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Journal of the Science of Food and Agriculture
J Sci Food Agric 80:861±879 (2000)
Review
Potential for increasing the content and
bioavailability of Fe, Zn and Ca in plants for
human nutrition
Emmanuel Frossard,1* Marcel Bucher,2 Felix Mächler,1 Ahmad Mozafar1 and
Richard Hurrell3
1
Plant Nutrition Group, Institute of Plant Science, Swiss Federal Institute of Technology (ETHZ), Research Station Eschikon, PO Box 185,
CH-8315 Lindau-Eschikon, Switzerland
2
Plant Biochemistry and Physiology Group, Institute of Plant Science, Swiss Federal Institute of Technology (ETHZ), Research Station
Eschikon, PO Box 185, CH-8315 Lindau-Eschikon, Switzerland
3
Human Nutrition Laboratory, Food Science Institute, Swiss Federal Institute of Technology (ETHZ), Seestrasse 72, PO Box 474, CH-8803
Rüschlikon, Switzerland
Abstract: This paper reviews the possibility and limits for increasing the content and bioavailability of
iron (Fe), zinc (Zn) and calcium (Ca) in edible parts of staple crops, such as cereals, pulses, roots and
tubers as a way to combat mineral de®ciencies in human populations. Theoretically, this could be
achieved by increasing the total level of Fe, Zn and Ca in the plant foods, while at the same time
increasing the concentration of compounds which promote their uptake (ascorbic acid), and/or by
decreasing the concentration of compounds which inhibit their absorption (phytic acid or phenolic
compounds). The content of Zn and Ca in grains and fruits can in some cases be increased through soil
and/or foliar applications of Zn and Ca fertilisers. Plant breeding and genetic engineering techniques,
however, have the greatest potential to increase Fe and Zn content in grains, roots and tubers. The
possibility of enhancing Ca and ascorbic acid content in plant foods by plant breeding and genetic
engineering remained to be explored. The critical factor is to ensure that the extra minerals have an
adequate bioavailability for man. Given the important role of phytic acid and polyphenols in plant
physiology, reducing the levels of these compounds in the edible parts of plants does not appear to be
wise although introduction of phytases which are active during digestion is an exciting possibility.
# 2000 Society of Chemical Industry
Keywords: review; human nutrition; plant nutrition; Fe; Zn; Ca; bioavailability; beans; cereals; roots; tubers;
fertilisers; plant breeding; biotechnology
INTRODUCTION
Increasing the amount of bioavailable micronutrients
in plant foods for human consumption is a challenge
which is particularly important for developing countries.1 Theoretically, this could be achieved by
increasing the total level of micronutrients in the
edible part of staple crops, such as cereals and pulses,
while at the same time increasing the concentration of
compounds which promote their uptake such as
ascorbic acid, and/or by decreasing the concentration
of compounds which inhibit their absorption, such as
phytic acid or phenolic compounds.2,3
The aim of this paper is to review the possibilities
and limits for increasing the content and bioavailability
of iron (Fe), zinc (Zn) and calcium (Ca) in plant foods
for human consumption. The focus has been set on
these micronutrients because Fe and Zn de®ciencies
are common not only in developing countries, where
the diet is essentially composed of cereals and root
crops, but also in some industrialised countries,1 and
because the low Ca intake is a risk factor for bone
fractures which are increasingly common in elderly
people.4 Since these minerals, as well as the compounds favouring or inhibiting their uptake in humans
(phytic acid, phenolic compounds, ascorbic acid),
have speci®c roles in plants, their uptake by the plant
from the soil as well as their concentrations in different
plant organs is strictly regulated.5,6 It might not be
possible therefore to alter their concentrations in the
plant without changing the entire physiological system.
In this review, we present the importance of Fe, Zn
* Correspondence to: Emmanuel Frossard, Plant Nutrition Group, Institute of Plant Science, Swiss Federal Institute of Technology (ETHZ)
Research Station Eschikon, PO Box 185, CH-8315 Lindau-Eschikon, Switzerland
E-mail: emmanuel.frossard@ipw.agrl.ethz.ch
(Received 7 December 1999; accepted 16 December 1999)
# 2000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2000/$17.50
861
E Frossard et al
and Ca for human nutrition, the mechanisms controlling their uptake by the plant from the soil, their
physiological role in the plant, their transfer to the
edible plant tissues, and the levels and physiological
role of ascorbic acid, phenolic compounds and phytic
acid in plants. We then discuss the possibilities and
limits of using fertiliser application, plant breeding
techniques or biotechnology to increase the content of
Fe, Zn and Ca in plant foods, to decrease phytic acid
or to increase ascorbic acid.
IMPORTANCE OF Fe, Zn AND Ca IN HUMAN
NUTRITION
Fe, Zn and Ca are essential nutrients that are often
lacking in human diets, either due to insuf®cient intake
or to poor absorption from food. In developing countries, de®ciencies of Fe and Zn lead to much suffering
and death. In industrialised countries, chronic Ca
de®ciency is one of the important causes of reduced
bone mass and osteoporosis in the elderly.7
Fe
The body requires Fe for the synthesis of the oxygen
transport proteins haemoglobin and myoglobin and
for the formation of haem enzymes and other Fecontaining enzymes which are particularly important
for energy production, immune defence and thyroid
function.8 The body normally regulates Fe absorption
so as to replace the obligatory iron losses of about
1±1.5 mg per day. There is no mechanism for Fe
excretion. Inadequate Fe absorption will ®rst lead to
the mobilisation of storage Fe, then to insuf®cient Fe
transport to the bone marrow, and ®nally to lower
haemoglobin levels or anaemia. Fe de®ciency anaemia
affects about one billion people world-wide and is
most prevalent in infants, children and women of
child-bearing age in developing countries, where some
50% or more of these population groups may be
anaemic.9 Fe de®ciency anaemia can decrease mental
and psychomotor development in children,10 increase
both morbidity and mortality of mother and child at
childbirth, decrease work performance and decrease
resistance to infection.11,12
The Fe content of individual foods has little
relevance, since Fe absorption from different foodstuffs varies considerably. There are two types of food
Fe: non-haem Fe which is present in both plant foods
and animal tissues, and haem Fe coming from the
haemoglobin and myoglobin in animal products.
Haem Fe represents 30±70% of the total Fe in lean
meat and is always well-absorbed.13 Non-haem Fe
from meat and vegetable foods enters a common nonhaem Fe pool in the gastric juice,14 the amount of Fe
absorbed from which depends to a large extent on the
presence of enhancing and inhibitory substances in the
meal and on the Fe status of the individual.
Haem Fe is absorbed by a different mechanism to
non-haem Fe. Its absorption is little in¯uenced by the
composition of the meal and varies from 15 to 35%
862
depending on the Fe status of the consumer. Although
haem Fe represents only 10±15% of dietary Fe intake
in populations with high meat intake, it could
contribute 40% or more to the total absorbed Fe.15
Many poorer regions of the world consume little
animal tissue however and rely almost entirely on nonhaem Fe.
The absorption of non-haem Fe is strongly in¯uenced by dietary components which bind iron in the
intestinal lumen. Non-haem Fe absorption can vary
widely from less than 1% to more than 90%, but is
usually situated in the region from 1 to 20%. The main
inhibitory substances are phytic acid from cereal
grains16 and legumes such as soy,17 and polyphenol
compounds from beverages such as tea and coffee.18
The main enhancers of Fe absorption are ascorbic
acid19 from fruits and vegetables and the partially
digested peptides from muscle tissues.20 Low Fe
bioavailability is considered a major factor in the
aetiology of Fe de®ciency anaemia.21
Although Fe is an essential nutrient, excess Fe could
be a potential health risk. The mechanism of cellular
and tissue injury with excess Fe is not fully understood
but is thought to be modulated via free radical
reactions since in vitro free Fe is a potent generator
of free radicals via the Fenton reaction. In vivo Fe is in
principle tightly bound to proteins. However its role in
disease has been suggested22 since free radical formation has been implicated in cancer, cardiovascular
disease, and arthritis.23 Hydroxy radical formation in
faeces due to a high content of forti®cation with Fe has
been hypothesised as a potent factor in the development of colon and rectal cancer. The evidence from
epidemiological studies in relation to both cancer and
cardiovascular disease is, however, contradictory and
requires further investigation.24
Zn
Most of the Zn in the human body is in the bone and
skeletal muscle. Zn acts as a stabiliser of the structures
of membranes and cellular components. Its biochemical function is as an essential component of a large
number of Zn-dependent enzymes, particularly in the
synthesis and degradation of carbohydrates, lipids,
proteins and nucleic acids. Zn also plays a major role in
gene expression.25 These biochemical functions of Zn
give it a unique role for growth and development.
