CHAPTER TWO

Agronomic Biofortification of
Cereal Grains with Iron and Zinc
Rajendra Prasad*,†,1, Yashbir S. Shivay†, Dinesh Kumar†
*Indian National Science Academy, New Delhi, India
†
Division of Agronomy, Indian Agricultural Research Institute, Pusa, New Delhi, India
1
Corresponding author: e-mail address: rajuma36@gmail.com

Contents
1. Introduction 56
2. Biofortification of Cereal Grains 58
3. Micronutrients in Human Nutrition 60
4. Functions and Deficiency of Fe and Zn in Humans 60
4.1 Iron 60
4.2 Zinc 61
5. Agronomic Biofortification of Cereal Grains 62
5.1 Rice 62
5.2 Wheat 67
5.3 Corn 69
5.4 Oats 69
6. Management Practices Other Than Fertilizer Affecting Fe and Zn Concentration
in Cereal Grains 69
6.1 Tillage 69
6.2 Water management 70
6.3 Interaction with other nutrients 71
6.4 Plant growth promoting rhizobacteria and mycorrhiza 72
7. Soil Factors Affecting the Availability of Fe and Zn to Plants 73
7.1 Amounts present in soil 73
7.2 Soil solution pH 73
7.3 Mechanisms of Zn fixation other than pH 75
8. Plant Factors Affecting Uptake of Fe and Zn 76
8.1 Root characteristics 76
8.2 Phytosiderophores 76
8.3 Organic acids 77
8.4 Zinc utilization at the cellular level 78
8.5 Translocation in plants 78
8.6 Mechanisms of Zn accumulation in grain 79
9. Conclusion 79
References 80

Advances in Agronomy, Volume 125 # 2014 Elsevier Inc. 55

ISSN 0065-2113 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800137-0.00002-9
56 Rajendra Prasad et al.

Abstract
Iron and zinc deficiencies in human nutrition are widespread in developing Asian and
African countries where cereal grains are the staple food. Effects are therefore underway
to develop cereal genotypes with grains denser in Fe and Zn by traditional plant breed-
ing or using genetic engineering techniques. This approach requires a long period and
adequate funds. However, the products of genetic engineering are not well accepted in
many countries. Also, there is a trade-off between yield and grain biofortification. Agro-
nomic biofortification offers to achieve this without sacrificing on yield and with no
problem of product acceptance. From the viewpoint of biofortification, foliar applica-
tion has been reported to be better than the soil application of Fe and Zn, and for this
purpose, chelated Fe and Zn fertilizers are better. When soil applied, water soluble
sources of Zn are better. Soil application of Fe is not recommended. Agronomic bio-
fortification depends upon management practices (tillage, water management, nutrient
interactions), soil factors (amounts present, pH, mechanisms of Zn fixation other than
pH), and plant factors (root characteristics, excretion of phytosiderophores and organic
acids by roots, Zn utilization at the cellular level, translocation within plant and mech-
anisms of Zn accumulation in grain). Genetic and agronomic biofortification are com-
plementary to each other. Once the genotypes having denser grains are developed,
they will have to be adequately fertilized with Fe and Zn. However, much more research
in agronomy, soil science, and plant physiology is needed to understand the complex
soil–plant–management interaction under different agroecological conditions under
which cereals are grown. The situation is more complex for rice, which is grown under
flooded, upland, and intermediate water conditions.

1. INTRODUCTION
Cereals are staple food in most developing low-income countries of
Asia and Africa, where they may contribute as much as 55% of the dietary
energy (Fig. 2.1). Rice feeds more than half the world population and meets
21% of energy and protein needs of human population globally (McLean
et al., 2002). About 90% of rice is grown and consumed in South, South-
east, and East Asia, where about 62.5% of the world’s total 925 million
hungry people reside; about 25.8% of world’s hungry people reside in
sub-Saharan Africa (FAO, 2010). Apart from hunger and malnutrition per
se, Fe deficiency anemia affects 88% of the pregnant women in South Asia,
contributing to maternal mortality and impaired mental development in vast
number of children (IRRI, 2006); in 2009, about 60% of the under-5-year
children were underweight in the 20% poorest group in this region
(UN, 2011). About 2 billion people suffer globally due to Fe deficiency ane-
mia, mostly in developing countries (Stolzfus and Dallman, 1998). As many
Agronomic Biofortification of Cereal Grains 57

Figure 2.1 Contribution of cereals to the dietary energy in high-income and low-
income countries. From FAO (2008).

as 79.1% of India’s children between the ages of 3 and 6 years, and 56.2% of
married women (15–49 years) are anemic (Krishnaswamy, 2009). Vitamin
A deficiency affects 169 million preschool children in South and Southeast
Asia (33% of all preschool children) and 104 million (32% of all preschool
children) in sub-Saharan Africa (IRRI, 2006). It is estimated that 60–70%
of population in Asia and sub-Saharan Africa could be at risk of low Zn defi-
ciency intake (Gibson, 2006); in absolute numbers, this translates to 2 billion
people in Asia and 400 million people in sub-Saharan Africa (IRRI, 2006).
More than one-third of the world’s population suffers from Zn deficiency
(Hotz and Brown, 2004; Stein, 2010), and Zn deficiency has been estimated
to be responsible for approximately 4% of the worldwide burden of morbid-
ity and mortality in under-5-year children and a loss of nearly 16 million
global disability-adjusted life years (Black et al., 2008; Walker et al.,
2009). Pirzadeh et al. (2010) reported that in Iran, rice provided 96% and
42% of the dietary Fe needs for male and female adults, respectively; the
values for Zn were only 30% and 22% for male and female adults, respec-
tively. Cereal grains are inherently low in concentration as well as bioavail-
ability of Zn, particularly when grown on Zn-deficient soils (Cakmak et al.,
2010a; Welch and Graham, 2004).
Wheat is the second staple cereal after rice in South Asia (India,
Pakistan, Nepal, and Bangladesh) (Prasad, 2005), China, and Turkey
(Chatrath et al., 2007) and is responsible up to 70% of the daily calorie
58 Rajendra Prasad et al.

intake of population living in rural areas in some of these countries

(Cakmak, 2008; Zhang et al., 2010a).
In Asia, China is the major corn-producing country, while in Africa it is a
major cereal in Egypt, South Africa, Zimbabwe, Malawi, and several other
countries. In 2010, the United States produced 37.8% of the total corn pro-
duced in the world, while China produced 20%. Of course in most corn-
producing countries, human nutrition is better, due to use of more meat,
poultry, fish, etc.

