RESEARCH ARTICLE

Evaluation and utilization of Aegilops and wild Triticumspecies for enhancing iron and zinc content in wheat

Nidhi Rawat Æ Vijay K. Tiwari Æ Neelam Singh ÆGursharn S. Randhawa Æ Kuldeep Singh ÆParveen Chhuneja Æ Harcharan S. Dhaliwal

Received: 20 October 2007 / Accepted: 28 April 2008 / Published online: 31 May 2008

� Springer Science+Business Media B.V. 2008

Abstract Grains of 80 accessions of nine species of

wild Triticum and Aegilops along with 15 semi-dwarf

cultivars of bread and durum wheat grown over

2 years at Indian Institute of Technology, Roorkee,

were analyzed for grain iron and zinc content. The

bread and durum cultivars had very low content and

little variability for both of these micronutrients. The

related non-progenitor wild species with S, U and M

genomes showed up to 3–4 folds higher iron and zinc

content in their grains as compared to bread and durum

wheat. For confirmation, two Ae. kotschyi Boiss.

accessions were analyzed after ashing and were found

to have more than 30% higher grain ash content than

the wheat cultivars containing more than 75% higher

iron and 60% higher zinc than that of wheat. There

were highly significant differences for iron and zinc

contents among various cultivars and wild relatives

over both the years with very high broad sense

heritability. There was a significantly high positive

correlation between flag leaf iron and grain iron

(r = 0.82) and flag leaf zinc and grain zinc (r = 0.92)

content of the selected donors suggesting that the leaf

analysis could be used for early selection for high iron

and zinc content. ‘Chinese Spring’ (PhI) was used for

inducing homoeologous chromosome pairing between

Aegilops and wheat genomes and transferring these

useful traits from the wild species to the elite wheat

cultivars. A majority of the interspecific hybrids had

higher leaf iron and zinc content than their wheat

parents and equivalent or higher content than their

Aegilops parents suggesting that the parental Aegilops

donors possess a more efficient system for uptake and

translocation of the micronutrients which could

ultimately be utilized for wheat grain biofortification.

Partially fertile to sterile BC1 derivatives with vari-

able chromosomes of Aegilops species had also

higher leaf iron and zinc content confirming the

possibility of transfer of required variability. Some of

the fertile BC1F3 and BC2F2 derivatives had as high

grain ash and grain ash iron and zinc content as that of

the donor Aegilops parent. Further work on back-

crossing, selfing, selection of fertile derivatives, leaf

and grain analyses for iron and zinc for developing

biofortified bread and durum wheat cultivars is in

progress.

Keywords Aegilops � Biofortification �Bread wheat � Grain � Iron � Zinc

Nidhi Rawat, Vijay K. Tiwari, and Neelam Singh have

contributed equally to the work.

N. Rawat � V. K. Tiwari � N. Singh �G. S. Randhawa � H. S. Dhaliwal (&)

Department of Biotechnology, Indian Institute of

Technology, Roorkee 247 667 Uttarakhand, India

e-mail: hsdhaliwal07@gmail.com; hsdhafbs@iitr.ernet.in

K. Singh � P. Chhuneja

Department of Plant Breeding, Genetics and

Biotechnology, Punjab Agricultural University,

Ludhiana 141 004, India

123

Genet Resour Crop Evol (2009) 56:53–64

DOI 10.1007/s10722-008-9344-8

Abbreviations

AAS Atomic absorption spectrophotometer

CIMMYT Centro Internacional de Mejoramiento

de Maız y Trigo

FAO Food and Agricultural Organization

Introduction

More than 2 billion people depending largely on

cereals, roots and tubers as staple food suffer from

disorders related to micronutrient iron and zinc

deficiencies commonly referred to as ‘hidden hunger’

(FAO 2002). Processing of some cereals like rice and

wheat further reduces the already limited content of

the micronutrients. A number of dietary inhibitors

such as phytic acid, food fibers, tannins and lignin’s

reduce the bioavailability of the micronutrients

(Welch 2002). Conventional and molecular breeding

and genetic engineering techniques have been con-

sidered to be the most feasible and cost effective

approaches for biofortification of cereals with high

iron and zinc content (Lonnerdal 2003; Bouis 1999).