Zn de®ciency in humans reduces growth, sexual
maturity and the immune defence system.26 Recent
studies from Chile, Vietnam and Guatemala reported
increased growth in Zn-supplemented infants and preschool children and a lower incidence of diarrhoea and
respiratory infections.27±29 Although body Zn homeostasis can be maintained over a wide range of Zn
intakes by increasing or decreasing both intestinal Zn
absorption and endogenous intestinal Zn excretion,
ultimately low Zn intake and/or bioavailability will
result in Zn de®ciency. Meat and seafood are good
sources of Zn, and 70% of Zn in the US diet is
provided by animal products.30 However, in many
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
parts of the developing world, most Zn is provided by
cereals and legume seeds. These plant foods are high
in phytic acid, which is a potent inhibitor of Zn
absorption.31
Ca
Ca is required for the normal growth and development
of the skeleton.32 Ca accumulates at the rate of about
150 mg per day during human skeletal growth until
genetically pre-determined peak bone mass is reached
in the early twenties. Bone mass is then stable until
about 50 years of age in man or until after menopause
in women. After this time, Ca balance becomes
negative and bone is lost from all skeletal sites. Bone
loss is associated with a marked rise in fracture rate in
both sexes, but particularly in women. Adequate Ca
intake during adolescence is critical in achieving
optimal peak bone mass and reducing the rate of bone
loss associated with ageing.7 Osteoporosis is characterised by a reduced peak bone mass and a microarchitectural deterioration of bone tissue, leading to
increased bone fragility and increased risk of fracture.33 There has been a large increase in the incidence
of osteoporosis in many countries over recent years.34
Milk and milk products are the most important
sources of Ca for people living in industrialised
countries, accounting for more than 50% of Ca intake,
with cereals, fruits and vegetables each making smaller
contributions.35 Foods of plant origin are not generally
rich sources of Ca so that individuals whose diet
contains no milk products would be expected to have a
lower than recommended Ca intake. This includes
many people living in developing countries. Oxalate is
a potent dietary inhibitor of Ca absorption36 with
phytic acid possessing a much smaller inhibitory
effect.37 However, low Ca intake from a low consumption of milk products, rather than low bioavailability, is considered the major factor resulting in an
inadequate supply of dietary Ca.
Strategies to combat micronutrient deficiencies
The three most widely recognised strategies for
reducing micronutrient malnutrition in human populations are supplementation with pharmaceutical
preparations, food forti®cation and dietary diversi®cation.38 Ca and Fe supplementation, and forti®cation
of foods with Ca and Fe, are successfully practised in
industrialised countries.4,39 Fe and Zn supplementation has also been useful in developing countries for
rapid improvement of Fe and Zn status in de®cient
individuals, but this strategy is relatively expensive and
often has poor compliance, particularly with Fe
because of unpleasant side-effects. Food forti®cation
is usually considered the best long-term strategy for
the prevention of micronutrient de®ciencies, but again
with Fe food forti®cation is dif®cult because of
technical problems related to the choice of Fe
compounds. Unfortunately, those Fe compounds of
relative high bioavailability, such as ferrous sulphate,
often provoke unacceptable colour and ¯avour
J Sci Food Agric 80:861±879 (2000)
changes in food vehicles. On the other hand those
compounds which are organoleptically inert, such as
elemental iron, are usually poorly absorbed.39
Food diversi®cation may also be impossible in
developing countries for economic or social reasons,
and people who avoid milk products in industrialised
countries usually do so because they dislike milk
products or are intolerant to these foods. For these
various reasons, the current strategies for combating
Fe, Zn and Ca de®ciencies have not always been so
successful.
A new strategy for combating micronutrient malnutrition could be to enrich food staples through the
application of fertilisers, plant breeding or genetic
engineering.40,41 This is a potentially more sustainable
strategy, and micronutrient-enriched foods could
reach larger numbers of people than nutrient supplements or forti®ed foods, and would also be less
expensive.
Fe, Zn AND Ca IN PLANTS
Fe, Zn and Ca availability from soils and their uptake
by plant roots
Nutrient availability for crops can be de®ned as the
quantity of a nutrient that can be taken up by a crop
from the soil (treated or not with fertilisers) during a
growing season. Plants take up most of their nutrients
from the aqueous soil solution in the form of free ions
(Fe2‡, Zn2‡, Ca2‡) via the roots. The availability of
these nutrients to the plant is a function of the soil
physicochemical and biological properties, which
govern their release from the solid phase of the soil
into solution, and of the plant properties that govern
nutrient uptake.
Physicochemical nature of Fe, Zn and Ca in the soils and
their availability to the plant
The availability of a mineral for a plant depends on its
speciation in the soil, where the mineral can be present
in many different physicochemical forms. Minerals in
soil solution can be in a free ionic form which is totally
available to the plant. Alternatively, the mineral may
be present in solution as a chelate, be adsorbed onto
mineral or organic surfaces, be present as precipitates
or in lattice structures, or be part of live or dead soil
biomass. These forms are either partly available or
completely unavailable to plants. The most important
soil properties governing mineral availability are soil
pH, redox conditions, cation exchange capacity, the
activity of micro-organisms, soil structure and water
content. Among these, soil pH which can vary in
agricultural soils from 4.0 to 9.0, exerts a strong
control over the free ion concentration in soil solution.
For example, in the pH range from 5.5 to 7.0, an
increase of one pH unit causes a 30±45-fold decrease
in aqueous Zn2‡ ion concentration. Similarly an
increase of one pH unit causes a 100- and 1000-fold
decrease in aqueous Fe2‡ and Fe3‡ ion concentration,
respectively.
863
E Frossard et al
In well-aerated soils, the concentration of free Fe2‡
or Fe3‡ in the solution is extremely low. Between pH 7
and 9, the total concentration of soluble Fe species in
the solution of aerated soils does not exceed 10ÿ10 M.
This is much lower than the concentration of 10ÿ6 to
10ÿ5 M which is required for optimal growth.6 Thus
when grown on well-aerated calcareous or alkaline
soils, many crops can suffer from Fe de®ciency. These
soils cover 25 to 30% of the land surface and are
distributed all over the world.42
The concentration of Zn2‡ ions in the soil solution
of non-polluted soils ranges from 10ÿ8 to 5 10ÿ7 M.
The transfer of Zn2‡ ions into roots with the
transpiration ¯ux of water through the plant (mass
¯ow) provides only a limited fraction of the plant total
Zn uptake.43 Most Zn moves towards the root by
diffusion, ie as the concentration of Zn2‡ ions
decreases in the vicinity of the roots following plant
uptake, Zn2‡ is released from the solid phase of the soil
into the solution and then diffuses through the soil to
the root surface.43 Zn de®ciencies in plants occur
mostly in calcareous and alkaline soils, especially
under arid and semi-arid conditions. Zn de®ciencies
also occur in highly weathered tropical soils such as
ferralsols, acrisols or lixisols (FAO soil classi®cation)42
which cover about 20% of the world's land surface in
the tropics and sutropics.42 Almost half of the
agricultural soils from India, one-third of the agricultural soils of China, 14 Mha in Turkey and 8 Mha in
Western Australia are considered as Zn-de®cient for
plants.44±47.
In most soils, the concentration of Ca2‡ in the soil
solution is so high (between 0.1 and 20 10ÿ3 M) that
the amount transferred to plant roots with the
transpiration ¯ux (mass ¯ow) is either suf®cient to
meet plant needs or, in neutral and calcareous soils,
largely exceeds plant needs, thus leading to Ca
accumulation in the vicinity of the roots or inside the
roots.6 Some soils however may be Ca-de®cient. Ca
de®ciency can occur in highly weathered tropical soils
because of their low total Ca content.48 In these soils
however, Ca de®ciency is dif®cult to separate from Al
toxicity which also strongly affects crop production in
tropical countries. Ca de®ciency in plants can also
occur in saline and sodic soils. These soils cover 260
Mha world-wide and are mostly found in the arid
subtropics.42 In these soils, excessive Na concentrations impair plant Ca uptake.
Uptake of Fe, Zn and Ca by roots
The uptake of Fe, Zn, and Ca by plants is in¯uenced
by the root architecture,49 the presence of mycorrhizal6 fungi and root physiology.6 Root architecture
and more speci®cally the distribution of root hairs and
mycorrhizal fungi determine the volume of soil from
which Fe, Zn, and Ca can be extracted, whereas root
molecular and physiological mechanisms control the
mineral uptake into the root.
Root hairs are extremely important organs with
respect to mineral uptake by the plant.6 These are
864
tubular outgrowths of rhizodermal cells located along
the nonligni®ed parts of the root system. Several
factors in¯uence root hair production, including soil
aeration, partial pressure of carbon dioxide, humidity,
physical resistance of the soil, nutrient supply, and
aqueous Ca concentration.50
Eighty percent of the terrestrial plant species can
establish a mutualistic interaction with mycorrhizal
fungi. In this interaction, the plant delivers carbohydrates to the fungi while the fungi provide to the
plant growth-limiting nutrients such as Zn and P.51,52
This improved nutrient uptake is related to the
formation of an extensive extraradical hyphae network
which allows the plant/fungi system to explore a soil
volume much larger than that accessed by the root
system alone. Although hyphal transport has also been
demonstrated for Ca by using radioisotopes, the
amounts transported are probably very small. The
same is probably true for Fe.6
Minerals enter the plant cells through transport
proteins located in the plasma membrane of the cell.
Membranes contain different types of transport
protein. ATPases or ATP-powered pumps, channel
proteins, and co-transporters. In plant cells, H‡ATPases pump protons across the plasma membrane
generating the proton motive force responsible for ion
uptake. Channel proteins facilitate the diffusion of
water and ions down energetically favourable gradients. Co-transporters, the third class of membranetransport proteins, can move solutes either up or down
gradients.53
Fe uptake. Plant species differ in the mechanisms they
have evolved to combat Fe de®ciency. These mechanisms are termed either `Strategy I' for dicotyledonous and non-graminaceous monocotyledonous
plants or `Strategy II' for graminaceous species.
