Accepted Manuscript

Soil and foliar zinc biofortification in field pea (Pisum sativum L.): grain accu-
mulation and bioavailability in raw and cooked grains

M.J. Poblaciones, Z. Rengel

PII: S0308-8146(16)30878-0
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.05.189
Reference: FOCH 19338

To appear in: Food Chemistry

Received Date: 15 February 2016

Revised Date: 30 May 2016
Accepted Date: 31 May 2016

Please cite this article as: Poblaciones, M.J., Rengel, Z., Soil and foliar zinc biofortification in field pea (Pisum
sativum L.): grain accumulation and bioavailability in raw and cooked grains, Food Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.foodchem.2016.05.189

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 Soil and foliar zinc biofortification in field pea (Pisum sativum L.): grain

accumulation and bioavailability in raw and cooked grains

M.J. Poblacionesa,b*, Z. Rengelb

a
Department of Agronomy and Forest Environment Engineering, University of

Extremadura Avda., Adolfo Suárez s/n, 06007 Badajoz, Spain

b
School of Earth and Environment, The University of Western Australia, 35 Stirling

Highway, Crawley, WA 6009, Australia

*Tel.: +34 924 289 300; Fax: +34 924 286 201. E-mail: majops@unex.es

1
ABSTRACT

To evaluate the potential of cooked field peas to be used in Zn biofortification

programs, all combinations of soil Zn application of 0, 4 and 8 mg ZnSO4·7H2O kg-1

and foliar Zn application of 0 and two sprays of 0.25% or 0.5% (w/v) ZnSO4·7H2O

before flowering and at early grain-filling stage were tested. Soil Zn application

increased Zn-DTPA concentration 3.7- to 5.6-times depending on the Zn soil

treatments. Grain Zn concentrations higher than 60 mg Zn kg-1 were obtained with all

foliar Zn applications, alone or in combination with soil Zn applications, and grain Zn

bioavailability was adequate (phytate:Zn ratios lower than 15). Processing (freezing and

cooking) caused a decrease of about 30% in grain Zn concentration and a 17%-increase

in phytate:Zn ratios (to ≤9.5). The combined application of 8 mg ZnSO4·7H2O kg-1 soil

+ 0.25% (w/v) ZnSO4·7H2O foliarly could be a good option for biofortifying field peas.

Key-words:

Agronomic biofortification

Foliar fertilization

Phytic acid

Zinc deficiency

Zinc fertilizers

1. Introduction

2
Zinc (Zn) is an essential nutrient in plants, animals and humans. In plants, Zn deficiency

reduces plant growth, pollen viability, flowering and grain production (Cakmak, 2000;

Pandey, Pathak, & Sharma, 2006). In humans, Zn deficiency is associated with severe

health complications, including impairments of physical growth, immune system and

learning ability, combined with increased risk of infections, DNA damage and cancer

development (Gibson, 2006; Levenson and Morris, 2011). However, more than 30% of

the world’s population is Zn deficient, with Zn deficiency being the 11th most important

factor causing disease or death in the world, and the 5 th most important factor in

developing countries only (WHO, 2011). In Australia, a study with 12,153 people using

a 24-h dietary recall method showed an average intake of 10.6 mg Zn per day (Auestad,

Hurley, Fulgoni, & Schweitzer, 2015), which is below the recommended intake of 15

mg per day established by the National Research Council Recommended Dietary

Allowances (2001). Gibson and Heath (2011) established that toddlers, adolescents

(especially those of Pacific and Aboriginal ethnicities), institutionalised elderly, and

possibly people with diabetes are the risks groups in Australia and New Zealand, whose

daily Zn intake should be increased to the recommended values.

Food consumption provides the principal route of Zn supply in most human

populations. A diet consisting of a high proportion of cereal-based food with low Zn

contents is considered one of the major reasons for the widespread occurrence of Zn

deficiency in humans, especially in developing countries (Gibson, 2006). Moreover,

plants grown in areas with low Zn availability in soils, such as south-west, east and

south-east of Australia (Alloway, 2008), suffer from Zn deficiency and show not only

poor yield but also low Zn concentration in edible parts. In Zn-deficient conditions,

agronomic biofortification has proved to be an effective and fast solution to increasing

3
Zn concentration in the edible parts of several crops, particularly cereals (Cakmak et al.,

2010; Ghasemi, Khoshgoftarmanesh, Afyuni, & Hadadzadeh, 2013; Gomez-Coronado,

Poblaciones, Almeida, & Cakmak, 2015). Biofortification via foliar Zn application has

been shown to be effective in increasing grain Zn concentration in crops grown in either

Zn-sufficient or Zn-deficient soils (eg. Hussain, Maqsoodab, Rengel, & Aziz, 2012).