Most of the Triticum aestivum L. and Triticum

turgidum L. ssp. durum (Desf.) Husn. cultivars have

lower grain iron and zinc content than the related

wild Triticum and Aegilops species (Chhuneja et al.

2006; Cakmak et al. 2000; Monasterio and Graham

2000). For the identification of useful variability for

wheat biofortification major emphasis has been on the

screening of progenitor species including diploid

wheat, T. monococcum L., Triticum turgidum L. ssp.

dicoccoides (Korn. ex Asch. et Graebn.) Thell.,

Triticum turgidum L. ssp. dicoccon (Schrank) Thell.,

Ae. tauschii Coss. etc. (White and Broadley 2005;

Cakmak et al. 2000, 2004; Monasterio and Graham

2000). Scientists at CIMMYT, Mexico in collabora-

tion with HarvestPlus have used synthetic hexaploid

wheat from crosses between T. turgidum ssp. durum

and Ae. tauschii with high iron and zinc contents in

breeding programs and have developed wheat lines

with higher level of these micronutrients which are

being tested in farmers’ fields in India, Pakistan and

other countries (Calderini and Monasterio 2003).

However the level of enhancement of iron and

zinc through the breeding program using wheat

synthetics remained low because of the limited

variability for iron and zinc in wild progenitor species

which were used as parents. Therefore, screening of

non-progenitor species comprising the secondary and

tertiary gene pools (Jiang et al. 1994) for additional

variability for micronutrients is very critical.

The A and D genome donors of hexaploid wheat

Triticum aestivum (AABBDD) have been unequivo-

cally identified as Triticum urartu Tumanian ex

Gandilyan (AA) and Ae. tauschii (DD) (Faris et al.

2002; Dvorak et al. 1993; McFadden and Sears 1946;

Kihara 1944) while Ae. speltoides Tausch (SS) or a

closely related species contributed the B genome

(Faris et al. 2002; Riley et al. 1958). A number of

genes for resistance against various wheat diseases

have been introgressed into wheat from related

progenitor and non-progenitor species (Kuraparthy

et al. 2007; Marais et al. 2005; Friebe et al. 1996)

and commercially exploited. It will be highly appro-

priate to identify diverse sources for high iron and

zinc content among related species and genera for

introgression and pyramiding of the desired variabil-

ity to achieve high level of iron and zinc content in

wheat. Among various approaches of induced homo-

eologous pairing mediated introgression in wheat

from non-progenitor genomes, the use of PhI gene

transferred from Ae. speltoides (Chen et al. 1994) has

been found to be very effective and feasible (Aghaee-

Sarbarzeh et al. 2002).

This article deals with the identification of

potential donors of useful variability for high iron

and zinc concentration in seeds of related wild

Triticum and Aegilops species including Ae. kotschyi

Boiss., Ae. peregrina (Hack.) Maire et Weill.,

Ae. geniculata Roth, Ae. ventricosa Tausch, Ae.

cylindrica Host by rigorous analyses with Atomic

Absorption Spectrophotometer (AAS) and the use of

selected donors for transfer of high iron and zinc

content into elite bread and durum cultivars through

interspecific hybridization.

Materials and methods

Experimental materials

The experimental material comprising eighty acces-

sions of nine related Aegilops and wild Triticum

species of wheat from different geographical origins

as well as 15 bread and durum wheat cultivars was

obtained from the Wheat Germplasm Collection

maintained at the Punjab Agricultural University,

54 Genet Resour Crop Evol (2009) 56:53–64

123

Ludhiana, India. The related wild species and bread

and durum wheat cultivars were grown at the

experimental fields of the Indian Institute of Tech-

nology, Roorkee for two consecutive seasons of

2004–2005 and 2005–2006 as unreplicated single

row of two meter length with plant to plant distance

of 10 cm and row to row spacing of 30 cm with

recommended fertilizers (50:25:25 kg/acre N, P2O5,

K) and irrigation as that of wheat. Grains, spikelets

and spikes were harvested and threshed from culti-

vars and wild accessions at physiological maturity.