Strategy I plants, such as beans, develop an
increased reducing capacity and an enhanced net
excretion of protons along the root surfaces to help
transform insoluble soil Fe3‡ compounds into soluble
Fe2‡ ions. They can also show an enhanced release of
reducing and/or chelating compounds such as phenolics, from the roots to the soil. Under Fe de®ciency
conditions, changes in root morphology and anatomy
can also occur such as the formation of transfer celllike structures.6 These transfer cells are presumably
the sites from which enhanced net excretion of protons
and reducing capacity as well as enhanced release of
phenolic compounds occur.6 One or more reductases
in the root-cell plasma membrane are capable of
transferring electrons from the cytosol to Fe3‡.54,55
This increases the reduction of Fe3‡ at the outer
surface of the plasma membrane which in turn
correlates with enhanced uptake rates of iron and
other cations.54 The reduction of Fe3‡ to Fe2‡ by
ferric chelate reductase is thought to be an obligatory
step in Fe uptake as well as the primary factor in
making Fe available for absorption by all strategy I
plants.56
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
Our understanding of the molecular basis of strategy
I-Fe uptake by eukaryotic cells has recently been
increased by the discovery and cloning of two genes,
one encoding the enzyme ferric chelate reductase and
the other encoding an iron transport protein. The
gene, FRO2, encoding the ferric chelate reductase is
expressed in Fe-de®cient roots of Arabidopsis thaliana.57 After Fe3‡ reduction, Fe2‡ is absorbed across
the plasma membrane of root cells bound to an Fe2‡
transport protein. The Fe2‡ transport protein has just
been reported in yeast58 and in Arabidopsis thaliana via
heterologous complementation of a yeast mutant
defective in iron transport.59 The protein has been
named IRT1 (iron-regulated transporter) and is
predicted to be an integral membrane protein. In
Arabidopsis, IRT1 is expressed in roots and is induced
by Fe de®ciency. It also exhibits altered regulation in
plant lines containing mutations that affect the Fe
uptake system. Subsequent biochemical studies with
IRT1 have led to this proposal that the protein
functions as a metal transporter with a broad substrate
range, suggesting that IRT1 mediates uptake and
transport in roots of Fe, Mn, Zn and Co.60
Strategy II plants, such as cereals, are characterised
by an enhanced release of non-proteinogenic amino
acids from roots, so-called phytosiderophores, in
response to Fe de®ciency. This response is rapidly
suppressed by the resupply of Fe.6 Phytosiderophores,
such as mugineic acid, form highly stable complexes
with Fe3‡. A second component of the Fe uptake in
strategy II plants is a highly speci®c Fe transport
system, which transports Fe3‡ phytosiderophores
across the plasma membrane into the cytoplasm of
the cell.61 Biosynthesis of the phytosiderophores
requires methionine as a precursor and nicotianamine
as an intermediate. Strategy II plants can grow on
calcareous soils that will not support the growth of
some dicotyledonous plants. This is possibly due to a
lower dependency of phytosiderophore Fe chelation
on rhizosphere pH. However, strategy II plants, and in
particular those with a low rate of phytosiderophore
release (eg sorghum, rice), may be affected by the
microbial breakdown of phytosiderophores in the
rhizosphere.
Zn uptake. When soils are Zn-de®cient, the plant
increases the exudation of low-molecular-weight
solutes from its roots. Whereas in dicotyledonous
species, amino acids, sugars, phenolics, and K‡
dominate in root exudates, in graminaceous species
the main solutes are the phytosiderophores.62
Although the phytosiderophores mainly chelate Fe,
they exhibit a low af®nity for Zn and the release of
these compounds may indirectly enhance the uptake
rate of Zn by increasing their transfer from the solid
phase of the soil to the solution and subsequently into
the plant apoplast. Zn uptake and translocation to the
shoot are inhibited by high concentrations of bicarbonate63 leading to Zn de®ciency in the plant.
In relation to Zn transport across the plasma
J Sci Food Agric 80:861±879 (2000)
membrane, a study of Zn in¯ux in giant algal cells
(Chara corallina) has suggested that two separate
transport systems, a high-af®nity and a low-af®nity
one, are involved.64 The same system could be present
in plants, since two Zn transporters from Arabidopsis
thaliana 65 have recently been cloned via heterologous
complementation of a yeast mutant that is disturbed in
Zn uptake66,67 with an A thaliana cDNA expression
library. The two genes, ZIP1 and ZIP3, are expressed
in roots in response to Zn de®ciency, suggesting that
they control the transport of Zn from the root surface
into the plant.65 The ZIP transport proteins probably
cannot transport Fe since they are unable to complement a Fe-de®cient yeast mutant and expression does
not respond to Fe de®ciency.65
Ca uptake. The main process of Ca uptake by the roots
is its binding to R-COOÿ groups located in the cell
wall of root tissues.6 Divalent cations such as Ca are
preferentially bound to these cation exchange sites in
comparison to monovalent cations. Most of the Ca
taken up by plant roots is still readily exchangeable
within 30 min and is almost certainly adsorbed on
R-COOÿ sites located in the apoplasm. In contrast,
K‡ is not readily exchangeable, because it crosses the
plasma membrane via speci®c transport processes and
passes into the cytoplasm and vacuoles.6 Dicotyledonous plants usually have a higher cation exchange
capacity in their roots and, as a consequence, they
normally take up more Ca from the soil than do
monocotyledonous plants. As a result, the Ca
concentration in dicotyledonous plants is generally
higher than the Ca concentration in monocotyledonous plants. Little is known however about
the molecular processes that control the uptake of Ca
in root cells.
The physiological role of Fe, Zn and Ca in plants and
their repartition in different plant tissues
The repartition of Fe, Zn and Ca between the various
parts of a plant depends both on the physiological roles
that are ful®lled by the three minerals and on the
physiological function of the tissues. In addition, the
repartition of these minerals is also affected by the
transport characteristics of the particular mineral
within the plant.
Occurrence and physiological roles of Fe, Zn, and Ca in
plant tissues
The concentration of free Fe2‡, Fe3‡, and Zn2‡ in
plant tissues is low because Fe- and Zn-cations are
either incorporated into enzyme proteins or complexed with low-molecular-weight organic compounds. Fe-proteins, such as cytochromes, are
involved in many metabolic processes. In redox
processes such as photosynthesis, respiration, nitrogen
and sulphur assimilation, Fe takes part by changing its
oxidation state (Fe2‡ $ Fe3‡). A great proportion of
the Fe in plants is needed for the photosynthetic
carbon assimilation in chloroplasts. Fe can be tem865
E Frossard et al
porarily stored in protein bodies such as phytoferritin,68 which is found in seeds, xylem, phloem and in
chloroplasts of leaves if they are kept in the dark.69,70
The exact contribution of phytoferritin to the Fe
content in these tissues is not known.
Zn-enzyme proteins are involved in many physiological activities and growth processes. High Zncontents are therefore found in meristematic tissues.
There is an important relationship between the
presence of Zn and membrane integrity. On the one
hand, Zn (together with Ca) may contribute to the
stabilisation of membranes, whereas on the other hand
Zn-de®ciency is associated with increased formation
and decreased detoxi®cation of oxygen free radicals
which may be harmful for membranes.71 Zn is
therefore important in plant tissues which are subjected to oxidative stress.
The content of free Ca is substantial in the cell walls
and in the vacuole but is very low in the cytosol of the
plant cells. A great proportion of Ca is bound to
negatively charged groups of structural carbohydrates
of the cell walls and contributes to their stability. Ca is
also important for the stabilisation of cell membranes
and is involved in the regulation of growth processes.
Excessive Ca is precipitated in the cell walls and the
vacuoles as Ca-oxalate.
The translocation of Fe, Zn, and Ca in plants
After minerals have been taken up by the cells of the
root tissues, they are transferred to the xylem vessels
for translocation to the shoots. Minerals are dissolved
in water, either as free ions or complexed to lowmolecular-weight organic molecules, and their transport in xylem vessels is due to the transport of water,
which is directed from the roots to the shoots and
which ends in the leaf blades, where the water is
released to the surrounding atmosphere by transpiration. The supply of minerals from roots to transpiring
leaves is therefore high, as long as transpiration is high
and acquisition by the roots is suf®cient. Ca as a
polyvalent cation may be withdrawn from the xylem
on its way to the leaves and bound to unsaturated
negatively charged cell wall components along the
vessels in stems and petioles. Fe and Zn are not bound
to negatively charged cell wall sites as long as they are
complexed with organic compounds.
Transport of nutrients in the phloem follows the
transport of photosynthetic carbohydrates, which is
directed from photosynthetic leaves to plant tissues
where carbohydrates are either consumed or stored for
later use. Phloem transportation of minerals depends
on the capacity of phloem loading. Phloem loading is
very low for Ca. The distribution of Ca to other plant
tissues is therefore very restricted. Fe is loaded into the
phloem when complexed with nicotianamine. Phloem
loading of Fe appears to be limited by the availability
of this Fe-chelator rather than by the presence of Fe.72
It is not known whether phloem loading of Zn is
dependent on the presence of a Zn-chelator.
The xylem transport of Ca ends in the leaf blades
866
where the xylem water is transpired. Ca entry into leaf
blades proceeds as transpiration continues and the Ca
accumulates in these tissues because Ca transport
from the leaves to other tissues via the phloem is low.