Secondly, soil Zn application was not effective in increasing grain Zn concentration, but

increased grain yield (Cakmak et al., 2010). Therefore, soil + foliar application is the

most effective method for increasing both grain Zn and grain yield (Cakmak et al.,

2010).

Little work was done on legumes regarding Zn biofortification. For example, foliar Zn

application resulted in increased grain yield and grain Zn concentration in dry beans

(Phaseolus vulgaris L.) (Ibrahim and Ramadan, 2015). However, legumes are an

excellent source of protein, complex carbohydrates, vitamins and minerals in the diets

of many millions of people, particularly in developing countries. Among legumes, field

peas (Pisum sativum L.) are the second largest legume crop worldwide, with annual

production of 17 million tonnes (FAOSTAT, 2014). In Australia, with field peas

production of approximately 0.3 million tonnes annually (FAOSTAT, 2014), grain Zn

concentration was between 10 and 11 mg kg-1 (Cunningham, Milligan, & Trevisan,

2001), suggesting that field peas may be a good candidate for Zn biofortification.

Indeed, much higher Zn concentrations have been found in field peas elsewhere in the

world, such as 24-37 mg kg-1 in Canada (Wang, Hatcher, & Gawalko, 2008), about 57

mg kg-1 in India (Pandey, Gupta, & Pathak, 2013) or 34-35 mg kg-1 in Pakistan

(Rafique, Yousra, Mahmood-Ul-Hassan, Sarwar, Tabassam, & Choudhary, 2015).

However, in all these cases, Zn concentrations were lower than the recommended

4
critical level, established by Huett, Maier, Sparrow, & Piggot (1997) to be 61 mg Zn kg-
1
field pea grains to achieve a sufficient Zn status in humans.

The grain Zn content is not the only important parameter. Presence of antinutritional

components, mainly phytic acid, is one of the major drawbacks limiting the nutritional

quality of legumes. Phytate (myo-inositol 1,2,3,4,5,6-hexakisphosphate) binds with Zn

and other metal cations to form insoluble complexes that hinder Zn absorption in the

human intestine. Accordingly, diets rich in phytate and low in mineral micronutrients

can cause health problems, typically Fe, Zn, Mg and Ca deficiencies (Raboy, 2002).

However, relatively low phytate concentration was found in field peas grown in the US

(4.9-7.1 g kg-1, Amarakoon, Thavarajah, McPhee, & Thavarajah, 2012) and Canada

(6.4-8.3 g kg-1, Wang et al., 2008).

To determine the bioavailability of minerals in food, one of the methods is measuring

the molar ratio of phytate:Zn (ratios greater than 15 were associated with Zn deficiency,

Sandberg, Anderson, Carlesson, & Sandstrom, 1987; Morris and Ellis, 1989). Most

cereal grains and their products contain high ratios, eg. between 25 and 34 (Morris and

Ellis, 1989). Foliar Zn application was effective in decreasing the phytate:Zn ratio and

thus increasing estimated Zn bioavailability (Hussain et al., 2012). Another important

factor to consider is that legumes are usually cooked before intake to improve flavour

and palatability. Cooking may improve nutritional value by decreasing concentration of

antinutrients, such as phytate or tannins, and increasing concentration of other

components such as protein and starch (Wang, Hatcher, Toews, & Gawalko, 2009;

Brigide, Canniatti-Brazaca, & Silva, 2014), probably due to the loss of soluble solids

during cooking, which would increase concentration or remaining grain components.

5
However, there is inadequate knowledge on how Zn biofortification influences phytate

concentration in grain, and how processing from raw to cooked grain affects content of

both Zn and phytate and thus Zn bioavailability. In order to increase Zn intake by

humans and clarify questions regarding the effectiveness of Zn application methods on

bioavailability of Zn in field pea grains, we tested the hypothesis that combined soil and

foliar application of Zn is effective in increasing both grain Zn concentration and Zn

bioavailability in cooked grains of field peas. The aims of the present study were (i) to

evaluate the potential of field peas to be used in the Zn biofortification programs and (ii)

to promote cooked peas as a Zn source. Such evaluation consisted of analysing the

effects of different soil and foliar Zn fertilization treatments on plant growth, nutrient

uptake and Zn accumulation in raw grains, subsequently frozen and boiled to estimate

Zn bioavailability.

2. Materials and methods

2.1. Site, experimental design and crop management

This study was conducted in a naturally-lit glasshouse located at The University of

Western Australia in Perth (31°57′ S, 115°47′ E), Australia, between May and August

2015. During the experiment, the average temperature in the glasshouse was 23 ± 4 ºC

during the day and 17 ± 4 ºC during the night. Light intensity varied between 250 and

1,100 µmol photon m-2 s-1, and relative humidity varied from 40 (midday) to 85%

(midnight).

The soil used in the experiment was topsoil (0-20 cm) collected from the area of Gingin

in Western Australia (31°35′S, 115°90′E) (Cambic Arenosol). The soil was disinfected

by heating to 60ºC for 1 hour and was then air-dried for 2 days and sieved to <5 mm.