Due to frequent shattering of spikes in various wild

species, collection of mature spikelets and spikes had

to be done repeatedly at different intervals over 2–

3 weeks. Due to tough glumes and hard threshing in

wild species the grains had to be taken out manually.

The genomic symbols and the number of accessions

of each of the species and cultivars are given in

Table 1.

Interspecific crosses

For transfer of useful variability for higher concen-

tration of iron and zinc from wild donors were

selected as based on their consistently and signifi-

cantly higher iron and zinc, for interspecific crosses

with bread and durum wheat cultivars as the maternal

parents. A bread wheat line ‘Chinese Spring’ with PhI

transferred from Ae. speltoides obtained from Dr.

B.S. Gill of Kansas State University, Kansas was

used for making crosses for induced homoeologous

pairing whereas other interspecific crosses were also

made with bread and durum cultivars without PhI

gene.

Chemical analyses

Grain analysis

For chemical analysis whole grain samples from

cultivated and wild accessions were rinsed with N/10

HCl for 1 min to remove any dust particle from seed

surface and dried in hot air oven at 80�C till constant

weight (0.5 g). Grain samples were digested in a

mixture of two parts of concentrated nitric acid and

one part perchloric acid as per the standard procedure

described by Zarcinas et al. (1987). Digestion was

continued till white residue was obtained. Required

volume was made after the completion of digestion

process and digests were analyzed by Atomic

Absorption Spectrophotometer (GBC—Avanta Garde

M). Concentration of iron and zinc was expressed

mg/kg (ppm) on dry weight basis. A minimum of five

replications of chemical analysis was made in each of

cultivars and wild accessions.

Grain ash analysis

One gram dried grains of two Ae. kotschyi acces-

sions, T. aestivum cultivars WL711 and CS (PhI), a

Table 1 Range and mean of grain iron and zinc content of bread and durum wheat cultivars and wild Triticum and Aegilops species

S.no. Species Number

of accessions

Genome Iron (mg/kg) Zinc (mg/kg)

Range Mean Range Mean

1 T. aestivum 13 ABD 21.26–30.59 27.69 14.88–19.33 22.15

2 T. durum 2 AB 21.91–25.60 23.58 13.68–19.60 18.79

3 T. boeoticum 19 Am 23.88–40.50 30.91 22.12–39.06 29.27

4 T. dicoccoides 17 AB 27.67–42.67 32.98 22.50–66.51 35.33

5 T. araraticum 6 AG 23.10–59.06 29.85 19.27–30.54 23.52

6 Ae. longissima 5 Sl 59.12–81.59 73.24** 24.99–50.52 41.66**

7 Ae. kotschyi 14 US 22.89–90.96 67.46** 22.29–58.61 49.27**

8 Ae. peregrina 10 US 34.37–82.32 52.85** 33.13–49.49 39.54**

9 Ae. cylindrica 3 CD 52.21–93.27 66.76** 32.38–52.18 38.51**

10 Ae. ventricosa 3 DN 55.41–93.52 65.75** 24.01–39.08 33.81**

11 Ae. geniculata 3 UM 52.25–81.97 69.95** 31.93–40.81 37.70**

**Significant at 0.01% level of probability

Genet Resour Crop Evol (2009) 56:53–64 55

123

BC2 and two BC1F2 derivatives each were cleaned

thoroughly and kept for incineration at 600�C for

10 h. The ash was further processed like the grains

for AAS analysis.