2. BIOFORTIFICATION OF CEREAL GRAINS

In view of globally widespread deficiencies of vitamin A, Fe, I, and Zn
in humans, the problem was first attended to by physicians and nutritionists.
A number of Vitamin A and Fe containing nutrition supplements are avail-
able off-the-shelf throughout the world; Zn has been recently added to
vitamin and mineral supplements (Haider and Bhutta, 2009; Hess and
King, 2009). National Institute of Nutrition, Hyderabad, India, has devel-
oped common salt fortified with both Fe and I (Sesikaran and Ranganathan,
2009). The other approach adopted by nutritionists is an enrichment of
cereal-based traditional foods with vitamins and minerals (Gibson, 2005;
Graham et al., 2007) either at home or in factories (Brown et al., 2010)
for products such as flour, bread, biscuits, etc. Vitamin A is added to several
cooking oils marketed in India. Recently, iodized salt is being marketed in
India to overcome iodine deficiency. Yet another approach is to improve
dietary composition by adding more red meat, fruits, and green vegetables
(Gibson and Anderson, 2009). The major disadvantage of these approaches
is that the major beneficiaries are urban people and rural poor masses are left
out in developing countries. As regards enrichment of traditional foods with
minerals, this requires a large number of workers/volunteers and continuous
supply of funds for demonstrations, which again may remain restricted to
urban or peri-urban people. Improving dietary composition with foods
richer in Fe, Zn, and vitamins requires money and in many developing
countries including India, poor purchasing power of a large part of human
population is a major issue (Swaminathan, 2002).
Considering Fe and Zn as an important nutritional problem, specially in
the developed countries (Graham, 2008), the need for genetic bio-
fortification of cereals by developing cultivars with denser Zn (and other
micronutrients) grains was mooted by Dr. Robin D. Graham of the Univer-
sity of Adelaide, Australia, Dr. Ross M. Welch of the U.S. Plant, Soil and
Agronomic Biofortification of Cereal Grains 59

Nutritional Laboratory, USDA-ARS, Ithaca, NY, USA, and Howarth,

E. Bouis of the International Food Policy Research Institute, Washington,
DC, USA (Bouis et al., 2011; Grahan et al., 2001). Genetic biofortification
may involve both traditional breeding as well biotechnological tools. Their
efforts led to the development of programs such as HarvestPlus, Golden
Rice, and African Biofortified Sorghum Project, focusing on the develop-
ment of crop varieties capable of producing grains denser in Zn and other
micronutrients (Stein, 2010). Golden rice has been engineered to express
beta carotene by introducing a combination of genes that code for biosyn-
thesis pathway for the production of provitamin A in the endosperm
(Ye et al., 2000). Enhancement of Fe content in rice has been also achieved
by improving the uptake from soil and by increasing the absorption and
storage of Fe (Murray-Kolb et al., 2002; Takahashi et al., 2001). Further,
Genetically Modified (GM) rice has been developed that produces both
beta-carotene and ferritin (Potrykus et al., 1996). However, there are prob-
lems in the acceptance of GM crops in several countries ( Jaffe, 2005).
A detailed discussion on this is beyond the scope of this review.
There is a large genotypic variation in grain Zn concentration of modern
wheat genotypes and its wild relatives (Cakmak et al., 2010a; Gome-Becerra
et al., 2010a,b). Similarly in screening of close to 1000 rice genotypes at the
International Rice Research Institute, Los Banos, Philippines, grain Zn
concentrations ranged from 15.9 to 58.4 mg kg
1 (Graham et al., 1999).
In most cases, there is an inverse relationship between grain yield and grain
Zn concentration (Garvin et al., 2006; McDonald et al., 2008) and higher
grain Zn concentration is most commonly associated with lower grain yield
(Fan et al., 2008; Oury et al., 2006). Breaking the trade-off between grain
yield and Zn concentration is an important issue in programs for breeding
for high Zn concentration in cereal grains (Bouis and Welch, 2010; Waters
and Sankaran, 2011; Zhao and McGarth, 2009). Such programs require
fairly long time and so far no Zn-rich cultivar in any cereal has become avail-
able. Further such cultivars may not be high yielding. Combining high grain
yield and high Zn concentration in grain needs still more time. Also, the
products from newly developed crops may not be acceptable to consumers,
who have their own preferences.
On the contrary, agronomic biofortification through Zn fertilization
(also referred to as ferti-fortification; Prasad, 2009) results in increased grain
production as well as higher Zn concentration in grains at the same time.
Also, even cultivars developed by genetic biofortification will need Zn
fertilization.
60 Rajendra Prasad et al.

3. MICRONUTRIENTS IN HUMAN NUTRITION

The basis of defining micronutrients in human nutrition is same as in
plant nutrition, that is, the nutrients that are required in small amounts.
However, in plant nutrition, these are only minerals (Fe, Mn, Zn, Cu, B,
Mo, Ni, Cl) (Prasad and Power, 1997), but the term micronutrients in
human nutrition include vitamins [A, D,E, K, C, B1(thiamine), B2 (ribofla-
vin), B3 (pantothenic acid), niacin, B6 (pyridoxal), folate, biotin, B12(cobal-
amin)], fatty acids linoleic and linolenic, and 17 minerals (Fe, Zn, Cu, Mn, I,
F, B, Se, Mo, Ni, Cr, V, Si, As, Li, Sn, Co (in B12); Gibson, 2005). Mineral
micronutrients make up only 0.05% of human body, whereas the macronu-
trients (C, H, N, O, Na, Mg, P, S, Cl, K, Ca) make up the rest 99.5% (Kotz
et al., 2006). All the micronutrients essential for plants are also essential for
humans and their concentration in cereal grains can be agronomically man-
aged. At present, only Fe and Zn deficiencies in humans have emerged glob-
ally, and Agronomists and Soil Scientists certainly have a role in alleviating
these deficiencies, and this is why this review.
An important point other than concentration per se in Fe and Zn in grains
is their bioavailability. In general, the efficiency of Zn absorption from a diet
ranges from 15% to 35%, depending upon the amounts consumed (decreases
with an increase in amounts consumed) and the presence of dietary phytate
(Hambidge et al., 2010). Phytate forms insoluble complexes with Zn that
cannot be digested or absorbed because of the absence of intestinal phytate
enzymes in humans (Iqbal et al., 1994). Diets with phytate/Zn molar ratios
above 15 are associated with Zn deficiency in humans (Bindra et al., 1986).
Gibson (2005) reported a Zn concentration(mg kg
1) of 22 in maize flour,
14 in sorghum flour, and 18 in white or polished rice and a phytate/Zn
molar ratio of 36 in maize flour, 32 in sorghum flour, and 5 in white rice.
Thus, Zn in rice was more absorbable.

4. FUNCTIONS AND DEFICIENCY OF Fe AND Zn IN

HUMANS
4.1. Iron
In humans, Fe is the key micromineral nutrient. As a component of hemo-
globin, it is involved in oxygen transport and storage and thus is responsible for
the very survival of the mankind. About 85% of Fe in human body is present as
a constituent of two heme proteins, namely, hemoglobin, responsible for the
Agronomic Biofortification of Cereal Grains 61

transport of oxygen from the lungs to other parts of the body, and myoglobin
that stores oxygen in the muscles (Bell and Dell, 2008).
Fe is also present in several nonheme proteins such as ferritin and
transferritin, which can transport Fe, and in metalloflavoproteins, ferrodoxins,
or FE-S proteins (Yip and Dallman, 1996). The clinical deficiency symptoms
include pallor (anemia), fatigue, weakness, dizziness, reduced intellectual per-
formance, and reduced work capacity (Lynch, 2003). The recommended
daily allowance (RDA) (mg Fe day
1) is 10 for children (4–8 years), 8 for adult
males, and 18 for adult females (USFNB, 2001).