6
Four subsamples of the sieved soil were analysed for various physico-chemical

properties. Soil texture (determined gravimetrically) was sandy. The soil had pH 6.5 (10

g soil:25 mL deionised H2O), electrical conductivity of 0.028 dS m−1, organic carbon

2.9 g kg−1 (Walkley-Black, 1947), available phosphorus 14 mg kg−1 and potassium <15

mg kg−1, nitrate nitrogen 1 mg kg−1 and ammonium nitrogen 2.5 mg kg−1 (extracted

with 1 M potassium chloride for 1 hour at 25°C and measured on a Lachat Flow

Injection Analyzer). Plant-available Zn was 0.26 mg kg-1 soil, determined according to

Lindsay and Norvell (1978) by extraction with DTPA (diethylenetriamine pentaacetic

acid) using a soil:solution ratio of 1:2 and shaking time of 2 hours. Extracted Zn was

determined by inductively-coupled plasma optical emission spectroscopy (Zarcinas,

1984) (ICP-OES, Vista-Pro Axial, Varian Pty Ltd, Mulgrave, Australia). Certified soil

reference material and blanks were included in each batch of samples. All the results

were reported on a dry weight basis.

To ensure Zn was the only nutrient limiting growth, the following basal nutrients (in mg

kg-1) were added to soil as solutions: 90.2 KH2PO4; 139.9 K2SO4; 40.1 MgSO4·7H2O;

95.2 NH4NO3; 150.3 CaCl2·2H2O; 10.0 MnSO4·H2O; 2 CuSO4·5H2O; 0.5

CoSO4·7H2O; 0.2 Na2MoO4·2H2O, 0.7 H3BO3. Soil Zn treatments (see below)

consisted of spraying Zn sulphate solution to the soil surface. After application of basal

nutrients and different soil Zn rates, the soil in each pot was thoroughly mixed.

Zinc treatments comprised all possible combinations of soil application of 0, 4 and 8 mg

ZnSO4·7H2O kg-1 soil (0Soil, 4Soil and 8Soil, respectively) and foliar application of

distilled-water spray (10 mL pot-1) (NoFoliar) and two sprays (10 mL pot-1 each) of

0.25% or 0.5% (w/v) ZnSO4·7H2O (0.25Foliar and 0.5Foliar, respectively), once before

7
flowering and the second time at early grain filling stage. Foliar Zn treatments were

applied in the late afternoon; spraying continued until all the leaves were wet. The pots

were covered with polythene at the base of the plants so that the solution did not trickle

to the soil. The treatments were arranged in completely randomized design with four

replications.

The field pea cultivar used was Twilight. Seeds were surface-sterilised by soaking in

80% v/v ethanol for 60 s, washed thoroughly with sterile water and sown in 20-cm-high

and 20-cm-wide pots containing 6.5 kg soil (nine seeds per pot). Seeds were inoculated

with a commercial inoculant (Becker Underwood Pty Ltd, Somersby, Australia)

following the manufacturer’s instructions. Two weeks after emergence, the pots were

thinned to leave three plants per pot. Soil moisture content was maintained around 60%

of the water holding capacity by watering plants every 2 days with deionised water.

Four drainage slits (2 cm wide) 2 cm above the pot base allowed drainage. There was no

incidence of pests or diseases during the study.

2.2. Plants analysis

Plants were harvested at maturity in early August. Roots, stems with leaves, and pods

including grains were separated. Fresh roots were used to determine root length, average

diameter and root surface. Roots were spread out on a Perspex tray and imaged using a

flat-bed scanner (EPSON Perfection V700/V750 operating resolution 400 dpi) and

WinRhizo 5.0 ATM software (Regent Instruments, Montreal, QC, Canada). Symbiotic

nodulation was estimated using a categorical scoring system based on the number and

colour (white = ineffective, pink = effective) of nodules: 0; no nodules, 1: ≤ 5 white

nodules, 2: 6-10 white nodules, 3: 10-20 white nodules, 4: > 20 white nodules, 5: ≤ 5

8
pink nodules, 6: 6-10 pink nodules, 7: 10-20 pink nodules, 8: > 20 pink nodules. Grains

and roots were subsampled for analyses that required fresh samples (see below); the

remaining subsamples and whole stems with leaves were air-dried at 60 ºC for 48 hours

in a forced-air cabinet until constant weight, and weighed.