Much of the excess Ca is sequestered as Ca oxalate
when leaf requirements for Ca are exceeded. The Ca
supply to plant tissues which have a low transpiration
(young leaves, fruits, seeds, tuber) is critical and these
tissues are the ®rst to show symptoms of Ca de®ciency
if supply of Ca to the plants is insuf®cient. Fe and Zn
are easily transported in the phloem and may easily
change from xylem to phloem vessels suggesting that
the distribution of Fe and Zn in plants is not restricted
as it is for Ca. However, the supply of Fe and Zn to the
growing tissues requires a continuous uptake by the
roots. Remobilisation and transfer of Fe and Zn from
well supplied older tissues to de®cient growing tissues
does not occur before senescence of the older tissues is
induced.6 Therefore, visible symptoms of Fe and Zn
de®ciency are ®rst apparent in young growing leaves.
Homeostatic processes that control uptake of
minerals by the roots, translocation through the plant,
and deposition in the various plant parts appear to be
strongly regulated. An increase in nutrient uptake may
not necessarily enhance its content in the edible plant
part to the same degree. This is especially true for Fe.
Fe uptake by the roots, Fe translocation through xylem
and phloem and Fe deposition in the various plant
parts as Fe proteins, Fe chelates or phytoferritin are
tightly balanced to minimise accumulation of toxic Fe
at all points within the plant system.5,73
Toxicity of Fe, Zn, and Ca in plant tissues
When taken up in too large quantities Fe, Zn and Ca
can be toxic to the plant. Iron toxicity is related to the
formation of oxygen free radicals in the presence of
Fe2‡ and may occur in plants grown in waterlogged
soils and under dry land conditions.74 The critical
toxicity level in leaves is 500 mg Fe kgÿ1 dry weight.
Zn toxicity is often related to concomitant de®ciency of Mg, Fe or Mn due to competition for uptake
between the polyvalent cations.75,76 The critical
toxicity level of Zn in leaves ranges between 100 and
300 mg kgÿ1 dry weight.
Most plant species can accumulate high Ca contents
in leaf blades (100 g kgÿ1 dry weight) without any
symptoms of toxicity (calcicole plant species). In these
species, excessive Ca is sequestered as insoluble Ca
oxalate and deposited either in the cell wall or in the
vacuole. However, some species may have insuf®cient
capacity for this mechanism of detoxi®cation (calcifuge species) and their growth is severely depressed at
high tissue contents of Ca.6
Contents of Fe, Zn, and Ca in various edible parts of
cultivated plants
Data presented in Table 1 are taken from the USDA77
and give some general information on the usual Fe, Zn
and Ca contents of different plant tissues. The
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
Table 1. Water, Ca, Fe, Zn, and ascorbic acid (AA) content in edible parts of cultivated plants Ref 77. Phytic acid content in cereals and
legumes (with the exception of cowpea) were collected from Ref 79, in vegetables from Ref 80, in yam and cassava (boiled samples) from
Ref 81and 82, and from Staubli F and Hurrell RF (pers comm) and in cowpea and plantain from Ref 81.
Water
(% fresh weight)
Ca
Fe
(mg kgÿ1 dry weight)
Leaf blades
Lettuce (loose leaves)
Lettuce (head)
Kale
Cabbage
94
96
85
92
12000
7000
9000
6000
250
70
110
80
50
40
30
25
Roots/tubers
Carrot
Potato
Yam
Cassava
Topinambur
Radish
Kohlrabi
88
79
70
60
78
95
91
2200
330
550
400
650
4000
2500
40
40
20
7
150
60
40
15 800
20 950
8 550
8 510
5 180
60 4400
3 7000
Seeds
Wheat
Wheat ¯our, white
Corn
Oat
Barley
Rye
Sorghum
Rice (brown)
Rice (white)
Cowpea
Soybean
Pigeon pea
11
12
10
8
9
11
9
12
13
77
8
11
350
170
80
600
350
370
310
380
34±317
5520
3000
1500
45
13
30
50
40
30
50
20
9±11
48
170
60
Fruits
Plantain
Tomato
Orange
65
94
87
86
800
3000
17
70
8
potential to increase the Fe, Zn and Ca content of
these tissues with various techniques is discussed later.
Leaf blades. The contents of Ca, Fe, and Zn in the leaf
blades on a dry matter basis are higher than in other
tissues. The leaf blades are the sites of light absorption,
photosynthesis and transpiration. Accordingly, they
are the sites where xylem transport ends and Ca is
accumulated. They are sites with high metabolic
activity and therefore exhibit a high demand for Fe
and Zn. About 80% of the leaf Fe is contained in the
chloroplasts78 and is needed for photosynthetic redox
reactions. Zn enzymes are also involved in photosynthetic processes and are important for the detoxi®cation of oxygen free radicals which are formed when
light energy is absorbed and photosynthetic redox
activity is high. Lower contents of Ca, Fe, and Zn are
found in leaf blades if they are covered by older leaves
and not directly exposed to light energy (head-lettuce,
cabbage). Such leaves are yellowish green and show
decreased transpiration and decreased photosynthetic
activity.
Roots, tubers, and bulbs. For most plant species the
J Sci Food Agric 80:861±879 (2000)
Zn
35
8
25
40
30
40
±
20
11±12
44
50
30
AA
3000
2000
8000
4000
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
109
65.0
0.0
4 529
15 3000
5 4000
Phytic acid
(g kgÿ1 dry weight)
0.1
0.9
0.1
0.4±1.6
1.5±2.2
3.9±13.5
4.9
8.3±22.2
7.0±11.6
7.5±11.6
5.4±14.6
9.1±13.5
8.4±8.9
3.4±5.0
9.4
10.0±22.2
7.1±70.0
1.8
0.4
contents of Ca, Fe, and Zn on a dry matter basis in
roots, tubers, and bulbs are lower than in other tissues
except perhaps fruits. The function of these tissues is
to store photosynthetic carbohydrates for later re-use.
Ca supply to storage tissues is very restricted since they
are supplied with nutrients through the phloem rather
than through the xylem. On the other hand, the low
metabolic activity of storage tissues may be the reason
for a low demand for Fe and Zn. Little is known on the
speciation of Fe, Zn and Ca in these tissues.
Fruits. The contents of Ca, Fe and Zn are low in the
fruits of most plant species. Ca supply to these tissues
is restricted due to low transpiration and low xylem
transport. Metabolic activity and demand for Fe and
Zn appear to be low as well. The contents of all of the
three minerals tend to be higher in vegetable fruits
compared to tree fruits and berries. For Ca, this is
possibly due to long xylem connections between roots
and fruits in shrubs and trees.
Seeds. Although the levels of Ca, Fe and Zn are
relatively low in seeds on a dry matter basis, their low
moisture content and relatively large consumption by
867
E Frossard et al
human subjects means that they provide a useful
amount of Fe and Zn intake. The Ca content in
legume seeds is higher than in cereal grains or in roots
and tubers. The Ca supply to these tissues is restricted
due to low transpiration and low xylem transport. The
Fe content is lower in cereal grains than in legume
seeds. Zn contents are similar to Fe contents in both
legume seeds and cereal grains, despite low metabolic
activity. Fe in seeds is stored as phytoferritin or phytate
for reuse during germination. Zn and Ca are also
stored as phytate and Zn may be in the storage protein
methallothionein. There is little quantitative information on the precise speciation of Fe, Zn and Ca in
seeds.
ORGANIC COMPOUNDS AFFECTING THE
BIOAVAILABILITY OF Fe, Zn AND Ca IN HUMAN
NUTRITION
Phytate
A detailed description of phytic acid (myo-inositol,
1,2,3,4,5,6-hexakisphosphate (InsP6)) metabolism is
provided by Raboy.83,84 InsP6 biosynthesis begins
with inositol (Ins) synthesis which itself starts with the
conversion of glucose-6-phosphate to L-Ins(1)P, followed by a dephosphorylation to myo-inositol. Starting
with L-Ins(1)P, most studies indicate that sequential
phosphorylation to phytic acid is catalysed by two or
more Ins phosphate or polyphosphate kinases or
phosphotransferases.83 In plants, animals and yeast,
the ®nal step in InsP6 biosynthesis from InsP5 occurs
via an InsP5 2-kinase.85,86 InsP6 has a high cationbinding af®nity, thus forming phytates, the salts of
InsP6. The sparingly soluble Ca-Mg salt of InsP6 is
phytin. Phytic acid metabolism plays a prominent role
in phosphate, Ins and mineral storage and in phosphate metabolism.84 An additional function of phytic
acid would seem to be in energy metabolism. A recent
study of a soybean InsP5 2-kinase has demonstrated
that the phosphorylation reaction from InsP5 to InsP6
is reversible and can lead to ATP regeneration, if the
concentrations of substrate and product are favourable, as might be the case during the initial stages of
seed germination.85 Indeed, the post-germinative
utilisation of phytate, the major phosphorus storage
compound in seeds, is essential for seedling growth.84
Phytic acid accumulates in cereal grains, nuts, and
legume seeds to signi®cant amounts (Table 1) and
may represent 50±80% of total phosphorus.79,80,84 In
cereals and legumes phytates are deposited in electrodense globoid crystals found inside membrane-bound
intracellular protein bodies. In cereal grains phytates
are mainly deposited in the aleurone layer. In legume
seeds phytates are in cotyledons and embryo axes.87,88
In seeds, phytic acid most typically accumulates as
mixed phytate salts of several cations, including K,
Mg, Ca, Mn, Zn, and Fe.88 Phytic acid phosphorus in
potato tubers may rise to values higher than 20% of
total phosphorus during early tuber development.89
Udoessien and Aremu81 found that the proportion of
868
P present as phytate in peeled plantain and yam was
close to 48% of the total P content.
