Maldonado et al. 2000). However, the complement
of amino acids present in different foodstuffs is
constrained by evolutionary heritage such that
cereal and vegetable crops contribute complimen-
tary amino acids to the diet (White and Broadley
2005).
1.5 Assessing Iron Bioavailability
from Biofortified Foods
Iron bioavailability from biofortified food can be
assessed through four different approaches:
(i) algorithmic approximations, (ii) in vitro diges-
tion and Caco-2 intestinal cell uptake assay, (iii)
animal studies, or (iv) human studies (Cockell
2007; Fairweather-Tait 2001). The algorithmic
method is the least suited to predict the effects of
new circumstances, such as the nutritional impact
of a new biofortified crop. By contrast, the choice
between the remaining methods should take into
account experimental costs, short- and long-term
responses, and differences in iron absorption
Table 1.1 Mineral nutritional enhancers and
antinutrients
Nutritional enhancers Antinutrients
b-Carotene (provitamin A) Oxalic acid
(oxalate)
Inulin Phytic acid
(phytate)
Long-chain fatty acids Polyphenols
Certain amino acids (cysteine,
lysine, etc.)
Tannins
Certain organic acids (ascorbic acid,
citrate, etc.)
Others
Vitamin D Others
Adapted from White and Broadley (2005); Welch and
Graham (2005)
14 U. Singh et al.
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between laboratory animals and humans (Cockell
2007; Walter et al. 2003). It is however important
to note that at least two types of human bioassays
for bioavailability have qualified as necessary:
efficacy and effectiveness trials. Efficacy trials
assess the beneficial effect of iron biofortification
under ideal conditions and thus depend only on
biological factors (such as in clinical trials); effec-
tiveness trials take into account behavioral factors,
because trials are performed under “real-life
situations” (Davidsson and Nestel 2004).
1.6 Biofortification: Strategic
Advantages
The biofortification strategy seeks to take advan-
tage of the consistent daily consumption of large
amounts of food staples by all family members,
including women and children who are most at
risk for micronutrient malnutrition. As a conse-
quence of the predominance of food staples in
the diets of the poor, this strategy implicitly
targets low-income households.
After the one-time investment is made to
develop seeds that fortify themselves, recurrent
costs are low and germplasm may be shared
internationally. It is this multiplier aspect of
plant breeding across time and distance that
makes it so cost-effective. Once in place, the
biofortified crop system is highly sustainable.
Nutritionally improved varieties will continue
to be grown and consumed year after year, even
if government attention and international funding
for micronutrient issues fade. Moreover, bioforti-
fication provides a truly feasible means of
reaching malnourished populations in relatively
remote rural areas, delivering naturally fortified
foods to people with limited access to commer-
cially marketed fortified foods, which are more
readily available in urban areas.
Biofortification and commercial fortification,
therefore, are highly complementary. Breeding
for higher trace mineral density in seeds will
not incur a yield penalty. In fact, biofortification
may have important spin-off effects for increas-
ing farm productivity in developing countries in
an environmentally beneficial way. Mineral-
packed seeds sell themselves to farmers because,
as recent research developments proved that
seeds rich in trace elements are stronger to resist
against biotic and abiotic stresses including
diseases and environmental stresses (Bouis
2003). Further, fortified or enriched seeds also
have more plant vigour, seedling survival, faster
initial emergence and grain yield.
1.7 Future Challenges
• Produce crops for human nutrition with
increased iron concentration. Biofortification
strategies alternative to reduction in concen-
tration of phytic acid or polyphenols should be
explored further, in order to increase iron
absorption without loss of their beneficial
effects. When overexpressing ferritin, such
crops should be tested for concentration of
various heavy metals, in laboratory as in
open-field trials, before releasing to the pub-
lic. Detailed knowledge on mechanisms
regulating iron compartmentalization in vari-
ous plant organs will offer a major contribu-
tion for reaching such goal.
• Expand research on prebiotics and iron absorp-
tion. Crops biofortified with prebiotics have the
potential to partially circumvent the “iron par-
adox” caused by host–pathogen competition
for iron, by favoring amelioration of gut health
and gut-associated immune defense.