Nitrogen content was determined in the raw grains as well as in grains after processing

(cooking, drying and grinding (see below)) using the Dumas combustion method (Leco

FP-428 analyzer). Grain protein was determined by multiplying the total N by 6.25 as a

conversion factor. In order to obtain cooked grains, the four replicates of each treatment

were bulked two by two. Each sample was frozen at -25ºC and after 24 h at that

temperature, 5 ± 0.5 g field peas were boiled for 5 min in 50 mL of deionised water in

Pyrex flasks previously cleaned with nitric acid (ultrapure, 10% v/v). The boiling time

of 5 min was determined following the typical producer’s instructions. Cooked grains

were dried at 60ºC for 48 h, and ground to fine powder (<0.05 mm size) using a

mechanical grinder; powder (0.2 g) was digested in a heated mixture of concentrated

nitric and perchloric acids. Grain digests as well as water in which grains were boiled

were analysed for total Zn, Ca, Fe and Mg by ICP-OES as described above.

To estimate grain Zn bioavailability, phytate concentration was determined in raw and

cooked grains. Protein content was also determined in cooked grains. The phytate assay

was based on precipitation of ferric phytate and measurement of iron (Fe) remaining in

the supernatant (Haug and Lantzsch, 1983). Phytate was extracted from about 0.2 g of

ground field pea grains in 10 mL of 0.2 M HCl (pH 0.3) after shaking for 2 h. One mL

of supernatant was treated with 2 mL of ferric solution (NH4Fe(SO4)2·12 H2O) in a

boiling water bath for 30 min. After cooling, samples were centrifuged, and 1 mL of

9
supernatant was treated with 1.5 mL of 0.064 M bipyridine (2-pyridin-2-ylpyridine,

C10H8N2) to measure Fe. After mixing, the solution was incubated for 10 min at room

temperature, and the light absorbance was measured with a spectrophotometer at 419

nm. The molar ratio between phytate and Zn, Ca, Fe or Mg was calculated.

2.3. Statistical analysis

Data were subjected to two-way ANOVA, including soil Zn (0, 4 and 8 mg

ZnSO4·7H2O kg-1) and foliar Zn (0, 0.25 and 0.5% w/v ZnSO4·7H2O) as well as their

interaction in the model. When significant differences were found in ANOVA, means

were compared using Fisher’s protected least significant difference (LSD) test at P ≤

0.05. Pearson correlation tests were performed between total Zn in grain and the other

measured parameters. All analyses were performed using Statistix v. 8.10 for Windows

(Analytical Software, Tallahassee, FL, USA).

3. Results

3.1. Soil DTPA Zn

The initial analysis of the soil used in the study showed that the DTPA-extractable Zn

was 0.26 ± 0.06 mg kg-1 (mean ± standard error). After plant harvest at maturity, soil

DTPA Zn was significantly influenced by both Zn application methods (foliar and soil),

but the interaction was not significant (Table 1). Soil DTPA Zn was significantly higher

in the 8Soil than 4Soil and 0Soil treatments (Table 2). Regarding foliar treatments, soil

DTPA Zn was significantly higher in 0.25Foliar and 0.5Foliar (0.94 and 1.04 mg

DTPA-Zn kg-1, respectively) compared with the NoFoliar treatment (0.81 mg DTPA-Zn

kg-1).

10
3.2. Effect of Zn treatments on root and shoot properties

All the studied root parameters (except nodulation) were significantly affected only by

the soil Zn application (Table 1), with significant increases recorded at both soil Zn

doses compared with the non-fertilised control (0Soil). Root DW was significantly

higher in the 4Soil than 8Soil treatment, but there were no significant differences

between these two treatments in root length, average root diameter and root surface

(Table 2). Symbiotic nodulation was not significantly affected by any of the studied Zn

treatments.

Shoot parameters were significantly affected only by the soil Zn treatment (Table 1).

The application of 4 mg of ZnSO4·7H2O kg-1 produced greater shoot dry weight than

the application of 8 mg of ZnSO4·7H2O kg-1, both being higher than the 0Soil treatment.

Regarding the number of pods per plant, grains per pod and 100-grain weight, the 4Soil

treatment was not significantly different from the 8Soil treatment, but they both tended

to be higher than the 0Soil Zn treatment (Table 2).

3.3. Effect of Zn treatments on raw and cooked grain

Protein content in raw grains was significantly influenced (P≤0.001) only by the foliar

treatment, but this effect was not significant for protein content in cooked grains (Table

1). Both foliar application rates significantly increased protein content in raw grains,

from 19.9% in NoFoliar to 22.0% in 0.25Foliar and 22.3% in the 0.5Foliar treatment.

Protein content in cooked grains decreased to 19.9% (averaged across all treatments

because of no significant difference among them)”

11
Total Zn concentration and phytate:Zn molar ratio in raw and cooked grains were

significantly influenced (P≤0.05) by the soil Zn x foliar Zn interaction. In contrast, for

phytate concentration, this interaction was significant only in raw grains (phytate

concentration in cooked grains was influenced only by the foliar treatment) (Table 1).