4.2. Zinc
It is estimated that in humans 2800–3000 proteins contain Zn prosthetic
group (Tapeiro and Tew, 2003). Zn is the only metal to be involved in
all six classes of enzymes: oxidoreductases, transferases, hydrolases, lysases,
isomerases, and ligases (Barak and Helmke, 1993). Further, Zn is required
for the activation of over 300 enzymes (Gibson, 2012). Zn ions are also neu-
rotransmitters and are present in the cells of the salivary glands, prostrate, and
immune system (Herschfinkel et al., 2007). Zn plays a key role in physical
growth and development, the functioning of the immune system, reproduc-
tive health, sensory functions, and neurobehavioral development. Zn is an
integral component of zinc finger proteins that regulate DNA transcription
(Levenson and Morris, 2011).
Zn is a “Type 2” nutrient, which means that its concentration in blood
does not decrease in proportion of the degree of deficiency (Alloway, 2009).
As a result, physical growth slows down and excretion is reduced to conserve
Zn. Thus, children suffering from Zn deficiency have reduced linear growth
(stunting) (Graham, 2008). Adverse health effects of Zn deficiency vary with
age: low weight gain, diarrhea (Bahl, 2002; Bhatnagar, 2004), aneroxia
(Bhandari, 2002), and neurobehavioral disturbances are observed during
infancy, whereas skin changes, blepharoconjunctivitis, and dwarfing are fre-
quent among toddlers and school children (Hambidge, 1997). Common
manifestations of Zn deficiency among the elderly include hypogeusia
(impaired taste sensitivity), chronic nonhealing leg ulcers, recurrent infec-
tions, and adverse pregnancy outcomes (Brown et al., 2004). Zn is also
essential for regulating intestinal absorption of Fe, and sufficient quantity
of Zn along with Fe in human body is crucial for treating Fe deficiency ane-
mia (Graham et al., 2012). The RDA (mg Zn day
1) is 5 for children (4–8
years), 11 for adult males, and 8 for adult females (USFNB, 2001).
62 Rajendra Prasad et al.

5. AGRONOMIC BIOFORTIFICATION OF CEREAL GRAINS

Essentiality of Fe in plants was reported by Sachs in 1860, while that of
Zn was established by Maze’ in 1916 (Bell and Dell, 2008). Zn deficiency
was later reported in citrus in the United States (Chapman et al., 1940).
A number of reviews on Zn in crop nutrition are available (Alloway,
2008; Hodgson, 1963; White, 1993). In India, Zn deficiency was first
reported in rice by Nene (1966), and was followed by that in wheat in
Punjab. Research on Zn in relation to crop production in India has been
thoroughly reviewed (Katyal and Rattan, 2003; Prasad, 2006; Shukla
et al., 2012). However, most work on Zn fertilization was done from the
viewpoint of increasing crop yield. Work on agronomic biofortification
of wheat was started in Turkey by Cakmak (2004), while in India it was ini-
tiated on rice by the authors of this review (Shivay and Prasad, 2012; Shivay
et al., 2007, 2008a,b,c). Most information on biofortification of cereal grains
with Zn is available on rice and wheat and is briefly reviewed.

5.1. Rice
5.1.1 Method of application
Zn could be applied to soil or foliage. The seed priming with Zn fertilizers
and dipping of rice seedlings in Zn fertilizer solutions have been tested and
recommended for increased yield, but no data are available on their effect on
biofortification of rice grains. Shivay and Prasad (2012) from New Delhi
showed that on Zn-deficient soils, application of Zn (as zinc sulfate
heptahydrate or ZSHH) significantly increased grain yield of rice as well
as Zn concentration in rice grain (Table 2.1). Soil application of Zn also
increased Zn harvest index by 2%, although this was not statistically
significant.
Shivay et al. (2010a,b) also reported that foliar application of only 1.2 kg
Zn ha
1 as compared with 5.3 kg Zn ha
1 as soil application gave similar
grain yield of rice but higher Zn concentration in grain. Zn harvest index
for soil and foliar application was similar, but agronomic efficiency of Zn
with foliar application was about four times of that for soil application; rate
of Zn application was much lower when applied on foliage. Dhaliwal et al.
(2010) from Ludhiana, India, showed that averaged on five rice cultivars
foliar-applied Zn (three sprays of 0.5% ZSHH solution) recorded a Zn con-
centration of 47.0 mg kg
1grain in brown rice when compared with
33.8 mg kg
1grain in no Zn check. They also reported a Zn concentration
Table 2.1 Effect of method, source, and rate of Zn application on grain yield of Basmati rice (Pusa Sugandh 5) (averaged over 2 years)
Zn uptake
Zn Zn Zn harvest index in
Polished concentration concentration Agronomic ((Zn uptake in polished
Unhusked rice (PR) in unhusked in polished efficiency of Zn grain/Zn uptake in rice
rice (t ha
1) rice (mg kg
1) rice (mg kg
1) (kg grain kg
1 grain þ straw)  (g ha
1) BREzn

1
Treatment (1) (t ha ) (2) (3) (4) (5) Zn applied) (6) 100) (7) (8) (9)
Check (no Zn)a 3.92 2.74 30.4 26.1 – 16.7 71.5 –
Soil application of 5.20 3.64 47.5 40.3 241.5 18.7 146.7 0.30
25 kg
ZnSO47H2O ha
1
(5.3 kg Zn ha
1)
One foliar 4.99 3.49 52.6 28.8 901.3 17.5 100.5 2.42
application of 0.2%
ZnSO47H2O
(1.2 kg Zn ha
1)
Soil application of 4.48 3.14 38.2 32.4 215.2 17.8 101.7 1.12
1% ZnO-coated
urea (2.6 kg Zn
ha
1)
Soil application of 5.13 3.59 44.7 37.9 232.2 19.6 136.1 1.24
2% ZnO-coated
urea (5.2 kg Zn
ha
1)
Continued
Table 2.1 Effect of method, source, and rate of Zn application on grain yield of Basmati rice (Pusa Sugandh 5) (averaged over 2 years)—cont'd
Zn uptake
Zn Zn Zn harvest index in
Polished concentration concentration Agronomic ((Zn uptake in polished
Unhusked rice (PR) in unhusked in polished efficiency of Zn grain/Zn uptake in rice
rice (t ha
1) rice (mg kg
1) rice (mg kg
1) (kg grain kg
1 grain þ straw)  (g ha
1) BREzn

1
Treatment (1) (t ha ) (2) (3) (4) (5) Zn applied) (6) 100) (7) (8) (9)
Soil application of 4.69 3.28 40.3 34.1 296.1 18.0 111.8 1.55
1% ZnSO47H2O-
coated urea (2.6 kg
Zn ha
1)
Soil application of 5.27 3.69 49.7 42.1 259.8 19.1 155.3 1.61
2% ZnSO47H2O-
coated urea (5.2 kg
Zn ha
1)
LSD (P ¼ 0.05) 0.45 0.31 4.5 NC 33.4 2.2 NC NC
NC, not computed.
Column 2—Computed on the basis of data reported by Shivay and Prasad (2012).
Column 3—Computed on 70% recovery of polished rice as reported by Fang et al. (2008).
Column 4—Based on the data reported by Shivay and Prasad (2012).
Column 5—Based on the data reported by Phattarakul et al. (2012).
Column 8—Zinc concentration (mg kg
1) in polished rice  polished rice yield (t ha
1).
Column 9—BREzn (Biofortification recovery efficiency ¼ 100{[Zn conc. in polished rice (mg kg
1)  polished rice yield (t ha
1) in þ Zn plots)]
[Zn conc. in
polished rice (mg kg
1)  polished rice yield (t ha
1) in
Zn plots)]  Zn applied in kg ha
1}.
a
All plots received 60 kg N as urea, 26 kg P as single super phosphate (SSP) and 33 kg K as muriate of potash per hectare at final puddling of rice field. Prilled urea or ZnO
or ZnSO47H2O-coated urea were applied in two splits, half 10 days after transplanting and the rest half at panicle initiation. Soil application of ZnSO47H2O was made
along with P and K at final puddling, while foliar application of ZnSO47H2O was made at panicle initiation. Use of SSP as a source of P also took care of S application in
all plots. DTPA-extractable Zn in initial soil sample 0.36 mg Zn kg
1.
From Shivay and Prasad (2012).
Agronomic Biofortification of Cereal Grains 65