Phytases (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) belong to the family of
histidine acid phosphatases which catalyse the hydrolysis of phytate, thereby generating myo-inositol
phosphates, myo-inositol and inorganic phosphate.
Phytases have been isolated from microbial, fungal,
plant and animal sources.90,91 Plant phytases have
been characterised in pollen,92,93 germinating
seeds,91,94,95 in roots94,96 and shoot tissue.97
Ascorbic acid
The content of ascorbic acid (AA) is different in the
various plant parts (Table 1). High contents of AA are
usually found in the leaf blades. Leaf cells contain AA
in the chloroplasts, the mitochondria, the cytosol, the
vacuoles and the cell wall. However, highest concentrations are found in chloroplasts.98 AA contents are
lower in stems and petioles. AA contents in storage
organs (roots, tubers, and bulbs) and in ¯eshy fruits
vary strongly depending on the plant species. Highest
contents in fruits and roots may exceed leaf contents
considerably. Low contents of AA or no AA are found
in the seeds.
AA in plants is synthesised from carbohydrates with
99
D-mannose and L-galactose as precursors.
Various
enzymes of the pathway of biosynthesis have been
identi®ed in plant tissues and genetic evidence for the
role of one of the enzymes, the GDP-mannose
pyrophosphorylase, has been recently provided for an
AA-de®cient mutant of Arabidopsis thaliana.100
AA is an important antioxidant in plant tissues and
protects plants against oxidative damage resulting
from photosynthesis and aerobic metabolism.101 The
role of AA in photosynthesis is especially important.
An excessive amount of absorbed light energy results
in increased photosynthetic electron ¯ow, increased
reducing power, over-excitation of photosynthetic
membrane structures and the formation of oxygen
free radicals, which are a threat to the plant cells. AA
contributes to the controlled dissipation of excessively
absorbed light energy by regulating the electron ¯ow
and it contributes to the detoxi®cation of oxygen free
radicals by removing hydrogen peroxide which is a
product of oxygen free radical transformations.102 In
its function as an antioxidant, AA is oxidised to
dehydroascorbic acid and can be regenerated in the
presence of reducing agents.103 Suf®cient reducing
power is usually available in photosynthetic tissues and
oxidised AA is easily regenerated. Thus, the storage of
high AA concentrations appears not to be necessary in
leaf blades despite its central role in the photosynthetic
process.
The demand for AA as an antioxidant is not
restricted to photosynthetic tissues. Oxygen free
radicals are generally formed in redox processes of
metabolically active tissues and in light-exposed
tissues and need to be detoxi®ed by AA. AA contents
are therefore increased when tissues are metabolically
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
active (eg in growing sections of potato tubers and of
carrot roots) and in the light-exposed peels of fruits.104
Oxidised AA may be less easily regenerated in the
absence of the photosynthetic reducing power and
storage of relatively high AA contents may sometimes
be necessary in these tissues although oxygen free
radical formation may be lower than in leaves.
A comparison between plant species shows highest
AA contents in leaves and roots in the plant family
`Brassicaceae'. The Brassicaceae have a special mechanism of protection against herbivores and AA plays an
important role in this mechanism. The Brassicaceae
produce glucosinolates and store them in the cellular
vacuoles together with AA.105 A further component in
this mechanism is the enzyme myrosinase which is
stored strictly separated from the glucosinolates and
the AA in special cells in an inactive form.105
Mechanical damage of tissues by herbivores results
in the release and contact of the myrosinase, the AA
and the glucosinolates. Myrosinase is activated by AA
and catalyses the cleavage of the glucosinolates and the
formation of toxic volatile compounds. They are
released and function in defence as feeding repellents.105
Phenolic compounds
More than 5000 compounds containing an aromatic
ring with one or more hydroxyl groups have been
identi®ed in the plant kingdom.106 Most of these
phenolic compounds are synthesised within the plant
aromatic pathway, which is sequentially composed of
three segments: (i) the shikimate segment that leads to
the production of the aromatic amino acids (phenylalanine, tyrosine, tryptophan), (ii) the phenylpropanoid segment which produces cinnamic acid
derivatives, and (iii) the ¯avonoid segment that
produces the different ¯avonoid compounds.107
Phenolic compounds play crucial roles in higher
plants. The lignins are indispensable for the maintenance of the vascular structure of plants, and
contribute to the protection of plants against pests.108
Anthocyanins are next to the chlorophyll as the most
important group of plant pigments visible to the
human eye. These compounds accumulate in leaves,
fruits and in ¯owers of all higher plants where they are
considered to act as `nectar guides' for pollinating
insects.109 Flavones, ¯avonols and cinnamic acid
derivatives are considered to be the main UV
protectants for plants because they strongly absorb in
the UV-B and UV-A regions of the light spectrum.
They are located in the plant epidermis, and they
accumulate rapidly following UV radiation.110 In
legumes, ¯avones, ¯avanones, chalcones (methoxychlacone) and iso¯avones released from the roots act
as signals for the nodulation genes of symbiotic
rhizobia.108 Similarly, the release of quercitin from
roots is a signal for spore germination and hyphal
growth of endomycorrhizal fungi.6 Low-molecularweight phenolic compounds are also exuded by roots
to increase their reducing capacity and to increase Fe
J Sci Food Agric 80:861±879 (2000)
uptake.6 In legumes, iso¯avonoids are a major class of
phytoalexins which are low-molecular-weight antimicrobial compounds produced by plants in response to
attacks by pathogens.108 Condensed tannins are
another major plant chemical defence against microorganisms and herbivores. Tannins have been repeatedly shown to inhibit the activity of a wide range of
fungal enzymes and so reduce fungal germination and
growth. Similarly, bacterial toxins may be bound by
tannins and inactivated.111 The inhibition of microorganisms by phenolic compounds is thought to be
due to hydrogen bonding to vital proteins such as
enzymes.112
Although phenolics can be potent inhibitor of Fe
bioavailability in human, they have so many important
roles in plant physiology (some known and many of
them still waiting to be discovered) that it would seem
unwise at the present time to attempt to decrease their
content in plant tissues.
IMPROVING BIOAVAILABILITY OF Fe, Zn AND Ca IN
PLANT EDIBLE PARTS
As stated in the introduction, this aim could be
achieved by increasing the total amount of minerals
in the edible parts of crops, by increasing the concentration of compounds, such as ascorbic acid,
promoting their uptake by humans, and/or by decreasing the concentration of compounds, such as phytic
acid, which inhibit mineral uptake.2
Increasing total quantity of Fe, Zn and Ca in plant
edible parts
The total concentration of Fe, Zn and Ca in edible
parts of plants can be increased in three ways: by
applying fertilisers to the soil or to the crop,113 by plant
breeding2,3 and/or by genetic engineering.73
Application of fertilisers
This section focuses only on the effect of fertiliser
application on Fe, Zn and Ca uptake by plants. The
effect of other agricultural practices such as crop
rotation on Fe, Zn and Ca uptake by plants has been
reviewed by Rengel et al. 113
Most research has focused on the use of fertilisers to
correct nutrient de®ciency and increase plant yield.
Little information exists on the ability of fertilisers to
increase mineral concentrations in edible plant parts,
and even less information exists on the effect of
fertilisers on improving mineral bioavailability for
human subjects.113
Fe. The application of fertilisers containing Fe
compounds, such as FeSO4, has in general shown a
very low ef®ciency in increasing crop yield. This is due
to the rapid and strong binding of the added Fe to soil
constituents mentioned above. For example application rates higher than 100 kg Fe haÿ1 to the soil may be
required to correct the Fe de®ciency in sorghum.114
Although soil application of Fe chelates, such as
869
E Frossard et al
Table 2. Effects of different Zn application methods on grain yield (t haÿ1)
and Zn content in grain (mg Zn kgÿ1 dry matter) of two bread wheat cultivars
(Gerek-79 and Bezostaja-1) grown on a Zn-deficient calcareous soil in central
Anatolia (adapted from Yilmaz et al119)
Yield (t haÿ1)
Treatment
Control 1
Soil 2
Seed 3
Leaf 4
Soil ‡ leaf 5
Seed ‡ leaf 6
LSD (5%)
Zn content in grain (mg
Zn kgÿ1 dry weight)
Gerek-79 Bezostaja-1 Gerek-79 Bezostaja-1
0.74
2.70
2.05
1.47
2.71
2.77
0.45
0.81
2.34
1.96
1.55
2.33
2.38
0.74
9
17
11
30
34
34
6
10
17
8
28
38
25
9
1 Control, no Zn application; 2: Soil application, 23 kg Zn haÿ1 as
ZnSO4 7H2O; 3: Seed application, 1.0 litre of 30% ZnSO4 7H2O for 10 kg
of seed; 4: Foliar application, 2 200 g Zn haÿ1 as ZnSO4 7H2O in 450 litre
during tillering and stem elongation stages; 5: Soil and leaf applications; 6:
Seed and leaf applications.