Flag leaf analysis

Flag leaves from selected potential donors, recipient

parents and their F1 hybrids were collected between

ear emergence and before anthesis, washed thor-

oughly with N/10 HCl, dried at 80�C for 8 h in oven

prior to digestion. Dried leaf samples were then

digested as a minimum of five replications using

mixture of nitric acid and perchloric acid (Zarcinas

et al. 1987). Iron and zinc concentrations in the

digests were analyzed by AAS.

Cytological studies

For meiotic analysis spikes of interspecific F1 plants

were fixed in Cornoy’s solution (6 ethanol: 3 chloro-

form: 1 acetic acid) for 24 h and transferred to 70%

ethanol. Anthers at various stages of meiotic division-

I were squashed in 2% acetocarmine and the Pollen

mother cells (PMCs) were scored for chromosomal

pairing in all the crosses. Photographs were taken with

a digital camera (Canon PC1049, No. 6934108049).

Statistical analysis

The data on iron and zinc concentration of grains of

all wild accessions, wheat cultivars and flag leaves of

selected parents and F1 hybrids were subjected to

statistical analyses. It included computation of mean

performance, analysis of variance, correlation coef-

ficient and regression. Genotypic and phenotypic

coefficients of variation were calculated using the

following formula (Burton 1952).

GCV ¼p

r2g

X� 100 PCV ¼

pr2p

X� 100

where GCV is the genotypic coefficient of variance,

PCV phenotypic coefficient of variance, r2g geno-

typic variance, r2p phenotypic variance, and X is the

general mean of characters.

Broad sense heritability (H2) was determined by

the following formula suggested by Lush (1940) and

Johnson et al. (1955)

H2 ¼ r2g

r2p� 100:

Results

The range and mean of grain iron and zinc content in

the cultivars of bread and durum and accessions of

various genomes of wild Aegilops and Triticum

species of wheat, grown over 2 years are given in

Table 1. All the 15 bread and durum wheat cultivars

recommended for commercial cultivation in northern

India, possess low level of grain iron and zinc content

with very limited variability, thus emphasizing the

necessity of their biofortification for high iron and

zinc content. On the basis of statistical analysis, wild

accessions differing significantly from cultivated

wheat were selected as donors. There were highly

significant differences among the cultivars and wild

relatives for iron and zinc content over the 2 years

(Table 3). Among the Aegilops species, Ae. longis-

sima (Sl) and Ae. kotschyi (US) had on an average

high grain iron and zinc content suggesting that the S

genome possesses useful variability for effective

uptake, translocation or deposition of the micronutri-

ents in the grains. Some other D genome Aegilops

species like Ae. cylindrica (CD) and Ae. ventricosa

(DN) and non-progenitor genomes such as Ae. geni-

culata (UM) also had high iron and zinc content. The

wild diploid and tetraploid Triticum species viz.,

T. dicoccoides, T. araraticum, and T. boeoticum had

however lower mean and limited variability for iron

and zinc content as compared to the Aegilops species.

Comparison of iron and zinc content among some

representative cultivars and accessions of wild

Triticum and Aegilops species shows that the mean

grain iron content is consistently higher than the mean

zinc content with the exception of T. dicoccoides

where the zinc content was higher than that of iron

(Table 2). Most of the accessions with high iron also

had higher zinc content.

Ae. kotschyi and Ae. longissima showed two to

three folds higher iron and zinc content as compared

to a very popular and high yielding semi-dwarf wheat

variety WL 711. T. dicoccoides accessions 4640 and

4641 with bold seeds also had nearly three fold

higher grain zinc content indicating that the higher

iron and zinc content in the wild relatives with

56 Genet Resour Crop Evol (2009) 56:53–64

123

Table 2 Grain iron and zinc content of bread and durum wheat cultivars and selected accessions of Aegilops and wild Triticumspecies over 2 years