of 29.1 mg kg
1 husk in Zn-sprayed crop as compared with 25.2 mg kg
1

husk in no Zn check.
In a multilocation study in China, India, Lao PDR, Thailand, and
Turkey, Zn concentration in unhusked rice grain was about 69% higher
with foliar application than with soil application; at some centers, it was
almost twice that of with soil application (Phattarakul et al., 2012). This
study also provided data on relative Zn concentration in unhusked (whole
grain with husk, known as paddy in India; most of the data on biofortifcation
of rice are on unhusked rice.), brown rice (whole caryopsis with husk
removed by hand), and white rice (outer layer of pericarpsis including peri-
carp, testa, mucella, and part of aleurone layer along with embryo removed
by polishing for 30 s in a standard laboratory mill). White rice is also known
as polished rice (the form in which rice is mostly consumed). When Zn is
foliar applied, only 53–54% of that in unhusked rice is found in polished or
white rice as compared with 84.8%, when Zn is soil applied (Table 2.2).
However, when Zn is soil applied, brown rice may contain a little more than
in unhusked rice. Thus, a greater portion of foliar-applied Zn remained in
husk. These data support the viewpoint of Jiang et al. (2007) that in rice Zn
absorbed from the root plays a major role, while mobilization from the
leaves plays a minor role. Using the data of Phattarakul et al. (2012) as
the base, it worked out that although unhusked rice contained 52.6% Zn,

Table 2.2 Grain yield and relative zinc concentration in unhusked, brown, and white
(polished) rice (averaged over nine site-years in China, India, Lao PDR, Thailand, and
Turkey)
Control (no Soil þ foliar
Characteristic Zn) Soil Zn Foliar Zn Zn Significance
Grain yield 6.7 7.0 6.9 7.0 NS
(t ha
1)
Zn in unhusked 18.7 19.1 32.3 34.7 P < 0.01
rice (mg kg
1)
Zn in brown rice 19.1 20.8 24.4 25.5 (73.5) P < 0.01
(mg kg
1) (102.1)a (108.9) (75.5)
Zinc in polished 16.1 16.2 17.7 18.4 (53.0) P < 0.01
rice (mg kg
1) (18.1)b (84.8) (54.8) (72.1)
(84.2)c (77.9) (72.5)
a
Zn in brown rice expressed as percentage of unhusked rice.
b
Zn in polished rice expressed as percentage of brown rice.
c
Zn in polished rice expressed as percentage of unhusked rice.
From Phattarakul et al. (2012).
66 Rajendra Prasad et al.

the polished rice from it is likely to contain only 28.8% Zn, when Zn was
foliar applied in the study of Shivay and Prasad (2012). As a contrast, when
Zn was soil applied in sufficient quantity (Table 2.1), Zn concentration in
unhusked rice was 47.5%, while it was 40.3% in polished rice. Total Zn
uptake by polished rice was also higher with soil-applied Zn; of course,
much more Zn was applied to soil (25 kg ha
1) as compared with that on
foliage (1.2 kg ha
1). Biofortification recovery (BREzn), the term suggested
by Impa and Johnson-Beebout (2012), with foliar application was about
eight times of that obtained with soil application. Saenchai et al. (2012) from
Thailand reported that the decrease in Zn concentration on milling of rice
ranged from 16.2% to 48.2% in rice genotypes, being more in long and slen-
der grain types. The range of Zn (mg kg
1) in polished rice was 9.6–40.2
(mean 20.6) when compared with 17.3–59.2 (mean 28.7) in brown rice.
It may be pointed out that in eastern India and in some other Asian coun-
tries rice is parboiled before milling. Parboiling is a hydrothermal process to
which unhusked rice is subjected before milling. It involves soaking in
water, steaming, and drying: the degree of soaking and steaming differs con-
siderably (Singh, 1999). As parboiled rice gives much better head recovery,
most millers practice parboiling to some degree. On soaking unhusked rice,
nutrients from the outer layer of endosperm (pericarp, seed coat, nucellus,
and aleuron layer) move into endosperm and the parboiled white rice is
much richer in vitamins and minerals and has a better storage quality. Thus,
the data on biofortification of rice grains depend very much on the milling
process adopted. Recently, Prom-u-Thai et al. (2011) reported that when
rice grains were soaked in Fe-EDTA þ ZnSO4 solutions, Fe and Zn pene-
trated across the husk and aleurone layer into endosperm, and when
parboiled, the polished rice retained 70–80.5% of Fe and Zn. Also, Fe
and Zn polished rice was highly bioaccessible.

5.1.2 Sources of Zinc

Shivay et al. (2008a,c,d) and Shivay and Prasad (2010) from New Delhi
reported that ZNSHH-coated urea was significantly superior to ZnO-
coated urea in increasing Zn concentration in unhusked rice (also in
polished rice when calculated on the basis of Phattarkul’s data). The supe-
riority of ZSHH was also recorded in succeeding wheat (Shivay et al.,
2008a,c,d); Zn was applied to rice only. Water solubility of zinc sources
is considered as an important criterion for Zn availability (Slaton et al.,
2005). Westfall and Gangloff (2001) observed that the effectiveness of six
granulated Zn fertilizers decreased as the percent of water-soluble Zn
Agronomic Biofortification of Cereal Grains 67

decreased in them, and calculated that at least 50% water-soluble Zn was

considered desirable. In the United States, Zn fertilizer manufacturers are
producing mixture of zinc sulfate and ZnO, which are known as Zn
oxysulfates. However, from the manufacturer’s viewpoint, ZnO is easier
to coat, because it forms a good emulsion with an oil. Kiekens (1995)
suggested that ZnO, Zn(OH)2, and ZnCO3 are about 105 times more
soluble than soil Zn and these materials could be used as fertilizers.
Naik and Das (2008) compared ZNSHH and Zn–EDTA for rice at
Pakyong, Sikkim. ZSHH was applied at 10 and 20 kg ha
1 as basal or in
two equal splits (half basal and the rest half at grand tillering stage). Zn–
EDTA was applied at 0.5 or 1.0 kg ha
1in single application as basal;
1 kg ha
1 was also applied in two equal splits. Zn concentration in rice grain
was significantly more (30.3 mg kg
1) with 0.5 kg ha
1 Zn–EDTA than
with 10 kg ha
1 ZSHH(25.5 mg kg
1). Split application was better than
a single application in ZSHH but not in Zn–EDTA. Zn-EDTA was better
than ZNSHH, but more expensive.
McGarth et al. (2012) reported from Rothamsted, UK, that sewage
sludge application to soil can increase Zn concentration in wheat grain in
noncalcareous soils, but not on a calcareous soil for at least 2–8 years after
application and was similar in effectiveness to zinc carbonate.