Total concentration of Zn in the raw field pea grains ranged between 34 mg Zn kg-1 in

the 0Soil/NoFoliar treatment to 107 mg Zn kg-1 in the 0Soil/0.5Foliar treatment (3.1-

fold increase, Table 3). However, in comparison with the 0Soil treatment, the effect of

foliar Zn application on total Zn concentration in raw grains was smaller in the 4Soil

(1.9-fold increase) and 8Soil treatments (1.3-fold increase), testifying to the

effectiveness of soil Zn application and thus making the interaction soil Zn x foliar Zn

significant. Similar relationships were recorded for cooked grains as well, with

corresponding increases in total Zn concentration due to foliar Zn application being 2.6-

, 2.0- and 1.4-fold in the 0Soil, 4Soil and 8Soil treatments, respectively (Table 3).

Phytate concentration in raw grains varied in a narrow range (6.3 to 7.0 g kg-1) across

the soil and foliar Zn treatments (Table 3), placing relatively low biological importance

on the significant soil Zn x foliar Zn interaction (Table 1). There was a small decreasing

trend in phytate concentration with the foliar Zn treatments (significant only for

0.5Foliar application in case of 0Soil treatments). In cooked grains, differences caused

by the foliar Zn treatment were significant (P≤0.05), but small, ranging from 6.6 g kg-1

in the NoFoliar treatment to 6.0 g kg-1 in the other two foliar treatments.

For phytate:Zn molar ratio in either raw or cooked grains, the soil Zn x foliar Zn

interaction was significant (Table 1) because foliar Zn application decreased the ratio

12
more in the 0Soil than the other two soil treatments (Table 3). That was caused by soil

Zn treatment increasing Zn concentration in raw as well as cooked grains, making foliar

Zn applications less effective in combination with soil Zn application than without any

soil Zn application.

Concentrations of Fe and Mg as well as phytate:Fe and phytate:Mg ratios in either raw

or cooked grains were significantly affected only by the soil Zn treatment; in contrast,

grain Ca concentration and phytate:Ca ratio were not influenced by any of the studied

treatments (Table 1). The Fe and Mg concentrations in either raw or cooked grains were

lower in the 8Soil than 4Soil and 0Soil treatments, resulting in the opposite trend in

phytate:Fe and phytate:Mg ratios (Fig. 1)

3.4. Effect of Zn treatments on nutrients in water after cooking field pea grains

Total Zn concentration found in the water after cooking grains was significantly

influenced (P≤0.05) by the soil Zn x foliar Zn interaction (Table 1). For all soils

treatments, the lowest Zn concentration was found without any foliar application and

progressively higher concentrations were found in water after cooking grains from the

increasing Zn foliar applications; this difference caused by foliar Zn treatments was

smaller in the 4Soil than the other two soil treatments (Table 3).

4. Discussion

The most useful indicator of Zn availability in the soil is DTPA-extractable Zn. In our

case, the initial soil DTPA-Zn, 0.26 ± 0.06 mg kg-1, was lower than the widely accepted

critical Zn concentration of 0.5 mg kg-1 (Sims and Johnson, 1991). Therefore, Zn

concentration observed in the soil might limit field pea growth and yield as well as

13
result in grain with Zn concentration below the minimal one recommended for human

daily intake, established by Huett et al. (1997) at 61 mg Zn kg-1. Under such conditions

of low Zn availability in soil, the implementation of a Zn biofortification program could

take on special importance in increasing the Zn content in commonly consumed plant

foods. A strong increase in the soil DTPA-Zn was achieved by soil Zn fertilization

(about 3.7-fold in the treatment with 4 mg ZnSO4·7H2O kg-1 and 5.6-fold with 8 mg

ZnSO4·7H2O kg-1) (Table 2). Working with a soil with low Zn availability, Zhao, Tian,

Cao, Lu, &Liu (2013) found similar increases in extractable soil Zn (3- to 3.7-fold) with

soil or combined soil+foliar fertilization. However, single Zn fertilization would have a

declining effect over time, with Brennan (2001) finding that the effectiveness of a single

Zn fertiliser treatment for wheat in a highly-deficient soil in Western Australia had

decreased by 50% over a period of 13 years. Similarly, Cakmak et al. (2010) found that

an application of 28 kg Zn ha-1 as zinc sulphate was sufficient to correct Zn deficiency

in wheat growing in calcareous soils for 4-6 years. Recently, Abid, Ahmed, Qayyum,

Shaaban, & Rashid (2013) concluded that a single application of 7.5 kg ZnSO4·7H2O

ha-1 is adequate for two cycles of a cotton-wheat production system. However, further

research with different crops and soil and climatic conditions should characterize the

residual effect of soil Zn application. This is a crucial point in establishing a successful

biofortification program.

