1.

Introduction:
Wheat, one of the most important staple food crops, and is the world’s second most important
cereal crops which represents 44% calorie of global cereal consumption. It is grown on about 225-
million-hectare land worldwide from the equator to latitudes of 60 0 N and 440 S and at altitudes
ranging from sea level to more than 3000 m above sea level. Approximately 600 million tons of
wheat is produced annually, roughly half of which is in developing countries (FAO,2013). Like
other cereals, wheat also contains sub-optimal quantities of micronutrients, especially zinc (Zn) and
Iron (Fe) which is mainly removed during milling. All living organisms requires essential
micronutrients to maintain proper metabolism and these nutrients are obtained from the diet of
human (Welch and Graham, 2004). In the regions where major dietary food consists of cereals are
low in micronutrient content. The World Health Organization (WHO) estimates that approximately
25% of world’s population suffers from anemia (WHO, 2008) and the Fe deficiency anemia caused
to the loss of over 46,000 disability adjusted life years (DALYs) in 2010 alone (Murray and Lopez,
2013).

2. Justification for biofortification:

Zinc and Iron are two of the well-known minerals important for human health .The concentration
of Zinc and Iron is very low in commercial wheat cultivars (Borill,2014) and these concentrations
are not sufficient for human nutrition in diets with wheat constituting the main source of essential
minerals. According to (Prasad,2013) there is no zinc storage system contained in our bodies, so
one has to consume iron and zinc rich diet every day for his/her own nutrition requirements.
Wheat being the major diet of people of developing countries, people suffer from nutrient
deficiency. Moreover, in present context, the global population is expanding at alarming rate and
the food demand is increasing. The main victims of malnutrition are the people from developing
nations whose major nutrition is cereal based. So, to combat the problem of food insecurity and
malnutrition, biofortification is one of the important measures because biofortified staple foods
though do not deliver high levels of minerals and vitamins per day compared to supplements or
fortified food products, they can increase micronutrient intake for the resource-poor people of
developing countries who consume them daily (Bouis,2011)

Biofortification is the process of increasing the content and bioavailability of essential vitamins and
minerals in staple crops, through plant breeding, transgenic techniques and agronomic practices, to
improve their nutritional status and to deal with alarming rates of malnutrition.

2.1. Role of zinc and iron in human physiology and health:

2.1.1. Zinc:
Zinc is present in all biological organisms where it has catalytic, structural, and regulatory
roles. It is required for the activity of over 300 enzymes involved in most metabolic pathways,
including the central nervous system, and as an integral component of zinc finger proteins that
 regulate DNA transcription (Cathy W. Levenson,2011). As a result, zinc is necessary for a
wide range of biochemical, immunological, and clinical functions. Zinc deficiency vary with
age. In early infancy, diarrhea is a prominent symptom. (Lazzerini and Ronfani, 2012) have
concluded that zinc supplementation in conjunction with oral rehydration therapy for diarrhea,
reduces the risk of mortality due to its protective effects. Since 2004, UNICEF and WHO have
recommended the use of zinc supplementation in the treatment of diarrhea. It also leads to
impaired cognitive function, behavioral problems, impaired memory, learning disability, and
neuronal atrophy. The major cause of Zinc deficiency is inadequate intake, increased
requirements and impaired utilization. According to (WHO,2013) zinc deficiency affect
approximately 155 million children globally.

2.1.2. Iron
Iron is a biologically essential component of every living organism. The body requires iron for
the synthesis of its oxygen transport proteins, in particular hemoglobin and myoglobin, and for
the formation of heme enzymes and other iron-containing enzymes involved in electron transfer
and oxidation-reductions (Abbaspour,2014). Fe deficiency causes various physiological
diseases, such as anemia and neurodegenerative disorders. A recent report based on the (WHO
2015) described that globally over 30% of the world’s population are anaemic, many due to
iron deficiency.

2.2. Contribution of wheat to zinc and iron intake:

According to a (WHO report 2002), the risk factors responsible for development of illnesses and
diseases, Zn deficiency ranks 11th and Iron deficiency ranks 9th.