Fe-EDDHA for calcareous soils or Fe-EDTA for acid
soils, have shown a better ef®ciency than simple Fe
salts, these compounds are expensive and they are not
always successful.114 Foliar application of FeSO4, or
Fe chelates, allows a direct uptake of Fe in the plant
through cuticular pores located around the guard cells
present on the leaf surface.6 Foliar fertilisation
ef®ciently corrects Fe chlorosis in beans, sorghum,
peanuts and fruit crops.114±116 Although foliar fertilisation increases crop yield to a greater extent than it
increases the Fe content of the grain, it might be the
only available fertilisation practice that can increase
the Fe content of grains.113,117
Zn. Application of Zn fertilisers such as ZnSO4 to the
soil ef®ciently increases the yield of cereals, maize,
sorghum and of beans in Zn-de®cient soils.114 It can
also signi®cantly increase Zn concentration in cereal
grains.45,113,118 Yilmaz et al 119 recently reported that
different methods of applying Zn to wheat affected
both the yield and the Zn content of the grain (Table
2). This work demonstrated that leaf application
induced the greatest increase in Zn concentration in
the grain.
Cakmak et al. 45 measured the phytate-to-Zn molar
ratio in the grains from a collection of 54 winter and
summer wheat cultivars grown in soils either strongly
or moderately Zn-de®cient for plants, in the presence
or absence of adequate Zn fertiliser applications. The
Zn fertilization
(mg Zn kgÿ1 soil)
Table 3. Yield and grain Zn concentration of wheat
cultivar Excalibur grown in a Zn-deficient soil in a
glasshouse as influenced by Zn fertilisation. Mean
(‡/ÿSE) (adapted from Rengel et al111).
870
0
0.05
0.2
0.8
3.2
mean phytate-to-Zn molar ratio ranged between 39 in
the grains with the highest Zn content (31 mg Zn kgÿ1)
and 137 in the grain with the lowest Zn content (8 mg
Zn kgÿ1). A ratio above 20 has been proposed as
necessary so as to reduce Zn absorption especially in
children.120 This ®nding con®rms the conclusion of
Welch121 who showed that increasing the Zn content
of grain through fertilisation did not change its
bioavailability to monogastric animals. In the study
of Cakmak et al,45 the phytate concentration in the
grain ranged from 7.4 to 14.3 mg gÿ1 dry matter. At a
typical phytate concentration of 10 mg gÿ1, a concentration of Zn in the seed higher than 40 mg kgÿ1 would
be needed to reach a phytate-to-Zn molar ratio lower
than 20. This Zn concentration is at the upper range of
the grain Zn content observed for various wheat
cultivars.1 It can be surpassed when an adequate
quantity of Zn fertilisers is applied (Table 3). Care
should be taken, however, before applying too large
quantities of metal onto soils since high metal loads
can have negative effects on plant growth122 and can
have negative effects on soil micro-organisms.123
Ca. Little information exists on the agronomic
practices which can lead to an increase in Ca uptake
and storage in the edible portions of plants grown in
neutral and calcareous soils where Ca is already
present in excess of plant needs. Even in calcareous
soils the low mobility of Ca in plants as outlined earlier
can lead to Ca de®ciency in organs which have a low
rate of transpiration such as roots, seeds or fruits. In
growing apples, this de®ciency can be ef®ciently
corrected by spraying CaCl2 or Ca(NO3)2 directly to
fruits while they are still on the tree.6 In acidic highly
weathered soils which are found throughout the
tropics, Ca de®ciency for plants is probably widespread and dif®cult to separate from Al toxicity. Ca
can be applied either as a water-soluble fertiliser, as
calcium carbonate or in a calcium phosphate form
such as apatite (rock phosphate) to correct both Ca
de®ciency and Al toxicity. Horst et al 124 reported that
increasing the concentration of Ca in a hydroponic
culture at pH 4.0 led to an increased concentration of
total Ca, water-soluble Ca and NaCl-extractable Ca in
the leaves of two cowpea cultivars. This indicates that
Ca fertilisation could perhaps increase the amount of
Ca to humans feeding on plants grown on Ca-de®cient
soils.
Interactions between nutrients. When aiming at improv-
Grain yield
(g dry weight plantÿ1)
Grain Zn concentration
(mg Zn kgÿ1 dry weight)
1.00 0.17
2.20 0.13
2.24 0.16
2.51 0.30
1.70 0.03
9.1 0.4
9.9 0.6
14 0.7
83 4
145 5
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
ing the Fe, Zn and Ca contents of edible plant parts
through the application of fertilisers, it is important to
consider the interaction of these minerals with other
nutrients, with soil pH and with different soil conditions (water excess, soil structure).
In nutrient-poor ecosystems, such as those encountered in most developing countries, plant production is
strongly limited by the lack of N, P or/and K. Under
these conditions, the application of N, P or/and K
fertilisers can increase root growth and result in a
higher transfer of Fe, Zn and Ca from the soil to the
plant.
Examples of negative interactions include excessive
liming or excessive use of P fertilisers. Excessive liming
can effectively lead to an increase in Ca content in the
crop but it can also cause Fe and Zn de®ciencies.
Excessive use of P fertilisers can lead to a lower rate of
root infection by myccorhizal fungi leading to a
decrease in the exploration of soil reserves as
described earlier. Excessive P fertilisation can also
result in a strong Zn de®ciency in maize, because of a
lower rate of Zn uptake from the soil, or/and because
of higher plant internal requirements for Zn.6,125
Nitrogen nutrition for the plant can also affect the
uptake of Fe, Zn and Ca, since N application as NH4
leads to an acidi®cation of the rhizosphere which
enhances the transfer of these metals from the soil to
the plant. Application of NO3, on the other hand, leads
to an alkalinisation of the rhizosphere and therefore to
a decreased transfer of Fe, Zn and Ca to the
plan.6,114,125
Plant breeding
A large project has recently been launched by the
Consultative Group for International Agriculture
Research (CGIAR) to increase the density of Fe and
Zn in edible plant parts through breeding.2,40,126
Reports from this project recently issued126 show that,
within the cultivars of a given species, there is a high
variability in the Zn and Fe content in the edible parts
of staple food crops (Table 4).
Research conducted on rice131 has shown that most
Table 4. Variability of the Fe and Zn content of the tuber or rhizome of
cassava and yam and in the seeds of wheat, bean, maize and rice
n
Fe
Zn
(mg kgÿ1 dry weight)
Cassava127
Yam species (peeled)128
Dioscorea alata
Dioscorea cayenensis/rotundata
Dioscorea dumetorum
Wheat129
Maize127
Common bean130
Brown rice131
Polished rice132
n: Number of cultivars tested in each study.
J Sci Food Agric 80:861±879 (2000)
162
23
27
23
170
126
>1000
1138
286
3±48
4±18
9±176
7±103
22±187
25±56
13±160
34±89
7±23
4±29
8±25
7±22
9±30
25±64
11±95
21±54
17±52
8±95
of the variation in Fe content in the seed is due to its
genetics component and that environmental effects
have only a small impact. Similarly, Beebe et al 130 have
shown that the genetic differences in Fe and Zn
contents of common bean seeds are expressed over
different seasons and in different environments. In
maize however, the impact of environmental factors on
the Fe and Zn content in the grain is strong and cannot
be easily separated from the genetic component.133
Little information exists on the heritability of Fe and
Zn content in tubers or rhizomes of yam or cassava and
more research is urgently needed in that respect. A
large number of reports show that there is a positive
relationship between the Fe and Zn contents in the
grain, indicating that efforts aimed at increasing the Fe
content may also increase the Zn content.127,129,130,134
Modern varieties of wheat and rice have a lower
concentration of Fe and Zn in grains than traditional
varieties.3,129,131 This is because breeding has been
mainly aimed at increasing yield, increasing disease
resistance etc, but not at improving the micronutrient
concentration in grain.3,129,131 It might be possible
therefore to increase the mineral concentrations in
grains by including in breeding programs traditional
varieties, or wild or primitive parents of cultivated
crops that have a higher ability to accumulate Fe and
Zn content in edible plant parts. Cakmak et al 134 have
recently shown that wild tetraploid wheats (ssp
dicoccoides), and more particularly their chromosomes
6A and 6B, are highly promising sources of the genes
determining high Fe and Zn levels in seeds. Such lines
could be used to increase the Fe and Zn grain content
in modern hexaploid wheat (ssp aestivum).
Graham and Welch2 have pointed out that seeds or
grains that are high in Fe, Zn and Mn will not only be
of interest for human nutrition but they may have also
signi®cant agronomic advantages. These advantages
include the demonstration that seeds high in minerals
produce more viable and vigorous seedlings, especially
in the low-fertility soils which cover large surfaces in
developing countries; they produce plants that are
more resistant to diseases, and they allow a better use
of soil and subsoil water content, which is a decisive
advantage in semi-arid climates. Finally, as the Fe and
Zn requirements of crops are rather low compared to
the relatively high total content of these minerals in
soils, growing plants which can accumulate higher
levels of Fe and Zn in their edible parts would not lead
to a signi®cant depletion of soil reserves.2
Because of the high genetic variability concerning
the Fe and Zn content of edible plant parts and the
relatively simple genetics governing their accumulation,2 there would seem to be considerable potential
for plant breeders to improve the nutritional quality of
crops. In addition, a high micronutrient concentration
in grains and seeds is bene®cial agronomically as well
as nutritionally and it is now possible to introduce such
a trait to high-yielding varieties. However, before
beginning extensive breeding programmes, it is essential to con®rm that the Fe and Zn in the existing Fe871
E Frossard et al
and Zn-rich varieties is suf®ciently well absorbed and
utilised by human subjects.