5.2. Wheat
Maqsood et al. (2009) from Pakistan reported that Zn concentration in
grains of 12 wheat genotypes varied from 40.0% to 54.1% in no Zn check
to 51.7% to 69.9% when 6 mg Zn kg
1 soil was applied. Soil applications are
reported to have, in general, small increases in grain Zn concentration in,
while foliar applications result in remarkable increases in grain Zn concen-
tration in wheat (Cakmak et al., 2010b; Yilmaz et al., 1997; Zhang et al.,
2010a,b). Hussain et al. (2012) from Pakistan also reported that foliar appli-
cation of Zn both at jointing and heading gave 25% higher Zn concentration
in wheat grain than soil application at 4.5 mg kg
1 soil; the increase was only
7% at 9 mg kg
1 soil application. Priming wheat seeds with zinc sulfate solu-
tion was not effective in increasing Zn concentration in wheat grain. Data
from 23 site-year trials in seven countries are in Table 2.3. The increase in
Zn concentration in wheat grain due to soil application varied from 0.4% to
28.1% (there was negative response at two centers) over no Zn control,
while the increase due to foliar application varied from 32% to 125% over
no Zn control. Thus, foliar application of Zn was more effective in
Table 2.3 Increase in Zn concentration (mg kg
1) in wheat grain due to Zn application at field capacity in seven countries (23 site-year trials)
(figures in parentheses are % increase over check)
Country Location (number of years) Variety Check (control) Soil Zn Foliar Zn Soil þ foliar Zn Significance
China Guzhou (2) Kenong 9204 28.6 36.6 (28.1) 45.7 (60.0) 52.9 (85.1) **
Yangzhou (2) Jonmai 47 19.1 21.5 (12.8) 28.9 (51.6) 28.2 (47.9) **
India Varanasi (1) HUW 234 29.0 32.0 (10.3) 44.0 (51.7) 47.0 (62.1) **
Kapurthala (2) DBW 17 40.2 41.1 (2.2) 57.9 (44.1) 57.2 (42.3) **
Ludhiana (2) DBW 17 26.4 33.5 (26.9) 59.6 (125.9) 58.9 (123.1) **
Kazakhstan Shortandy (1) Akmela 2 21.5 29.5 (37.2) 66.5 (209.3) 76.5 (255.8) **
Mexico Yaqui Valley (1) Kronstad 26.0 25.0 (19.0) 43.0 (104.8) 45.0 (114.3) **
Pakistan Ayub (1) Sehar 2006 27.0 25.3 (
6.2) 48.2 (78.5) 44.6 (65.2) *
Faislabad (1) Auqab 2000 29.0 29.0 (0.0) 60.0 (106.9) 59.0 (103.4) **
Muridke I (2) Sehar 2006 36.6 35.8 (
2.0) 48.3 (32.1) 48.1 (31.4) *
Muridke II (2) Sehar 2006 38.9 46.4 (19.2) 52.2 (34.2) 52.0 (33.6) *
Turkey Eskisehir (2) Bezostaya 1 25.8 26.0 (0.7) 43.4 (68.4) 43.3 (68.0) **
Konya (2) Bezostaya 1 12.8 12.9 (0.7) 25.7 (101.1) 27.3 (113.7) **
Zambia Chisamba (1) Rorrie II 23.0 24.0 (0.4) – 43.0 (86.9) **
Average 27.4 30.5 (11.3) 48.0 (75.2) 49.0 (78.8) **
Significance at *P ¼ 0.05, **P ¼ 0.01, or less.
Soil pH ranged from 7.5 to 8.2, except at Chisamba, where it was 5.7.
Soil Zn application: 50 kg ZnSO47H2O; foliar: two applications; 0.05% (w/v) aqueous solution at 600–800 L ha
1 in the later afternoon; soil þ foliar: combination of
soil and foliar application.
A significant increase in grain yield of wheat due to Zn fertilization was recorded in Pakistan only.
From Zou et al. (2012).
Agronomic Biofortification of Cereal Grains 69

increasing Zn concentration in wheat grain. A combination of soil þ foliar

application of Zn was significantly superior to foliar application only at two
centers and thus in general was no better than foliar application alone. Yang
et al. (2011) also reported that Zn concentration in wheat grain was 24%
larger with soil þ foliar application than the foliar application alone. They
also observed that the foliar application of Zn gave best results when it
was done at grain filling stage.

5.3. Corn
Not much information is available on agronomic biofortification in corn.
Small holder farmers in South Africa, Zimbabwe, and other African coun-
tries use very little amounts of chemical fertilizers and use of Zn fertilizers is a
far cry. A study in Zimbabwe showed that the application of cattle manure
(supplying 113 g Zn ha
1) þ NPK and leaf litter (supplying 430 g Zn
ha
1) þ NPK significantly increased Zn concentration in corn grain over
NPK (Manzeke et al, 2012). Welch and Graham (2004) observed that
expected increase in Zn (and iron) from plant breeding is likely to be lesser
in corn than in rice and wheat.

5.4. Oats
In a recent study, Shivay et al. (2013) reported that coating Zn as ZnO or
zinc sulfate onto oats grains at 2 kg per 100 kg (required for sowing 1 ha)
gave a zinc concentration of about 32 mg kg
1 as compared with about
25 mg kg
1 obtained with soil application at the same rate of application.
For soil application, zinc sulfate was better than ZnO.

6. MANAGEMENT PRACTICES OTHER THAN FERTILIZER

AFFECTING FE AND ZN CONCENTRATION IN CEREAL
GRAINS
6.1. Tillage
At present, there is a considerable emphasis on reduced or zero tillage in rice,
wheat, and other crops (Lal, 2007; Sharma et al., 2012). However, the
reduced tillage is known to increase soil compaction (Larney and
Kladivko, 1989; Prasad and Power, 1991), which can create serious prob-
lems for root proliferation (Busscher and Sojka, 1987). In a over 50-year
study on a Walla Walla silt loam in Oregon, residue burial resulted in a
well-defined tillage pan at 19–29 cm depth (Rasmussen and Smiley,
1989). Similarly, Hill and Curse (1985) reported an increased soil bulk
70 Rajendra Prasad et al.

density with NT (no tillage) as compared to CT (conventional tillage).

A large number of researchers in the United States reported accumulation
of P in surface soil in NT plots (Follet and Peterson, 1988; Hargrove and
Hardcastle, 1984; Weil et al., 1988). The subject has been reviewed by
Prasad and Power (1991). This can lead to reduced Zn uptake (Lavedo
et al., 2001). Stipesevic et al. (2009) reported that in winter wheat Zn con-
centration in the plant tissue at the beginning of heading did not differ due to
tillage treatments in the first 2 years, but in the third year it was 11.7 mg kg
1
in the conventional tillage plots and only 6.4 mg kg
1 in the zero till plots.