Although field peas is categorized as having relatively low sensitivity to Zn deficiency

(Alloway, 2008), our study (Table 2) as well other reports in the literature indicate

substantial plant growth and grain yield depression in conditions of low Zn supply. The

reduction in dry matter yield due to low Zn supply might be attributed to the distorted

14
formation of apices and abnormal floral development, with reduced number of flowers

per plant and premature shedding of floral organs (Pandey et al., 2006).

In the present study, foliar Zn application did not improve growth. In contrast, Pandey

et al. (2013) found a positive effect of foliar application of 0.5% w/v ZnSO4 at the

initiation of bud formation in field pea on grain yield when soil Zn availability was low.

In our study, soil Zn fertilization before sowing increased plant growth and grain yield

(Table 2). Root development followed the same tendency as the aerial parts, with soil

fertilization before sowing exerting a positive effect, and foliar application having no

effect, probably due to its late timing (Table 3). Similar results were found in cereals

such as barley (Genc, Huang, & Langridge, 2007). The majority of Zn transport in soil

is by diffusion; therefore, efficiency of Zn acquisition would be influenced by root

morphology and root exploration of new soil volumes (cf. Genc et al., 2007). An

increase in root surface area would increase acquisition of Zn as well as other nutrients,

such as Cu, Fe and Mn (Dong, Rengel and Graham, 1995).

In the non-treated soil, total Zn concentration in raw grains was 34 mg kg-1 (Table 3),

much higher than that found by Cunningham et al. (2001) in Australia (10 to 11 mg kg-
1
), but similar to those found by Wang et al. (2008) in Canada (24-37 mg kg-1) and

Rafique et al. (2015) in Pakistan (34-35 mg kg-1); however, Zn concentration in the raw

field pea grains in the non-treated soil in the present study was lower than 57 mg Zn kg-
1
found in India by Pandey et al. (2013) and lower than the level considered by Huett et

al. (1997) to be sufficient (61 mg Zn kg-1). Bioavailability of grain Zn, expressed as

phytate:Zn ratio, was 19.9 in the non-fertilized plants (Table 3), lower than the critical

target level of 15 to ensure adequate bioavailability (Sandberg et al., 1987).

15
Based on the reports published by Graham et al. (2007) and Cakmak et al. (2010) in

wheat, documenting that an agronomic biofortification practice could be considered to

have had a measurable biological impact on human health when it produced an increase

of at least 10 mg Zn kg-1 grain, all the treatments reported in the present paper were

effective (Table 3). However, the target level of 61 mg kg-1 grain (see Huett et al. 1997)

was achieved only when a foliar Zn treatment was included. In the present study, foliar

treatments were more effective than soil treatments (Table 3), likely due to foliar

applications facilitating Zn loading into the phloem. The increases in grain Zn

concentration in field peas due to foliar Zn fertilization were higher than those reported

in other crops, such as wheat (Hussain et al., 2012; Gomez-Coronado et al., 2015).

Hence, it appears that legumes may accumulate a greater amount of Zn in the seeds than

cereals, which could be due to the lower protein content in cereal than legume grains,

with grain Zn being mainly associated with proteins (Cakmak, 2000).

Regarding grain concentration of phytate, the principal Zn antinutrient, the range found

in this study (6.3-7.0 g kg-1, Table 3) was in accordance with the data reported by

Amarakoon et al. (2012) in the US and Wang et al. (2008) in Canada. Generally, there

was a trend of foliar Zn treatments resulting in decreased raw grain phytate

concentration compared with non-treated control (Table 3, also in other crops, Hussain

et al., 2012; Ghasemi et al., 2013). The phytate:Zn molar ratio is considered a better

indicator of Zn bioavailability than total acidity in the diet (Ghasemi et al., 2013). In the

study presented here, only the control (0Soil/NoFoliar) and the 4Soil/NoFoliar treatment

resulted in the ratio higher than 15 in raw field pea grains (Table 3), suggesting poor Zn

bioavailability (cf. Sandberg et al., 1987; Morris and Ellis, 1989).

16
Cooking of field pea grains resulted in decreased protein content (by about 7%). Other

reports found cooking increased protein concentration in lentil grains (Wang et al. 2009)

and grains of some field pea cultivars (Brigide et al. 2014). Zn concentration in cooked

grains decreased by about 32% and phytate concentration by about 6%. These decreases

caused a 17%-increase in phytate:Zn molar ratio. Nevertheless, the target Zn

concentration (higher than 61 mg kg-1) and phytate:Zn ratio (lower than 15) were

achieved in any treatment, except 0Soil/NoFoliar and 4Soil/NoFoliar (Table 3). As

Brigide et al. (2014) reported, cooking of field pea grains before intake improved their

nutritional value by reducing concentration of antinutrients, such as phytate. Based on

the results presented in Table 3, the intake of 100 g of frozen and subsequently cooked

field pea grains fertilized with the 4Soil/0.25Foliar treatment may provide about 7.6 mg

Zn, which is about 50% of the Zn recommended dietary allowance (RDA) for humans

(National Research Council, 2001), and about 2.4-times more than the same intake of

non-biofortified grain.