Species Variety/

accession

Year 1 Year 2

Iron Zinc Iron Zinc

Mean ± SD Mean ± SD Mean ± SD Mean ± SD

T. aestivum WL 711 22.01a ± 1.65 19.28bc ± 1.67 26.09b ± 2.24 18.15ab ± 0.99

T. aestivum PBW343 25.39b ± 1.44 18.25bc ± 1.16 30.59cd ± 2.023 19.33b ± 1.73

T. aestivum UP2338 27.07b ± 3.98 16.64b ± 1.09 28.77c ± 2.65 16.32a ± 0.67

T. aestivum ‘Chinese

Spring’ (PhI)

21.86a ± 3.25 14.88a ± 1.18 23.45a ± 0.50 16.91ab ± 1.53

T. aestivum UP2382 23.08ab ± 2.65 16.75b ± 0.96 22.76a ± 1.25 15.19a ± 1.46

T. aestivum UP 2565 21.26a ± 2.90 16.02ab ± 1.22 25.89b ± 1.72 16.94ab ± 1.49

T. durum PDW274 21.94a ± 2.93 13.68a ± 0.90 23.62a ± 2.72 16.31a ± 1.31

T. durum PDW233 21.91a ± 1.27 19.60c ± 0.62 25.60b ± 0.76 15.85a ± 1.88

T. boeoticum 4873 37.61ef ± 2.12 27.97ef ± 3.18 40.5g ± 2.87 29.27ef ± 2.68

T. boeoticum 4874 23.88ab ± 2.59 22.12cd ± 1.58 26.43b ± 2.20 24.27de ± 2.27

T. dicoccoides 4630 38.03ef ± 2.51 35.74hi ± 2.46 42.67h ± 3.07 32.88fg ± 2.98

T. dicoccoides 4640 34.37de ± 2.02 52.12op ± 2.20 39.50gh ± 3.69 52.05m ± 0.81

T. dicoccoides 4641 37.87ef ± 2.18 65.62st ± 2.98 40.09g ± 5.06 66.51qr ± 2.60

T. dicoccoides 4772 27.67bc ± 0.67 43.93lm ± 2.23 33.25ef ± 3.11 48.95k ± 2.06

T. araraticum 4770 58.59jk ± 2.09 27.32ef ± 1.62 59.06mn ± 5.60 30.54f ± 4.04

Ae. longissima 3507 75.00p ± 2.39 50.52no ± 2.16 81.59u ± 2.61 49.95kl ± 4.06

Ae. longissima 3506 59.12jk ± 1.94 35.95hi ± 2.31 69.66q ± 3.25 35.15gh ± 3.19

Ae. longissima 28 65.06ml ± 3.12 43.08l ± 1.62 69.96q ± 5.28 38.48hi ± 2.49

Ae. longissima 3819 57.66jk ± 3.05 24.99de ± 2.76 67.21pq ± 4.01 27.69e ± 2.51

Ae. longissima 3770 78.60q ± 5.08 43.14l ± 1.83 76.52st ± 2.48 39.99i ± 3.86

Ae. kotschyi 3774 82.42r ± 3.83 36.61hi ± 2.12 82.92uv ± 6.25 45.82k ± 2.60

Ae. kotschyi 3790 73.01no ± 2.67 36.96i ± 2.93 76.08st ± 1.79 46.37kl ± 3.91

Ae. kotschyi 3573 82.28r ± 3.27 50.43mn ± 3.01 90.96wy ± 4.75 52.77m ± 2.43

Ae. kotschyi 387 69.97mn ± 6.57 45.17kl ± 2.01 73.46sr ± 4.85 49.93kl ± 1.48

Ae. kotschyi 388 67.53mn ± 3.90 54.57p ± 2.79 68.18q ± 5.48 50.43lm ± 3.21

Ae. kotschyi 389 22.89ab ± 1.27 48.08l ± 2.18 30.45cd ± 2.19 51.70m ± 2.18

Ae. kotschyi 390 70.48mn ± 3.74 22.29cd ± 2.38 69.06q ± 3.39 24.44d ± 3.05

Ae. kotschyi 393 66.47lm ± 3.95 23.11d ± 2.70 66.43p ± 6.15 26.91de ± 2.09

Ae. kotschyi 394 61.16k ± 5.55 25.75e ± 3.02 74.45s ± 2.97 51.45lm ± 3.37

Ae. kotschyi 396 78.49p ± 3.23 48.76m ± 2.08 75.49s ± 2.60 53.38m ± 3.43

Ae. kotschyi 400804 78.42p ± 2.23 44.23kl ± 1.50 82.51v ± 2.92 40.62j ± 3.34

Ae. kotschyi 401021 37.43e ± 4.59 52.41n ± 1.64 40.22g ± 4.90 58.61o ± 3.10

Ae. peregrina 13772 61.28l ± 1.43 41.71jk ± 1.26 68.76r ± 1.39 40.19i ± 2.47

Ae. peregrina 3477 56.21j ± 1.83 38.92ij ± 2.61 62.19o ± 2.45 40.75i ± 2.34

Ae. peregrina 3519 49.43ih ± 1.79 38.00ij ± 1.59 54.24l ± 2.45 38.47ij ± 0.98