6.2. Water management

Rice suffers from Zn deficiency both under flooded or anaerobic conditions
(Fageria et al., 2003; van Breeman and Castro, 1980) as well as under aerobic
conditions (Farooq et al., 2011; Gao et al., 2006). However, opinions differ
on this, and Kirk and Bajita (1995) observed that submerged conditions as
obtained in the conventional lowland rice cultivation result in increased Hþ
extrusion by rice roots, which increases Zn availability. Under prolonged
submergence, Fe could get precipitated as plaques of ferric hydroxide
(Zhou et al., 2007), which are primarily lepidocrocite or goethite and have
a high capacity to bind metal ions, such as Zn2þand Cu2þ, resulting in their
reduced uptake by rice roots (Chen et al., 1980a,b). In calcareous soils, mod-
erately anaerobic conditions have higher Zn availability than aerobic con-
ditions (Gao et al., 2006), but Zn availability decreases when soils
become very aerobic (Johnson-Beebout et al., 2009).
Fe deficiency is observed in rice nurseries and in rice grown under
upland conditions, when rains fail and irrigation is delayed (Pal et al.,
2008; Reddy and Siva Prasad, 1986; Synder and Jones, 1988). Zn deficiency
in rice occurs after transplanting and is a widespread phenomenon limiting
productivity under lowland conditions (Neue and Lantin, 1994: Quijano-
Guerta et al., 2002). Although reports are not available, Zn deficiency could
be a hidden hunger in cereals like pearl millet (Pennisetum glaucum L.) and
sorghum (Sorghum bicolor L.), which are mostly grown under dryland agri-
culture conditions.
In view of acute global water shortage and the fact that rice requires too
much water (3000–5000 L kg
1 in grain as compared to only 400 L kg
1 in
wheat), considerable research is underway to encourage aerobic rice pro-
duction (Bouman et al., 2005; Prasad, 2011). Most studies on aerobic rice
focus on grain yield and water saving (Yang et al., 2005). Under aerobic
Agronomic Biofortification of Cereal Grains 71

conditions, nitrates are dominant ion and rice roots release more OH
ions,
resulting in the precipitation of Zn (Gao et al., 2006) and reduced Zn
uptake. Chen et al. (2008) reported that population and activity of Fe oxi-
dizing/reducing bacteria may increase under aerobic conditions with a sig-
nificant impact on Zn concentration and speciation in soil solution.
More research is needed to find out the optimum water management
techniques for providing the desired Fe and Zn uptake by cereals.

6.3. Interaction with other nutrients

Both Fe and Zn interact positively with N and inversely with P. A positive
N  Zn interaction in cereals was reported by a number of researchers
(Lakshmanan et al., 2005). This is due to improvement in root uptake
and translocation of Zn due to nitrogen (Kutman et al., 2010). Xu et al.
(2012) reported that grain Zn concentration in winter wheat increased with
an increase in N application rate; Zn concentration (mg kg
1) was 21.5,
25.1, 30.9, and 37.0 with 0, 99, 198, and 297 kg N ha
1. Kutman et al.
(2011a,b) also reported that Zn concentration in wheat grain increased with
high rates of N application. A close relationship between the seed concen-
tration of Zn, Fe, N, and P was found in different germplasms of wheat
(Gomez-Becerra et al., 2010a,b; Morgounov et al., 2006; Zhao et al., 2009).
A negative P  Zn interaction has been widely studied and reviews on
the subject are available (Loneragan and Webb, 1993; Subba Rao and
Rupa, 2003). Recently, Zhang et al. (2012a,b) reported that as the
P application rate increased from 0 to 400 kg ha
1, the Zn concentration
in wheat grain decreased from 29.5 to 13.0 mg kg
1. The P/Zn molar
ratio in wheat grain also increased with increased P application; it increased
from 187 in no P plots to 537 in plots receiving 400 kg P ha
1. Reduced Zn
concentration in wheat grain due to P application has been reported by a
number of workers (Goh et al., 1997; Grant et al., 2002; Ryan et al.,
2008). There are a number of possible reasons for reduced uptake of Zn
due to application of large amounts of P. These include: (1) decreased con-
centration of Zn in soil solution (Norvell et al., 1987), (2) enhanced shoot
growth resulting in dilution of Zn concentration (Loneragan and Webb,
1993), and (3) reduced mycorrhizal (VAM) infection resulting in reduced
Zn uptake (Loneragan and Webb, 1993). Recently, Huang et al. (2000)
pointed out that in Zn-deficient plants, expression of genes decoding a high
affinity P transport protein breaks down leading to P accumulation in plants.
Zhang et al. (2012a,b) reported that Fe concentration in wheat grain
72 Rajendra Prasad et al.

increased with an application of P. Fe and Zn also interact with each other.

Metabolic functions of Fe in plants are in some way connected with the sup-
ply of Zn (Narwal and Malik, 2011), and Zn deficiency may accentuate Fe
uptake to toxic levels. Zn supply improved the growth of Fe-deficient soy-
bean and Fe application helped to overcome Zn toxicity effects (Fontes and
Cox, 1998). A number of workers, however, reported a negative Fe  Zn
interaction (Brar and Sekhon, 1976; Dangarwala et al., 1983; Safaya,
1976). A critical study of these data shows that the Fe  Zn interaction is
positive at low concentrations and negative at high concentrations of these
nutrients.
Zn also reacts negatively with Mn (Gupta and Gupta, 1984; Safaya, 1976)
and Cu (Brar and Sekhon, 1978; Langin et al., 1962). Zn application can
reduce B toxicity (Mishra and Singh, 1996).

6.4. Plant growth promoting rhizobacteria and mycorrhiza

Plant growth promoting rhizobacteria (PGPR) include a wide variety of soil
bacteria, which when grown in association with a host plant, results in stim-
ulation of growth of the host plant due to increased mobility, uptake, and
enrichment of nutrients in the plant (Cakmakci et al., 2006; Glick, 1995).
A major role of PGPR is through increasing the availability of nutrients
in the rhizosphere region of the plant. In a study at New Delhi, Rana
et al. (2012b) found that a combination of Bacillus sp. and Providencia sp.
resulted in a significant increase in Zn and Fe concentration of wheat grains.
Results with Providencia sp. were confirmed in another study by the same
group of researchers (Rana et al., 2012a). A number of other researchers
have also reported the role of PGPR in increasing Zn and Fe content in
wheat (Karthikeyan et al., 2007; Manjunath et al., 2010). The colonization
of arbuscular mycorrhizal fungi (AMF) has been shown to enhance nutrient
uptake through roots of upland plants due to increased surface area for soil
exploration (Smith and Read, 1997). AMF are aerobic organisms, and under
anaerobic conditions, their growth and effectiveness are decreased (Sylvia
and Williams, 1992). Nevertheless, short-time survival (colonizaion of
56–58% of roots) of aerobically inoculated AMF on rice after transplanting
and flooding has been demonstrated; however, the AMF colonization
decreased to 30% at panicle initiation stage (Purkayastha and Chhonkar,
2001; Solaiman and Hirata, 1997, 1998). Inoculating rice seedlings in a
wet or dry nursery with AMF increased Zn concentration in brown rice
by 2 mg kg
1.
Agronomic Biofortification of Cereal Grains 73

As agronomic biofortification of cereals is through soil–crop plant sys-

tem, a discussion on factors affecting the availability of Fe and Zn in soil
and their uptake by plants is essential and is briefly reviewed.