Little information is available at the present time on
how to increase Ca accumulation in edible plant parts
through plant breeding. Genetic variability has been
demonstrated in vegetables and pulses between
cultivars in their response to soil Ca de®ciency.124,135
However this response can be due to the ability of
given cultivars to take up more Ca from Ca-de®cient
soil (higher nutrient acquisition ef®ciency) or/and to
the ability of such cultivars to produce more dry matter
for a given quantity of Ca (higher nutrient use
ef®ciency). Unfortunately, having plants growing
better in a soil low in a given nutrient (Ca, Zn¼) is
not a guarantee that they will accumulate more of this
nutrient in grains or seeds. For instance, high Zn
ef®ciency in cereals grown in Zn-de®cient soils in
Turkey was associated with higher uptake of Zn from
the soil but not with increased accumulation of Zn in
the grain.45
Genetic engineering
As outlined above, molecular genetic tools have given
the researchers the possibility to identify key regulatory
steps in the acquisition of Fe and Zn by plants. The
transfer of the corresponding genes to agriculturally
important crops might therefore allow to increase their
nutrient uptake capacity. However, increasing the Fe,
Zn and Ca content of leafy vegetables, tubers or seeds
does not only necessitate an increase in minerals into
the plant, but also require modi®cations in mineral
partitioning within the plant.
Manipulating Fe and Zn uptake. The uptake of Fe in
transgenic tobacco was increased by constitutively
expressing the yeast FRE2 gene encoding a ferric
reductase.136 This was related to a higher rate of Fe3‡
reduction along the entire length of the roots and in the
shoots which is typical for the strategy I mechanism for
Fe acquisition (see above). Transgenic plants were
more tolerant to iron de®ciency and exhibited 50%
higher Fe concentrations in younger leaves than nontransformed plants when cultivated in a Fe-de®cient
medium. This suggests that the FRE2 gene may be
used to improve Fe uptake in crop plants. A greater
increase in Fe concentration in the plant may involve
the expression of several components of the Fe
acquisition system including Fe transporters,59,60
proteins involved in the synthesis of the Fe status
signalling molecule,137,138 and transcriptional activators of iron starvation inducible genes.139 Similarly,
expression of Zn transporters may lead to increased Zn
absorption in roots.140 A thorough understanding of
phytosiderophore (PS) biosynthesis and of the PScation complex transport mechanism into the cytoplasm may allow the generation of dicotyledonous
plants exhibiting strategy II uptake and thus improved
Fe and Zn uptake.141
Manipulating Fe and Zn storage. A complementary
872
Table 5. Iron content in transgenic rice seeds expressing ferritin
mg Fe gÿ1 fresh weight
Wild-type rice seed144
Transgenic rice seed144
Wild-type rice seed145
Transgenic rice seed145
8.6
13.3
10.0
7.6
(3.3) to 14.3
(2.8) to 38.1
(0.4) to 10.7
(0.0) to 22.1
(3.0)
(4.5)
(0.6)
(0.7)
approach is to express a mineral storage protein in the
organ where increased Fe should be stored in an
available form. High Fe-containing transgenic plants
have been produced by expression of cDNAs coding
for ferritin under the control of either constitutive or
seed-speci®c promoters.142±144 A threefold greater Fe
content in rice seeds was obtained.144 Similar results
although at a somewhat lower seed Fe concentration
were obtained by Lucca,145 demonstrating that this
approach could contribute to solving the global dietary
Fe de®ciency41 (Table 5). Further investigations are
now needed to demonstrate the stability of this trait
under different environmental conditions and in
subsequent generations of the transgenic plants.
Furthermore, it remains to be shown that Fe accumulates in the seeds in a form bioavailable to human
subject. This type of approach could also be evaluated
for Zn by increasing the level of methallothionein.146
Manipulating Ca uptake and storage. As suggested in
the section 3.1.2. an increase in the RCOOÿ concentration in the cell wall of root tissues could lead to a
higher uptake of Ca by the plants. This could be
achieved by increasing the concentration of pectins in
the cell wall via a modulation of the amount and
activity of enzymes responsible for pectin synthesis or
degradation.147,148 Similarly, the storage of Ca in roots
or seeds might also be achieved by modulating the
amount and activity of enzymes governing the pectin
biosynthesis in these organs. Such an approach might
allow storage of higher concentrations of Ca which will
remain available to human subjects.
Decreasing the amount of phytin in plant edible
parts: possibilities and limitations
A substantial decrease of phytin in edible parts of food
crops could improve Fe, Zn and Ca availability to the
consumer. Two approaches can be implemented to
reach this goal: modify phytase activity in the plant
edible part so that the enzyme remains active after food
preparation in the human digestive tract, and/or
decrease the phytin concentration in the edible plant
part. Genetic engineering offers a great potential to
achieve both goals.
The phytase genes from Apergillus niger and Aspergillus fumigatus have been inserted into plant transformation plasmids under control of constitutive and
seed-speci®c promoters.145,149±151 Transgenic rice
seeds and transformed cell-suspension cultures both
synthesised and secreted functional microbial phytase.
Microbial phytases were introduced because they have
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
been shown to substantially improve radio tracer Fe
absorption in humans.152 It is important that the
enzymes which are introduced should be able to
withstand the cooking temperatures that occur during
food preparation. A critical point is the refolding
capacity of a protein after heat denaturation. This
character has been studied for phytases of different
species.153 Molecular biological methods such as
random or site-directed mutagenesis were used to
generate improved phytases. A heat-stable phytase
able to withstand temperatures up to 100 °C over a
period of 20 min, with a loss of only 10% of the initial
enzymatic activity has been produced.154
Using a different approach, chemically induced
mutants of barley and maize with reduced levels of
phytic acid have been generated.155,156 These singlegene, low phytic acid (1pa) mutations, which are nonlethal for the plant, cause the seed to store most of the
phosphorus as inorganic phosphorus instead of as
phytate phosphorus. For example, 1pa1-1 from maize
contains a 65% reduction in grain phytic acid and is
accompanied by a molar-equivalent increase in inorganic P. This mutant was backcrossed into elite maize
inbred lines and the resulting hybrids did not exhibit
any marked alterations of important agronomic traits.
Some, but not all, 1pa1-1 hybrids had yield reductions. The biochemical phenotype of 1pa1 mutations
may be caused by a mutation in one of the steps that
leads to phytic acid biosynthesis, namely in myoinositol 1-phosphate synthase.157 Consumption of
genetically modi®ed, low-phytic-acid strains of maize
may improve iron and zinc absorption in human
populations that consume maize-based diets.158
However, care should be taken when developing
plants with a lower phytin content in the seeds. This is
because phytin is a source of P, energy and minerals
such as Fe, Zn, Ca for the seedling, and a decreased
phytin content might affect seedling vigour especially
in the low-fertility soils. This was con®rmed in a recent
study conducted by Long and BaÈnziger133 in Africa in
a soil de®cient in P. They showed that low-phytate
maize lines had signi®cantly lower shoot dry weights
than high-phytate lines two weeks after emergence.
These differences disappeared 3.5 weeks after emergence. Before decreasing the amount of phytate in
edible plant parts, it is advised to carefully evaluate the
possible consequences especially for plants growing in
soils with a low fertility.2
Increasing the concentration of ascorbic acid in
edible plant parts: possibilities and limitations
Increasing the AA concentration can be achieved by
modulating the application of N fertilisers and by plant
breeding. Since the biosynthetic pathway of AA is in
the process of being elucidated,99 genetic engineering
does not yet appear to be a viable method to increase
AA in edible plant parts, but might become an
interesting option in the future. Rather than increasing
AA concentration in the plant, another approach
might be to minimise AA losses after harvest.
J Sci Food Agric 80:861±879 (2000)
Excessive application of N fertilisers has a pronounced negative effect on the AA content in fruits
and vegetables.104,159,160 This may be due to biochemical reasons and/or canopy structure changes.
With excessive use of these fertilisers, plants may
produce many leaves. This will result in some leaves
and fruits being progressively shaded by other leaves
and thus containing less AA because of being deprived
of enough light intensity (see above). Lower amount of
available NO3 in the soil, may be one of the reasons
why organically grown fruits and vegetables in certain
(but not all) instances have been found to contain
higher AA acid than those grown with chemical
fertilisers.104,159,161
A large genotypic variation of AA has been observed
by Chavez et al 162 in cassava roots and leaves. From a
collection of 530 accessions they observed that the AA
content of cassava tubers varied between 0 and
375 mg kgÿ1 fresh weight, with a median value of
80.9. In another survey Treche163 observed that the
AA content of tubers of Dioscorea alata, a widely
cultivated yam species, can vary between 0.07 and
1.31 g kgÿ1 dry weight. In a recent review Mozafar104
also showed that a high genotypic variability exists in
the AA content of fruits and vegetables. These results
suggest that there is a potential to breed root and tuber
crops, fruits and vegetables with higher AA content.
Similarly given the relatively high AA content in
legume seeds (Table 1), there might also be a potential
to breed these crops for higher AA content. However a
lot of work is now needed to understand the
heritability of this trait, to identify the number of
genes involved and to propose suitable breeding
techniques. Such a potential seems unfortunately
non-existent at present in cereals, since cereal grains
do not contain any AA at all (Table 1).
Effects of post-harvest conditions during transport,
storage, freezing, display in market, for storage at
home, bruising and shredding prior to consumption
on the vitamin content in fruits and vegetables have
been extensively studied.164,165 In general, AA content
in vegetables is very sensitive to temperature and
humidity during storage and the degree of water loss
and wilting of plant tissues. For example, a 4-h
exposure of vegetables in a Nigerian market to sun at
35 °C was found to reduce the AA content in okra
(Hibiscus esculentus) by 63%, and in African Spinach
(Amaranthus hybridus) by 82%.166 Decreasing these
losses through a better protection of fruits and
vegetables, could help to increase the ®nal amount of
AA available to humans.