Ae. peregrina 3791 78.14q ± 1.37 33.13h ± 1.47 82.32uv ± 4.60 35.75gh ± 1.99

Ae. peregrina 1155-1-1 46.35gh ± 4.19 42.57jk ± 1.55 51.77l ± 3.64 45.23k ± 0.91

Ae. peregrina 1155-2-2 34.37e ± 1.84 35.04hi ± 2.46 41.72gh ± 3.18 37.66h ± 1.11

Ae. peregrina 1155-2-4 37.74e ± 0.89 33.80gh ± 3.67 42.69gh ± 3.55 35.65gh ± 2.76

Ae. peregrina 1155-4-1 40.66f ± 2.10 38.35ij ± 1.68 47.14ij ± 2.67 38.54hi ± 2.35

Ae. peregrina 1155-2-8 35.21ed ± 1.60 35.54hi ± 1.71 41.93gh ± 3.34 38.57hi ± 2.30

Genet Resour Crop Evol (2009) 56:53–64 57

123

smaller seeds is probably due to their superior genetic

system(s).

In the analysis of variance for iron and zinc

content over replicated chemical analysis, highly

significant differences were found among wild

accessions and cultivars (Table 3). Based on the

highly reproducible results, potential donors of useful

variability for high iron and zinc content among

Aegilops species were selected for crosses with bread

and durum wheat cultivars for their biofortification.

Genetic variability of grain iron and zinc content

for different genetic parameters studied is given in

Table 4. High genotypic coefficient of variation was

observed for both iron and zinc content. The geno-

typic coefficient of variation was found to be only

slightly lower than the phenotypic coefficient of

variation suggesting very little genotype 9 environ-

ment interaction component in the expression of

micronutrient content. This observation is also sup-

ported by the high heritability values.

To confirm whether the accessions with high iron

and zinc content in the grains also had higher content

of these micronutrients in leaves, iron and zinc

content in the flag leaves of some of the selected

Aegilops donors were analyzed during flowering but

before anther dehiscence. Significant positive corre-

lations were found between leaf and grain iron

(r = +0.82) and zinc content (r = +0.92) for 17

accessions of these species analyzed (Fig. 1). This

indicates that all the selected accessions with high

iron and zinc content in grains also had higher iron

and zinc content in their flag leaves.

Mean and range of induced chromosome pairing in

F1 hybrids of Ae. kotschyi with ‘Chinese Spring’

(PhI) is given in Table 5. Due to high univalent

frequency (13–35) all the F1 hybrids were completely

Table 3 Analysis of variance for grain iron and zinc content

Source of variation Degree of freedom Mean sum of square

Grain iron Grain zinc

Year 1 Year 2 Year 1 Year 2

Replication 2 25.33 6.48 2.63 16.31

Accession 50 1382.10** 1413.13** 373.16** 381.57**

Error 100 8.13 11.29 4.70 5.28

**Significant at 1% level of probability

Table 2 continued

Species Variety/

accession

Year 1 Year 2

Iron Zinc Iron Zinc

Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Ae. peregrina 1155-5-3 55.97j ± 2.51 49.49m ± 2.36 56.57lm ± 3.98 47.94kl ± 2.45