Wheat are genetically low in Zn and Fe concentration, with reduced bioavailability as amino
acid is relatively low in wheat (Bouis ,2003). About half of the world’s wheat production come
from soils low in plant available micronutrient (Bal R. Singh,2018), leading to poor quality of
wheat grain with respect to Zn content. The situation is similar, concerning Fe deficiency in
wheat. At present, bread wheat alone is the staple food for 35% of the world’s population
(Cakmak,2017).The concentration of minerals vary within the grain of wheat i.e. major
concentration of Fe and Zn accumulates in the embryo and aleurone layer than in endosperm of a
wheat grain (Singh,2018). Hence, the consumption of whole grain wheat rather than white wheat
flour has been recommended to increase the daily Fe and Zn intake. Generally, the concentration
of zinc is 27mg/kg and iron is 42mg/kg (Cakmak,2010) .The concentration of wheat needs to be
increased by 10 or 20 mg in order to combat the problem of malnutrition.

Due to the low amount of Zinc and Iron in the wheat grain, vulnerable people are mainly suffered
from hidden hunger. So, Nitrogen fertilization is known not only to increase wheat grain yield
 but also to facilitate the uptake of Fe and Zn in wheat grain (Cakmak,2010). According to
(Kutman ,2010) the uptake and transport of Fe and Zn to grain is probably facilitated by metal
chelating compounds, such as 2-deoxymugineic acid (DMA), mainly for the translocation of Fe
and Zn from flag leaves to grain in wheat (Barunawati,2013). Improving N status of plants from
low to sufficient resulted in threefold increase in shoot Fe content of wheat plants
(Ackisoz,2011). Similarly,(Erenoglu,2011) demonstrated that N is a critical player in the uptake
and accumulation of Zn in plants and thus deserves special attention in biofortification of food
crops with Zn.

2.3. Dilution of zinc and iron increase yield: (green revolution and food security)
In most cases, there is an inverse relationship between grain yield and grain zinc concentration
(Mcdonald,2008) with higher grain zinc concentrations being most commonly associated with
lower yielding genotypes (Mcdonald,2008). Moreover, some studies reveal that
(Habib,2009) there was a significant positive correlation between grain yield and grain zinc i.e.
an increase in grain zinc concentration substantially enhanced grain yield. According to
(Muhammad U. Chattha,2017) zinc application improves yield and yield components through
various mechanisms, for example, it improves chlorophyll content and activates photosynthetic
activity and auxin synthesis which lead to better growth and development of the crop, thus
effectively expanding yield and yield components . Studies in the past have mentioned that
dilution of Zn and Fe in wheat grain occurs at increased grain yields (Badhakshan,2013).
Similarly, (Zhao ,2008)  reported that Zn- concentration of wheat grain correlated negatively with
grain yield, but the correlation with grain weight was weak. A positive correlation of Zn- and Fe-
uptake (i.e., total uptake in grain) suggested that higher concentrations of Zn and Fe in wheat
grain were due to the increased uptake from soil or translocation of Zn and Fe from vegetative
parts to the grain (Cakmak,2010).

Food insecurity and malnutrition is the major problem of whole world, especially of developing
countries due to poor diet. So, considering the ever-growing global demand for food and
widespread occurrence of zinc and iron malnutrition, increasing grain Zn and Fe concentration in
high-yielding wheat cultivars is important (Graham,2007) as population is expanding and food
demand is increasing. Food insecurity and malnutrition is the major problem of whole world,
especially of developing countries due to poor diet.

2.4. Localization and bioavailability of Zn and Fe in wheat grain:

The wheat grain is a single‐seeded fruit, called a caryopsis. It contains a small embryo, which
forms the new plant on germination, and a large storage tissue (the endosperm), which comprises
mainly starch (a source of energy) and protein. Iron and zinc, together with other minerals, are
concentrated in the embryo and in the outer layer of endosperm cells, called the aleurone. The
two minerals, iron and zinc, differ slightly in distribution between the two tissues, with iron being
more concentrated in the aleurone and zinc in the embryo because the starchy endosperm cells
 die (Maga 1982.Neal et al), during the later stages of grain maturation (Todd E. Young). The
starch and protein are mobilized as sugars and amino acids respectively after lytic digestion by
enzymes secreted from the aleurone layer and embryo. Hence, the two minerals differ in
distribution between the two tissues, with iron being more concentrated in the aleurone and zinc
in the embryo.