Field pea is an excellent source of carbohydrates, protein, dietary fibre, vitamins and

minerals (Wang et al., 2008). Compared with the present study (Tables 1 and 3),

Amarakoon et al. (2012) reported similar concentrations of Ca, lower of Fe and higher

of Mg. All samples obtained in the present study had phytate:Ca ratios higher than 0.24

(data not shown), indicating low bioavailability of Ca.

Soil application of 8 mg of ZnSO4·7H2O kg-1 reduced significantly the grain

concentration of total Fe and increased phytate:Fe ratio (Fig. 1). Molar ratios of

phytate:Fe above 10 lead to reduced human Fe bioavailability (Engle-Stone, Yeung,

17
Welch, & Glahn, 2005). In this study, these ratios ranged between 6.0 and 8.2 in raw

grains and were slightly lower in cooked grains (Fig. 1), suggesting good bioavailability

to humans. These ranges were lower than those found by Amarakoon et al. (2012) (9-11

in field pea grains) and comparable with whole cooked Fe-dense common bean (6.3-

12.2; Ariza-Nieto, Blair, Welch, & Glahn, 2007). A consumption of 100 g of cooked

field peas, fertilized with the 4Soil/0.25Foliar treatment, with concentrations of 0.5 g Ca

kg-1, 71 mg Fe kg-1 and 0.8 g Mg kg-1 would cover more than the 80% of the RDA of Fe

and about 33% of Mg, with acceptable bioavailability (phytate:Fe ratio of 6.0 and

phytate:Mg ratio of 0.24), but only about 5% of Ca with low bioavailability (phytate:Ca

ratio of 0.8).

5. Conclusions

The present study showed that field pea may be a good candidate to be included in Zn

biofortification programs because its grain could efficiently increase Zn content in the

human food chain. The combined application of 8 mg ZnSO4·7H2O kg-1 soil + 0.25%

(w/v) ZnSO4·7H2O foliarly increased grain Zn concentration by more than 40 mg Zn

kg-1 with good bioavailability. Therefore, this combination could be an interesting

option for biofortification. The cooking process caused a loss of Zn of about 30%, but

bioavailability remained good. For practical biofortification, further work is required in

the field, especially regarding residual value of Zn fertilisers applied to soil.

Acknowledgments

The authors would like to acknowledge the financial support provided by the Ministry

of Education, Culture and Sport of Spain (Program: José Castillejo) during Dr.

18
Poblaciones’s stay in the School of Earth and Environment of the University of Western

Australia.

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Figure captions

Fig. 1. Grain Fe and Mg concentrations (top panels) and phytate:Fe and phytate:Mg

molar ratios (bottom panels) in raw and cooked grains as affected by the soil Zn

treatment.

23
 Bars with different lowercase letters (for raw grains) or different Greek letters (for

cooked grains) were significantly different (P ≤ 0.05) according to the Fisher’s

protected LSD test for raw grains.

Table 1: Two-way ANOVA for the parameters measured

Soil Zn Foliar Zn Soil x Foliar

DF 2 2 4

Soil DTPA Zn (mg kg-1) 228*** 7.6** 1.4

24
Root DW (g plant-1) 12*** 0.6 1.4
Root length (cm) 11*** 0.3 0.1
Average root diameter (mm) 16*** 0.9 0.1
2
Root surface (cm ) 6.7** 0.3 0.5
Nodulation 2.3 0.7 0.5
-1
Shoot DW (g plant ) 21*** 2.7 1.2
-1
Pods plant 5.0* 3.5 0.6
-1
Grains pod 4.3* 2.4 1.4
100-grain weight (g) 9.5** 1.8 1.3
Protein content (%) 0.5 9.7*** 1.6
Grain Zn concentration (mg kg-1) 7.5* 44*** 4.5*
Grain Ca concentration (mg kg-1) 0.1 1.1 0.6
Grain Fe concentration (mg kg-1) 31*** 0.4 1.3
Grain Mg concentration (mg kg-1) 7.5*** 0.2 1.5
Phytate (g kg-1) 27*** 29*** 3.0*
Phytate:Zn ratio 4.8* 65*** 8.2***
Phytate:Ca ratio 0.7 0.6 0.9
Phytate:Fe ratio 27*** 2.3 0.9
Phytate:Mg ratio 13*** 0.7 1.3
Zn concentration in water after cooking
105*** 1023*** 27***
grains
Protein content (cooked grains) (%) 0.6 2.3 1.2
Grain Zn concentration (cooked) (mg kg-1) 95*** 638*** 33***
Grain Ca concentration (cooked) (mg kg-1) 2.0 3.9 3.2
Grain Fe concentration (cooked) (mg kg-1) 74*** 1.3 0.9
Grain Mg concentration (cooked) (mg kg-1) 39*** 0.5 3.0
Phytic acid (cooked grains) (g kg-1) 0.4 7.6* 1.1
Phytate:Zn ratio (cooked grains) 25*** 192*** 16***
Phytate:Ca ratio (cooked grains) 1.4 0.73 3.0
Phytate:Fe ratio (cooked grains) 44*** 7.0* 0.2
Phytate:Mg ratio (cooked grains) 12** 4.3* 0.2
DF. Degree of freedom; F. Statistic F. Level of significance: * p ≤ 0.05; ** p ≤ 0.01;