DISCUSSION
Fe and Zn de®ciencies are common in human
populations particularly in developing countries.
These trace mineral de®ciencies can severely limit
the physical and intellectual capacity of the people as
well as adversely affect their health and well-being. Ca
is another mineral where low intakes can be detri873
E Frossard et al
mental to health, since low Ca intake is one of the risk
factors in the bone diseases osteoporosis. Human
populations which exhibit de®ciencies or low intakes
of Fe, Zn or Ca are usually nourished primarily by
plant foods such as cereals, roots, tubers and pulses.
Increasing the levels of Fe, Zn and Ca in such plant
foods would therefore be expected to have a positive
effect on human health, provided that the minerals are
present in absorbable forms.
Bioavailability of these minerals for man is clearly a
critical factor since mineral absorption by man from
plant foods is often low. This appears to be mainly due
to the presence of phytic acid167 which is especially
high in cereal grains and legume seeds and also present
in other major starchy staples such as yam and cassava.
Phytic acid has been shown to decrease the absorption
of Ca in man but is a particularly potent inhibitor of Fe
and Zn absorption.167 The phenolic compounds
present in many leaves are another strong inhibitor
of Fe absorption18 and any Ca stored in leaves in the
form of Ca oxalate would be expected to have a low
absorption.36 Mineral bioavailability is therefore as
important as mineral content when evaluating the
usefulness of Fe, Zn and Ca in plant foods in relation
to human nutrition and, in order to predict bioavailability more accurately, it is important to know in
which form the mineral is present in the plant food.
We have discussed three different approaches to
increasing the Fe, Zn and Ca levels in plant foods.
These are the application of fertilisers containing the
respective mineral, the use of plant breeding techniques to introduce high Fe, Zn or Ca traits into highyielding crops and genetic engineering. While soil
application of fertilisers would seem to be a useful
approach to increasing the Zn levels in plant foods
such as cereals, they would seem to have much less
potential in relation to Fe and Ca. This lack of
ef®ciency is related to the strong and rapid binding of
Fe added with the fertilisers to soil compounds which
renders it unavailable to plants, and to the low phloem
transport within the plant of Ca and in some cases of
Fe. The application of Zn or Ca fertilisers directly on
leaves or fruits has, on the other hand, a much higher
ef®ciency in enhancing mineral content in plant foods.
This is probably also the case for Fe. Little information
is however available on the effect of fertilisation on
mineral bioavailability for human subjects. It is not
possible to state in which form the extra mineral is
stored in the plant tissue. The concern with increasing
the Zn level in cereals by the application of fertilisers
would be that Zn bioavailability would still be low due
to the naturally high phytate concentrations in cereal
grains. However, if the Zn concentration in the grain
was increased several-fold, the absorbed Zn should
also be increased several-fold even assuming a similar
low absorption in the high-Zn plant as in the low-Zn
plant. Similarly, it is not known whether the additional
Ca entering the plant after the application of foliar
fertilisers is stored as Ca oxalate or bound to cell wall
polysaccharides. In addition, the application of foliar
874
Ca fertiliser will probably have a limited effect on root,
tuber and seed Ca content.
There would seem to be much potential for the
introduction of high Fe or Zn traits into wheat, rice,
maize, cassava, yam and beans using plant breeding
techniques (see Table 4). The question arises however
as to the bioavailability of the mineral in these high Fe
and Zn plant foods, since many of the foods concerned
are naturally high in phytic acid and in addition
speciation of the mineral is largely unknown. If Fe was
present as phytoferritin for example, bioavailability
might be low. It has been reported that part of the Fe
contained in the ferritin molecule is not released into
the gastro-intestinal tract during digestion and is thus
not available for absorption. Human studies with
extrinsically radiolabelled animal ferritin indicated
that the Fe contained within the ferritin molecule
added to the meal was only about half as well absorbed
as vegetable Fe168,169 or as FeSO4.170 There are
unfortunately no human studies with plant ferritins. It
is not easy to predict the bioavailability of food Fe or
Zn in man. Animal tests are considered to be of little
use and in vitro models, such as dialysability or uptake
by Caco-2 cells also have their limitations.25 Human
studies, in which the plant foods are labelled intrinsically with either stable or radioisotopes would appear
to be necessary.
Genetic engineering approaches could be used to
increase both the level and bioavailability of Fe, Zn
and Ca in plant foods. Increasing mineral uptake by
the root, and increasing the distribution of mineral
from the leaf to the edible plant parts via the phloem,
would seem to be obvious targets. While certain genes
coding for key proteins involved in Fe and Zn uptake
and transport have been identi®ed and offer the
possibility of genetic manipulation, more information
needs to be obtained on the mineral transport system
in the phloem and on its downloading into the storing
organ. Another approach would be to introduce
storage proteins into edible plant parts, such as grains,
and to assume that enough mineral is present in the
whole plant to enable the storage protein to be ®lled
with mineral. Phytoferritin from beans has been
expressed in rice grains using genetic engineering
techniques and the Fe content of the rice grain has
been increased by two-to three-fold.144,145 However,
as mentioned earlier, the bioavailability to man of Fe in
phytoferritin may be only half that of other non-haem
food iron. Further investigations are also needed to
demonstrate the stability of the high Fe content of rice
seeds expressing soybean ferritin under different
environmental conditions and in subsequent generations of the transgenic plants.
Phytic acid is the major inhibitor of Fe, Zn and Ca
absorption from plant foods and decreasing the
content of phytic acid in meals of plant origin could
greatly improve absorption of these minerals. One
possibility would be to produce low-phytate plant
foods by genetic engineering or traditional plant
breeding techniques.155,156 The dif®culty with this
J Sci Food Agric 80:861±879 (2000)
Increasing content and bioavailability of Fe, Zn and Ca in plants for human nutrition
approach is that phytic acid is a potent inhibitor of
mineral absorption in human subjects even at very low
levels. As an example, it has been reported that
decreasing the phytic acid level in spray-dried soy
bean protein isolate from 9.9 g kgÿ1 to 3.7 g kgÿ1 did
not improve iron absorption when fed to human
subjects as part of a liquid formula meal.17 Absorption
only improved when phytic acid was reduced below
1.0 g kgÿ1 in the spray-dried material. Most whole
cereal and legume grains contain about 10 g kgÿ1 dry
weight phytic acid (Table 1) and the phytic acid
content would need to be reduced considerably in
order to improve Fe absorption.
It should be remembered that removal of the bran
from wheat or rice grains by milling or polishing
reduces phytic acid by about 90%, and degerming
maize can remove phytic acid almost completely.
Finally, decreasing the phytic acid content in seeds
and grains to such an extent might result not only in a
decrease in P reserves in these organs but also in a
decrease in Fe, Zn and Ca content since these are
stored as phytic acid±mineral complexes. Altogether
this lower level of mineral reserves might have a
negative impact, especially in low fertility soils, on the
seedlings growth obtained from these grains which
would force farmers to buy new seeds every year. A
second possibility would be to insert a microbial
phytase into cereal grains. Unlike cereal phytases,
microbial phytases remain active during digestion in
the human gastro-intestinal tract and degrade phytic
acid. When a phytase from Aspergillus niger was added
to a high-phytate bread roll just prior to consumption,
Fe absorption by human subjects was doubled.152
Recently a microbial phytase has been expressed in
rice grains145 and it would seem possible that such
phytases could be designed to be stable during food
preparation and cooking.154 Theoretically it should be
possible to degrade all the phytic acid in a rice meal,
including that present in other ingredients such as
beans, and so substantially improve the absorption of
Fe, Zn and Ca.
Ascorbic acid can enhance the uptake of Fe by
human subjects. Increasing the AA content of plant
foods might therefore contribute to alleviating Fe
de®ciencies in human populations. Although no
agricultural practices leading to an increase in AA
could be identi®ed, there appears to be some potential
for breeding pulses, fruits, vegetables, root and tuber
crops with a higher AA content. This potential does
not exist for cereals since their grains contain no AA.
Recent advances made in the understanding of the
biosynthesis pathway of AA99,100 in plants open up the
possibility that genetic engineering techniques in the
future might be a possible way to increase the AA
content of plant foods including in cereal grains.
In conclusion, several different strategies are possible to combat the widespread mineral de®ciencies that
occur in human populations. In addition to the usual
strategies of supplementation with pharmacological
preparations, food forti®cation and dietary diversi®caJ Sci Food Agric 80:861±879 (2000)
tion, we must now add a fourth strategy of increasing
the native Fe, Zn and Ca content of plant foods. This
would be possible for Zn by the application of Zn
fertilisers to the soil or to the leaves and fruits, for Ca
by the application of Ca fertilisers to the leaves and the
fruits, and for Fe and Zn by both plant breeding and
genetic engineering techniques. The possibility of
enhancing the Ca and ascorbic acid content in plant
foods by plant breeding and genetic engineering
should be explored. The critical factor is to ensure
that the extra minerals have an adequate bioavailability
for man.
ACKNOWLEDGEMENTS
The authors warmly thank RD Graham (University of
Adelaide, Glen Osmond), P Lucca (ETH Zurich), I
Rao (CIAT, Cali), F Staubli (ETH, Zurich) and S
TreÂche (IRD, Montpellier) for the fruitful discussions
during the preparation of this manuscript.
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