Iron and zinc are transported in plants as soluble forms chelated by small organic molecules and
reach the developing grain through specialized vascular tissues (xylem and phloem).The
enhancement of amino acid promotes the bioavailability of micronutrient and decreases the anti-
nutrient substance (phytic acid ) which inhibit the micronutrient bioavailability (Welch and
Graham,2004).The intercellular part of wheat contains phosphate in the form of phytic acid.
Phytic acid has a cyclic structure with six phosphate groups, each of which can bind a metal ion
such as iron and zinc. Thus, due to its binding properties, the high concentration of phytic acid in
cereal-based foods is a major cause of zinc and iron deficiency in humans. Also, the organic acids
and malate facilitate the transport of iron in the xylem while nicotianamine facilitates iron and
zinc transport in the phloem and intracellularly (Connorton et  al. 2017a). According to
(Balk,2018) nicotianamine enhances the bioavailability of iron and zinc. So, to combat the
deficiency of zinc and iron, use of nicotianamine and amino acids play the important role.

3. A
g r
o
n
o
m
y

Staining and localization of Zn in a bread wheat Perl’s Prussian Blue staining for Iron in wheat
grain: Without foliar application of zinc (Left) and grains : Control line (Top) and High-iron line
with foliar application of Zinc (Right) (Bottom).

biofortification:
Zinc and Iron deficiency is the major problem, mainly of nutrient deficient soil areas of world
leading to hidden hunger. Agronomic biofortification of wheat is aimed to increase the
micronutrient concentration so as to reduce dietary deficiency. Soil and foliar application of
 fertilizer can be applied to reduce the deficiency problem. Also, agronomic biofortification with Fe
and Zn seems to be a very cost-effective, fast and practical approach to improve Fe and Zn
concentration in wheat (Cakmak 2007) to insure food security. Application of Iron sulphate
(FeSO4) and zinc sulphate (ZnSO4) and as alone or in combination either with soil and foliar
application increased the economic yield and concentration of Fe and Zn. Various research has
been performed to establish evidence towards the use of foliar application of zinc sulphate and iron
sulphate.

A study has shown that the foliar application and timing of Zinc supply is one of the important
factor enhancing the concentration of Zinc in wheat. Application of Zinc both as soil treatment
(5kg/ha) and foliar supply (1% ZnSO4) increases the concentration of Zinc in wheat grown in all
types of soils (Khattak et al,2016). Also, (Zhou et al 2012) proved that foliar Zn supply alone or in
combination with soil increases the concentration by 84% and 90%. Along with the method of
application, timing of application is also important factor for enhancing the concentration.
According to (Cakmak et al 2010) foliar Zn application especially at the later growth stages (e.g.
early milk stage and dough stage) was most effective in ZN concentration of both whole grain as
well as of endosperm in wheat while as soil Zn applications remained less effective. It enhanced
grain Zn concentration by 2- or 3- fold depending on the plant availability of Zn in soils, in wheat.
Irrespective to the zinc availability in wheat after application, Fe is known to rapidly convert into
unavailable forms upon application to soils and poses poor mobility in Phloem, soil or foliar Fe. It
is for this reason that Fe is attributed to be less effective than Zn for enrichment of wheat (Riaz et
al 2017). Foliar application of FeSO4 or Fe chelates increases grain Fe concentration by only 36%
in wheat. Application of Fe fertilizers either in inorganic (FeSO 4) or in chelated form (e.g. Fe-
EDDHA, Fe-EDTA or Fe-citrate) had only a small positive impact on increasing Fe concentration
in grain while as soil N application enhanced by 47% (Aciksoz 2011).

Hence, it is suggested that the foliar application at the right time and at the right amount increases
the activity of Zn and Fe in the flag leaf and the stem during grain filling period. A number of
researches have been reported that the combined application of Zn and Fe (0.5% ZnSO 4 and 1%
FeSO4) through foliar and spray is recommended to enhance the productivity of wheat crop with
good quality of grains. (Zhao 2011) Fe and Zn when supplied together increases the chlorophyll
content and also overcomes the deficiency.

4. Genetic biofortification:
Genetic biofortification involves both traditional breeding as well as biotechnological tools.
Transgenic approach is maximum researched but of minimum utilization. In this approach, a
content of wheat has been enhanced by expressing bacterial PSY and carotene desaturase genes [
CrtB, Crt1 (76, 77)]. The iron content in wheat has been enhanced by expression of ferritin gene
from soybeans (78) and wheat [TaFer1-A (79)] (Monika Garg). Rather than transgenic techniques,
 plant breeding can be used more successfully to enrich the concentration in grains. The efforts led
to the development of programs such as HarvestPlus focusing on increasing the concentration of
zinc and iron in wheat (Bouis). Plant breeding for biofortification is conducted within the
agricultural research partnership known as the CGIAR, while HarvestPlus, a program within the
CGIAR, sets the micronutrient targets for breeding. HarvestPlus is directed at using plant breeding
as an intervention strategy to address micronutrient malnutrition by producing staple food crops
with enhanced levels of bioavailable essential minerals and vitamins that will have measurable
impact on improving the micronutrient status of targeted populations, primarily resource poor
people in the developing world. Impressive progress has been made at meeting the goals of the
HarvestPlus program set forth at its inception in 2003, but much remains to be done (Bouis, 2009).
Therefore, Conventional breeding has been the primary focus of programs to enhance staple food
crops with sufficient levels of Fe, Zn, to meet the needs of at risk populations in the Global world.