*** p ≤ 0.001

25
Table 2: Soil DTPA-Zn, root DW, root length, average root diameter, root surface, shoot DW, number of pods plant-1, number of grains pod -1

and 100-grain weight as affected by the soil Zn treatment. Means ± standard error.

Soil DTPA-Zn Root DW Root length Average root Root surface Shoot DW Number of Number of 100-grain
(mg kg-1) (g plant-1) (cm) diameter (mm) (cm2) (g plant-1) pods plant-1 grains pod-1 weight (g)

0Soil 0.27 ± 0.02c 0.17 ± 0.02c 936 ± 21b 0.35 ± 0.02b 120 ± 5b 2.9 ± 0.2c 6.9 ± 0.6b 6.4 ± 0.1b 28.6 ± 0.8b

4Soil 0.99 ± 0.06b 0.27 ± 0.03a 1329 ± 59a 0.41 ± 0.02a 155 ± 10a 4.0 ± 0.2a 8.4 ± 0.2a 6.8 ± 0.1a 32.5 ± 0.8a

8Soil 1.52 ± 0.07a 0.21 ± 0.02b 1213 ± 37a 0.43 ± 0.01a 160 ± 9a 3.4 ± 0.1b 7.6 ± 0.3ab 6.6 ± 0.1ab 31.2 ± 0.5a

Means in a column with different lowercase letters were significantly different (P ≤ 0.05) according to the Fisher’s protected LSD test.

26
Table 3: Total Zn concentration in raw and cooked grains, phytate in raw grains, phytate:Zn molar ratio in raw and cooked grains and total Zn in

water after cooking grains as affected by the Zn soil and foliar treatments.

Total Zn concentration Phytate in raw Total Zn boiled

Phytate:Zn ratio
Treatment (mg kg-1) grains water
Raw grains Cooked grains (g kg-1) Raw grains Cooked grains (mg kg-1)

0Soil
34 ± 3 d 31 ± 2 e 6.9 ± 0.1 ab 19.9 ± 0.8 a 20.1 ± 1.7 a 0.43 ± 0.02f
NoFoliar
87 ± 1 bc 63 ± 3 c 6.3 ± 0.7 d 7.1 ± 1.4 d 9.5 ± 0.8 c 1.21 ± 0.03 c
0.25 Foliar
107 ± 3 a 81 ± 4 a 6.4 ± 0.4 d 5.8 ± 0.8 d 7.5 ± 0.9 c 1.68 ± 0.04a
0.5 Foliar

4Soil
44 ± 3.33d 31 ± 3 e 6.9 ± 0.5 ab 15.9 ± 0.8 b 21.1 ± 1.2 a 0.38 ± 0.02 f
NoFoliar
80 ± 3.58bc 62 ± 5 c 6.7 ± 0.3 c 8.5 ± 0.5 cd 9.5 ± 0.8 c 0.88 ± 0.07 e
0.25 Foliar
84 ± 3.09bc 64 ± 3 c 6.6 ± 0.3 c 7.8 ± 1.1 d 8.9 ± 0.7 c 1.08 ± 0.06 d
0.5 Foliar

8Soil
70 ± 2c 51 ± 4 d 7.0 ± 0.4 a 10.9 ± 0.5 c 12.3 ± 1.2 b 0.39 ± 0.04 f
NoFoliar
94 ± 3ab 76 ± 7 b 6.9 ± 0.3 ab 7.2 ± 0.5 d 8.0 ± 0.6 c 1.04 ± 0.02 d
0.25 Foliar
92 ± 4ab 73 ± 6 b 6.8 ± 0.6 bc 7.7 ± 0.8 d 8.4 ± 1.8 c 1.50 ± 0.04 b
0.5 Foliar
Means in a column with different lowercase letters were significantly different (P ≤ 0.05) according to the Fisher’s protected LSD test for the

interaction soil Zn x foliar Zn.

27
 Highlights

• Soil and foliar Zn fertilization was tested for biofortification of field pea grains

• Foliar application prompted increases above 60 mg Zn kg-1 in grain

• Processing (cooking) caused a decrease of about 30% in grain Zn concentration

• Adequate bioavailability of Zn in raw and cooked field peas was achieved

28