7. SOIL FACTORS AFFECTING THE AVAILABILITY OF Fe

AND Zn TO PLANTS
7.1. Amounts present in soil
Fe is the fourth most abundant (41,000 mg kg
1) in the earth’s crust and
comes only after oxygen (474,000 mg kg
1), Si (277,000 mg kg
1), and
Al (82,000 mg kg
1) (Kotz et al., 2006). As compared to this, the amount
of Zn in the earth’s crust is only 75 mg kg
1. The general range for total
Zn in soils is 10–300 mg kg
1 (Swaine, 1955; White, 1993). Zn in soils is
present in different forms including soluble solution Zn, exchangeable
Zn, organic matter-associated Zn, and Zn coprecipitated as secondary min-
erals or associated with sesquioxides and as structural part of primary minerals
(Shuman, 1991). About 30% of world soils are deficient in available Zn and
most of these are calcareous (Alloway, 2008; Fig. 2.2). There is a close over-
lap between global distribution of Zn deficiency in soil and humans
(Cakmak, 2004, 2008). Both Fe and Zn deficiencies are caused not only
by the low total content of these nutrients in soils but also by their low bio-
availability (Alloway, 2009). DTPA-extractable Zn (Lindsay and Norwell,
1978) is the most widely used method for determining Zn availability in
soils; however, Cakmak et al. (2010b) and Karami et al. (2009) found it
to be a poor predictor of the changes in Zn concentration in wheat grains.
Pirzadeh et al. (2010) from Iran also reported a nonsignificant Pearson’s cor-
relation coefficient of 0.12 between rice grain Zn concentration and 0.10
between grain Fe concentration and DTPA-extractable soil Zn.

7.2. Soil solution pH

The availability of both Fe and Zn decreases with an increase in soil solution
pH. Fe in aqueous solutions is generally surrounded by six molecules of water
and is present as FeðH2 OÞ6 3þ (Baes and Mesmer, 1976). An increase in pH
reduces Hþ from the hydrated water and gives rise to molecules with varying
degree of hydration (Table 2.4).Thus, soil solution pH has an important role
in determining the solubility of Fe, which is quite lower in pH range 7.5–8.5.
At pH 8.0, soil solution in equilibrium with soil Fe contains mostly FeðOHÞ3 0
giving a soluble Fe concentration of 10
10.4 M, which is not adequate for
74 Rajendra Prasad et al.

Figure 2.2 Zinc-deficient soils in the world. Alloway (2008). With permission from Inter-
national Zinc Association and International Fertilizer Association.

Table 2.4 Dominant iron species in soil solution at different pH

Soil solution pH Dominant species
1 FeðH2 OÞ6 3þ
2 Fe2 ðOHÞ2 4þ
4 Fe3 ðOHÞ4 5þ
6 FeðOHÞ2 þ
8 FeðOHÞ3 0
10 FeðOHÞ4

From Baes and Mesmer (1976).

plant growth; most plants need an Fe concentration of 10
8 M of soluble Fe

(Schwab and Lindsay, 1989).
The solubility of Zn is also highly pH dependent and decreases 100-fold
with each unit increase in pH (Lindsay, 1991). The predominant species in
Agronomic Biofortification of Cereal Grains 75

soil solution are: Zn2þ below pH 7.7, Zn (OH)þ between pH 7.7 and 9.0,
and Zn(OH)2 above pH 9.1 (Lindsay, 1991). Caroll and Loneragan (1969)
reported the critical level of Zn for plant growth in continuous flow cul-
ture at 10
7 to 10
8 M. Zn deficiency in plants can be expected in alkaline
soils, where Zn concentration in soils is between 10
8 and 10
10 M
(Lindsay, 1991). Rupa and Tomar (1999) reported a sharp decrease in soil
Zn with an increase in pH from 4.25 to 6.75, beyond which all Zn was
sorbed. This is why liming of acid soils decreases the availability of Fe
and Zn. Zn deficiency is widespread in alkaline calcareous soils
(Alloway, 2008; Cakmak, 2009; Prasad, 2006). The effects of calcium car-
bonate on Zn availability are threefold. First, an increase in calcium car-
bonate increases pH and thus reduces Fe and Zn availability; second, Zn
is directly sorbed on precipitated calcium carbonate, and third, Ca in cal-
cium carbonates forms insoluble calcium zincate (Prasad, 2007). Donner
et al. (2012) observed that reduction in CaCl2-extractable Zn over time
is a function of pH; Zn fixation was higher during the first week of incu-
bation and declined later.
Rice differs from other cereals, because it is grown under sub-
merged conditions. Rice roots tend to acidify the rhizosphere by two
mechanisms: (1) release of O2 by the roots oxidizes Fe2þ(which is present
under anaerobic conditions) to Fe3þ releasing Hþ ions (Ahmed and Nye,
1990) and (2) taking up excess NHþ 4 (again dominant N ion species
under submerged conditions) over anions resulting in release of Hþ to
maintain neutrality (Begg et al., 1994). Rhizosphere acidification strongly
influences the ability of organic acids to release Zn from soils (Yang et al.,
1993, 1994).

7.3. Mechanisms of Zn fixation other than pH

There are a number of mechanisms other than pH that are responsible for the
fixation of Zn. These include complex formation with organic matter
(McLaughlin, 2001; Tye et al., 2003), occlusion in minerals through precip-
itation of other phases (McLaughlin, 2001; Tye et al., 2003), diffusion into
micropores and interparticle spaces (McLaughlin, 2001), solid-phase diffu-
sion (Sparks, 1998; Tye et al., 2003), and precipitates including
coprecipitation with other metals (Almas and Singh, 2001; Sparks, 1998).
Precipitation of Zn as insoluble franklinite (ZnFe2O4) (Sajwan, 1985) and
ZnS in acidic soils and ZnCO3 in calcareous soils (Bostick et al., 2001)
has been reported.
76 Rajendra Prasad et al.

8. PLANT FACTORS AFFECTING UPTAKE OF Fe AND Zn

8.1. Root characteristics
The uptake of Fe and Zn as well as other nutrients depends largely on root
characteristics (Broadley et al., 2007). Of the various root characteristics, mor-
phology (length, diameter, density, volume) and architecture (special config-
uration) are functionally important in acquisition of nutrients (Lynch and
Whipps, 1990; Rengel, 1993). Cereal cultivars that produce finer roots (diam-
eter <0.2 mm) can explore large volume of soil and hence can scavenge Fe
and Zn more efficiently. Growing longer and thinner roots in the total root
mass early in growth period is associated with Zn efficient wheat genotypes
(Dong et al., 1995). Mechanistic simulation models of nutrient uptake based
on soil chemistry, kinetics of nutrient uptake, and root architecture and mor-
phology have been developed (Rengel, 1993). It is therefore highly desirable
to breed cereal species with a more efficient root system that is capable of
mobilizing Zn in Zn-deficient soils (Graham et al., 1992).
Under continuous submergence, there could be high concentration
of bicarbonates due to anaerobic decomposition of organic matter
(Ponnamperuma, 1972). Under high bicarbonate, (>10 mM L
1) root
growth of Zn-inefficient genotypes is strongly inhibited, whereas that of
Zn-efficient types may remain unaffected or may even be enhanced
(Hajiboland et al., 2003). Yang et al. (1993) also found that bicarbonate
inhibited the uptake of Fe and Zn in Zn-inefficient genotypes but not in
Zn-efficient types.