The concentration of Zinc and Iron in wheat is influenced by numerous genes that is absorbed by
roots, translocated to shoots, remobilized in vegetative parts and stored in grains. Similarly,
environment and gene interaction also favored to alter the concentration of minerals in grains
(Bouis, 2010). Henceforth, the harvest plus program have strongly marked these gene interactions
and overwhelm the problems of nutrient deficiency. The biofortified seeds will be disseminated by
HarvestPlus through established partnerships with country agencies and the seeds will be available
to the farmers and, ultimately, to the consumer groups. The dissemination program will be
conducted by HarvestPlus in several stages. First, the seeds will be multiplied and test the
biofortified lines at multiple locations in trials throughout the target country to determine
environmental × genetic interactions on expression of the high micronutrient traits in the
biofortified crops by National Agricultural Research and Extension Programs. Selected promising
lines from this trait will be formally submitted to the Varietal Release Committees for further
testing and, once approved, will be officially released within the target country. The completion of
this process may take up to 8 years. Once implemented, baseline nutritional studies will be
compared to post dissemination impact and effectiveness studies in both control and intervention
locations to establish if biofortified crops can improve the micronutrient status of people in target
populations.

5. Advantages of Biofortification

Biofortification of wheat have several complimentary advantages. The major expected and intended advantages of
biofortification is to increase micronutrient intakes among the poor, thus improving their nutrition and health status. Non-
staple food prices have increased steadily and substantially, making it more difficult for the poor to afford a good quality diet
(Bouis et al. 2011). Thus, biofortification can be more cost-effective in the long term and reach underserved rural
populations who may have limited access to diverse diets or other micronutrient interventions. Tying micronutrients to staple
crops also reduces people’s nutritional vulnerability, because, when economic shocks occur, the poor tend to reduce their
consumption of higher-value food commodities that are naturally rich in micronutrients. Furthermore, biofortification could
be more sustainable than alternative micronutrient interventions. Since biofortified seeds can easily be reproduced, poor
farmers in remote rural areas, with limited access to formal seed markets, could also be reached. unlike other micronutrient
interventions, which require large funds on an annual basis, biofortification could produce a continuous stream of benefits
with minimal recurrent costs. Biofortification promises to be a pro-poor and pro-rural approach, which could complement
existing interventions. Biofortification provides a truly feasible means of reaching malnourished populations in relatively
remote rural areas, delivering naturally- fortified foods to population groups with limited access to commercially-marketed
fortified foods that are more readily available in urban areas (H.E. Bouis, 2003). There is no requirement of behavioral
changes in uptake of desired staple food since it can provide desired nutrients. Biofortified wheat crops cannot deliver as high
a level of minerals and vitamins per day as supplements or industrially fortified foods, but they can help to bring millions
over the threshold from malnourishment to micronutrient sufficiency. Similarly, the main advantage of biofortification is, it is
sustainable approach to micronutrient enrichment as compared to commercially-marketed fortified foods because seeds are
self-fortifying.

6. Challenges:
6.1. Economic and Environment concerns of fertilizer
The success of agronomical biofortification depends on a number of factors viz, mineral
mobility, mineral accumulation within the plant species, soil composition at the specific
geographical location of each crop. Agronomic biofortification involves recurrent cost because of
continuous application of inputs (fertilizers) at regular intervals and is also labor intensive.
Besides, it is not always possible to target the micronutrient into edible plant part like seed/grain
or fruit and can sometimes accumulate in non-desired plant parts, resulting in resource wastage
and rendering the whole activity as futile. Moreover, the biggest of all constraints is that of the
environmental degradation due to accumulation of fertilizers in the soil and water.

6.2. Adverse soil conditions

6.3. Releasing of high cultivar and large offtake of nutrients.

7. Conclusion

8. Acknowledgement

9. Abstract
Wheat being the predominant source of nutrient in developing countries, mainly of Asia and
Africa, contains low level of micronutrients such as iron and zinc.