Ae. cylindrica 3472 84.85rq ± 2.34 32.38gh ± 2.61 93.27yz ± 2.67 35.74gh ± 2.95

Ae. cylindrica 3511 78.8p ± 1.92 52.18n ± 1.36 82.85uv ± 3.12 51.78lm ± 2.22

Ae. cylindrica 3705 52.21I ± 2.60 46.77l ± 1.75 53.61kl ± 2.74 47.83kl ± 1.83

Ae. ventricosa 401027 92.23st ± 4.32 37.97i ± 1.91 93.52yz ± 3.18 39.08hi ± 1.66

Ae. ventricosa 401447 60.89k ± 1.63 25.06de ± 2.22 67.97pq ± 1.70 24.01cd ± 1.02

Ae. ventricosa 3520 55.41j ± 2.75 37.10i ± 1.90 55.88l ± 2.24 38.70hi ± 2.71

Ae. geniculata 3800 79.29p ± 3.93 31.93g ± 2.23 81.97u ± 4.72 34.93gh ± 1.36

Ae. geniculata 3548 52.64i ± 4.35 40.17j ± 2.18 52.25k ± 3.78 40.43i ± 1.07

Ae. geniculata 3565 76.10op ± 2.30 40.81j ± 1.48 72.58r ± 4.86 37.58h ± 1.39

Note: Similar lower case alphabet letters as superscripts within each column indicate non-significant differences in the means of

different accessions for micronutrient contents

58 Genet Resour Crop Evol (2009) 56:53–64

123

male sterile and no selfed seed set was obtained. The

extent of induced homoeologous paring varied among

the hybrids (Fig. 2) indicating that various accessions

of Ae. kotschyi in crosses with ‘Chinese Spring’ (PhI)

stock had different genetic interactions for control-

ling intergenomic chromosome paring. The hybrids

of ‘Chinese Spring’ (PhI) with Ae. kotschyi acc. 396

showed up to eleven bivalents, three trivalents and

least number of univalent frequency.

The iron and zinc content of flag leaves of sterile

F1 hybrids was also analyzed and compared with their

wheat and Aegilops parents for each of the hybrids

(Fig. 3). The flag leaves of all the F1 hybrids had

higher iron content than their wheat and some of the

Aegilops parents, whereas only three hybrids had

lower zinc content than either of the parents. This

suggests that almost all the selected wild donors with

high grain iron and zinc content possess useful

genetic systems for efficient uptake and better

transport of high iron and zinc content to leaves

which could ultimately be mobilized to grains. Iron

content in flag leaves of about half of the hybrids

exceeded the level of flag leaves in the Aegilops

parents suggesting a synergistic interaction between

wheat and Aegilops. In all the hybrids, bread wheat

lines ‘Chinese Spring’ (PhI) or WL711 were used as

the female parents. Higher level of iron and zinc in

majority of the F1 hybrids suggests that the superior

genetic system of Aegilops is partially due to

dominant gene(s) and is capable of expression in

Table 4 Genetic parameters for iron and zinc among bread and durum wheat cultivars and related wild species over 2 years

Micronutrient (mg/kg) Genotypic

variance (r2g)

Phenotypic

variance (r2p)

Genotypic coefficient

of variation (GCV)

Phenotypic coefficient

of variation (PCV)

Heritability

(H2)

Year 1 Year 2 Year 1 Year 2 Year 1 Year 2 Year 1 Year 2 Year 1 Year 2

Iron 458.14 449.75 466.27 461.05 40.18 37.89 40.55 38.39 98.25 97.54

Zinc 122.81 125.43 127.52 130.71 30.62 29.07 31.20 29.67 96.31 95.96

10

30

50

70

30 40 50 60 70

Leaf zinc (mg/kg)