• Promote initiatives supporting large-scale
prospective studies on the effects of iron
biofortified crops on effectiveness of the
adopted biofortification strategy in relieving
iron deficiency anemia and in improving gen-
eral health.
• Improve the efficiency with which minerals
are mobilized in the soil.
• Improve the efficiency with which minerals are
taken up from the soil into the roots of the plant.
• Improve the transport of minerals from the
roots to storage tissues, such as grain.
• Increase the capacity of storage tissues to
accumulate minerals in a form that does not
impair plant vegetative growth and develop-
ment, but remains bioavailable for humans.
• Reduce the level of antinutritional compounds
such as phytic acid, which inhibit the absorp-
tion of minerals in the gut.
1 Biofortification: Introduction, Approaches, Limitations, and Challenges 15
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Glossary
Anemia condition in which the number of red
blood cells or their oxygen-carrying capacity
is insufficient to meet physiological needs.
The number of red blood cells is dependent
on age, gender, and altitude and is altered by
smoking, or during pregnancy the hemoglobin
threshold used to define anemia is <11 g/dL
Antinutrient a substance that impairs the
absorption of an essential element by the gut
Bioavailability measure of fractional utilization
of orally ingested nutrient and also defined as
the proportion of a particular nutrient that can
be used by the body to provide its associated
biological function. In simple words it is the
amount of an element in a food constituent or
a meal that can be absorbed and used by a
person eating the meal. Bioavailability also
refers to the portion of an ingested nutrient
that can be absorbed in the human gut. The
bioavailability of minerals can be reduced or
enhanced by the consumption of food rich in
antinutrients (inhibitors of absorption) or
nutritional enhancers, respectively
Biofortification process for improving the
nutritional value of the edible parts of the
plants, through mineral fertilization, conven-
tional breeding, or transgenic approaches. It
can also be defined as the process of increas-
ing the bioavailable concentrations of an ele-
ment in edible portions of crop plants through
agronomic intervention or genetic approaches
Disability-adjusted life year (DALY) a time-
based parameter for assessing global burden
of disease that combines years of life lost due
to premature mortality (years of life lost, YLL)
and years of life lost due to time lived in states
of less than full health (years lived with dis-
ability, YLD): DALY¼YLL+YLD
Hidden hunger the term “hidden hunger” has
been used to describe the micronutrient mal-
nutrition inherent in human diets that are ade-
quate in calories but lack vitamins and/or
mineral elements
Hunger the physical sensation of desiring food.
When politicians, relief workers, and social
scientists talk about people suffering from
hunger, they usually refer to those who, for
sustained periods, are unable to eat sufficient
food to meet basic nutritional needs
Food fortification with iron a way to increase
iron concentration in food by adding an iron
compound (e.g., ferrous sulfate heptahydrate,
ferrous gluconate, and sodium FeEDTA
among others) to processed food (e.g., infant
formula or cereals, wheat flour products, and
corn meal among others)
Fortification the addition of an ingredient to
food to increase the concentration of a partic-
ular element
Malnutrition the condition that results from
eating a diet in which certain nutrients are
lacking, in excess (too high in intake), or in
the wrong proportions. The verb form is
“malnourish”; “malnourishment” is some-
times used instead of “malnutrition.” A num-
ber of different nutrition disorders may arise,
depending on which nutrients are under- or
overabundant in the diet. In most of the
world, malnutrition is present in the form of
undernutrition, which is caused by a diet
lacking adequate calories and protein and not
enough food, and of poor quality. Extreme
undernourishment is starvation, and its
symptoms and effects are inanition. While
malnutrition is more common in less devel-
oped countries, it is also present in
industrialized countries. In wealthier nations
it is more likely to be caused by unhealthy
diets with excess energy, fats, and refined
carbohydrates (WHO 2001)
Promoter a substance that stimulates the
absorption of an essential element by the gut
Reference nutrient intake (RNI) the amount of
an element that is enough, or more than
enough, for most people in a group (usually
at least 97 %). If the average intake of a group
is at the RNI level, then the risk of deficiency
in the group is small
Regulators organic compounds, either natural
or synthetic, are able to modify or control
plant growth and/or development. Also, plant
growth regulators (PGRs)
Supplementation the addition of an element to
the diet to make up for an insufficiency
16 U. Singh et al.
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