8.2. Phytosiderophores
Phytosiderophores (PS) are organic substances (nicotinamine, mugeniec
acid, avenic acid, etc.) produced by plants under Fe- or Zn-deficient
conditions, which can chelate Fe or Zn and increase their uptake by plants
(Ueno et al., 2007). PS have received considerable attention in the recent past.
In cereal genotypes, tolerance to Fe deficiency depends upon the amount of
Fe-siderophores (FeSP) of mugineic acid (MA) family (Marschner and
Romheld, 1994; Romheld and Marschner, 1990). MA and other members
of its family are hexadental ligands with aminocarboxylate and
hydroxycarboxylate functional groups; the ligands are sythesized by hydroxyl-
ation of nicotinamine (Ma and Nomoto, 1996). Nicotinamine synthase and
Agronomic Biofortification of Cereal Grains 77

nicotinamine transferase are the chief enzymes associated with the release of PS
in rice (Huguchi et al., 1999; Kanazawa et al., 1994). Studies on PS also tend to
explain why Fe deficiency appears in young rice seedlings. All Fe in young rice
seedlings is transported to phloem sap as deoxyMA-Fe, because phloem sap has a
high pH (about 8.0) and under such conditions Fe will preferentially bind with
MA rather than with malate or other organic acids (Takagi et al., 1988). Thus, Fe
uptake by rice seedlings depends on MA released, which may not be enough due
to a short root system.
ZnPS (Zn phytosiderophores) possess greater ability to complex Zn and
enhance its mobility in the rhizosphere (Clarkson and Sanderson, 1978) and
root apoplast (Zhang et al., 1991). ZnPS have similar structural confirma-
tions as FeSP and a similar regulatory mechanism for the biosythesis and/or
release of PS under Zn and Fe deficiency (Rengel and Romheld, 2000);
however; stability of FePS is much higher than ZnPS (Murakami
et al., 1989).
DMA (20 -deoxymugineic acid) is the dominant PS released from the
roots of rice plants (Bashir et al., 2006). DMA exudation is reported to occur
at a faster rate in Zn-deficiency tolerant lines than in susceptible lines but did
not increase under Zn-deficient conditions in either group of genotypes
(Widodo et al., 2010). There is a strong gradient of PS concentration away
from the root surface with an average of 1 mM within the first 0.25 mm dur-
ing the period of maximum exudation rates (Romheld, 1991). Plants can
take up Zn–Ma complex as a whole, but Zn2þ is preferred for root uptake
(Suzuki et al., 2008). Mathematical modeling supports the conclusion that
the observed rate of DMA secretion from rice roots, though not observed as
a Zn-deficiency response adequately accounts for observed rates of Zn
uptake. The genes involved in DMA biosynthesis in crop plants have been
reviewed by Impa and Johnson-Beebout (2012).

8.3. Organic acids

Zn deficiency increases root exudation of organic acids, such as oxalate, cit-
rate, and malate. However, the amounts of these acids secreted and their rel-
ative role differ from genotype to genotype; citrate was found to be more
relevant to Zn deficiency than oxalate in seven rice genotypes (Hoffland
et al., 2006), while malate was more relevant than citrate in two genotypes
(Widodo et al., 2010). Rose et al. (2011) also showed that Zn-efficient
genotypes had enhanced exudation of malate and also confirmed that high
tolerance of rice cultivars to Zn deficiency was most likely a result of malate
78 Rajendra Prasad et al.

exudation. However, in another study, malate was not correlated with Zn

deficiency in six genotypes (Gao et al., 2009).

8.4. Zinc utilization at the cellular level

Zinc utilization efficiency defined as dry matter production per unit of Zn
present in dry matter is reported to be linked with the activity of two
Zn-regulated enzymes, namely, carbonic anhydrase (CA) and superoxide
dismutase (SOD). Activity of CA is reported to decrease as a consequence
of Zn deficiency in a number of plant species and it is suggested as a measure
of physiologically active Zn in the leaf tissue (Reed and Graham, 1980).
Under extreme Zn deficiency, CA activity may be almost absent. Generally,
CA is present in excess of what may be required for photosynthetic activity
in C3 plants, such as wheat (Makino et al., 1992). When Zn was supplied to
Zn-deficient plants, Zn-inefficient wheat genotypes lost the ability to
increase CA activity, while Zn-efficient genotype Warigal showed a very
high increase in CA activity (Rengel, 1995). This permits maintaining
the photosynthetic activity at a desired rate in Zn-efficient plants and con-
sequent increase in dry matter production due to Zn application in
Zn-efficient genotypes.
Zn is essential for normal functioning of Cu–Zn–SOD (Marschner,
1995). Under Zn deficiency, Cu–Zn–SOD activity is much lower and is
controlled by Zn supply. Gokhan et al. (2003) showed that Cu–Zn–SOD
gene expression was upregulated in Zn-efficient but not in Zn-inefficient
genotypes.
An efficient utilization of Zn at the cellular level therefore appears to be a
major factor determining the expression of Zn efficiency in cereals grown
under Zn-deficient conditions.

8.5. Translocation in plants

Hajiboland et al. (2001) found that Zn deficiency tolerance of a Zn-efficient
rice genotype is related to its ability to retranslocate Zn from older to emerg-
ing leaves. Haslett et al. (2001) reported that Zn is highly mobile within the
plant system and foliar-applied Zn is translocated to leaves both above and
below the treated leaf as well as to the root tips. The enhanced capacity of
genotypes for Zn translocation from root to shoot and its utilization under
reduced Zn supply has been shown to contribute to Zn efficiency in wheat
genotypes (Cakmak et al., 1996).
Agronomic Biofortification of Cereal Grains 79

8.6. Mechanisms of Zn accumulation in grain

In wheat and barley, the xylem discontinues at the base of the grain, so Zn
has to enter the grain only through phloem (Palmgren et al., 2008), while
there is no such discontinuation of xylem in rice and Zn can enter the grain
directly through the xylem (Stomph et al., 2009). Therefore in wheat and
barley, a large proportion of Zn in grain comes through remobilization of
Zn from the leaves during the grain filling stage (Zee and O’Brien,
1970). Using 67Zn, Hegelund et al. (2012) showed that in barley Zn fluxes
derived from root uptake and remobilization were about equal in plants with
low Zn status, while at high Zn status remobilization delivered four times
more Zn to the developing grain than did root Zn uptake. On the other
hand in rice, mobilization of Zn through leaves may not be necessary, as long
as the Zn supply to the roots is sufficient. There are conflicting reports about
the relative importance of the two pathways of Zn accumulation in rice
grain. Jiang et al. (2007) observed that translocation through xylem was
the primary mechanism, while Wu et al. (2010) observed that
remobilization was important for Zn accumulation in rice genotypes that
had previously been identified as having consistently higher Zn and Fe con-
centration in grain. Genotypes with accelerated senescence might show
higher remobilization to grain than genotypes with slower senescence
(Speretto et al., 2009). Rice genotypes with a shorter grain filling period
tend to take up less Zn that is supplied after flowering (Jiang et al., 2007).
A genotype that has the ability both to take it directly and to remobilize
is less likely to be affected by the environment (Impa and Johnson-
Beebout, 2012).

9. CONCLUSION
Agronomic biofortification is the easiest and fastest way for bio-
fortification of cereal grains with Fe, Zn, or other micro mineral nutrients
in developing Asian and African countries, where cereals are the staple food.
Agronomic biofortification is the only way to reach the poorest of the poor
rural masses, who will never have money to buy mineral supplements nor
can afford to improve the components of their diet by incorporating animal
products. From the biofortification viewpoint, foliar application is better and
requires lesser amount of Fe and Zn fertilizers than their soil application.
When cultivars or GM crops with grains denser in Fe and Zn are developed,
adequate Fe and Zn fertilization will be necessary. The genetic and
80 Rajendra Prasad et al.

agronomic approaches are therefore complementary to each other and

should progress in tandem. However, a better understanding of the various
reactions that micro mineral nutrients undergo in soil and the mechanisms
involved in their absorption and translocation in plants, specially to grains, is
required. This calls for adequate funding of Agronomy, Soil Science and
Plant Physiology departments in agricultural institutes in developing as well
as in developed countries.

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