Gra

in z

inc

(mg

/kg

)

WL711(NN)Ae. kotschyiAe. peregrinaAe. longissima

r = 0.92

20

40

60

80

100

40 60 80 100 120 140 160

Leaf iron (mg/kg)

Gra

in ir

on

(m

g/k

g)

WL711(NN)Ae. kotschyiAe. peregrinaAe. longissima

r = 0.82

Fig. 1 Correlation between

leaf and grain

micronutrients

Table 5 Mean and range (within parenthesis) of induced homoeologous pairing configuration of F1 hybrids between ‘Chinese

Spring’ CS (PhI) and different accessions of Ae. kotschyi

Crosses 2n PMCs Mean ± SE (range) Mean ± SE (range) Mean ± SE (range)

Univalent (I) Bivalent (II) Trivalent (III)

CS (PhI) 9 Ae. kotschyi 3790 35 100 29.40 ± 0.42 (23–35) 2.86 ± 0.28 (0–6) 0.16 ± 0.02 (0–1)

CS (PhI) 9 Ae. kotschyi 3573 35 100 29.38 ± 0.29 (21–35) 2.52 ± 0.14 (0–7) 0.20 ± 0.04 (0–1)

CS (PhI) 9 Ae. kotschyi 396 35 100 25.69 ± 0.61 (13–35) 4.17 ± 0.24 (0–11) 0.32 ± 0.06 (0–3)

CS (PhI) 9 Ae. kotschyi 390 35 100 31.88 ± 1.68 (24–35) 2.19 ± 0.23 (0–4) 0.15 ± 0.07 (0–1)

CS (PhI) 9 Ae. kotschyi 393 35 100 31.63 ± 0.56 (26–35) 1.5 ± 0.23 (0–3) 0.13 ± 0.07 (0–1)

CS (PhI) 9 Ae. kotschyi 395 35 100 32.74 ± 0.39 (26–35) 1.0 ± 0.16 (0–3) 0.09 ± 0.05 (0–1)

Genet Resour Crop Evol (2009) 56:53–64 59

123

association or in the background of cultivated wheat,

and hence could be transferred and exploited.

The sterile F1 hybrids between wheat and Aegilops

were extensively backcrossed with the recurrent or

other wheat parent to get BC1 seeds. The male sterile

F1 hybrids on backcrossing as female parent with

wheat cultivars gave a very low backcross seed set

(0.53%) suggesting that the hybrids had only partial

female fertility. As positive and significant correla-

tion between leaf and grain iron and zinc content is

observed and also due to less number of seeds of BC1

plants, digestion of their leaves was done. Chromo-

some number of some of the BC1 plants and iron and

zinc content in their flag leaves is given in Table 6.

Again most of the BC1 plants with variable number of

chromosomes from Aegilops parents had higher iron

content. Some of the BC1 plants in certain crosses

had even higher iron content in their leaves than those

of the wild donors, indicating the synergistic inter-

action between parents and the possibility of

obtaining transgressive segregants for high iron.

Comparatively lower level of zinc in the flag leaves

among the BC1 progenies may be due to epistatic

effect of increasing wheat background on the uptake

and translocation of zinc to the leaves or failure of

transmission of the critical Aegilops chromosome

carrying the gene(s) for zinc content.

Some of BC1 plants were self fertile giving

sufficient BC1F2 seeds while others with partial

fertility were further backcrossed with recurrent

wheat cultivars to get BC2 seeds. Most of the

BC1F2 and BC2 plants had high tillering, comparable

biomass and seed set as that of the wheat cultivars.

Seeds of these plants were as bold as or even bolder

than their wheat parents and had iron and zinc

concentrations in the range of 26.8–79.8 and 22.1–

50.2 mg/kg, respectively (Table 7).

The ash content of Ae. kotschyi accessions was

higher than that of the two wheat cultivars used as the

recurrent parent whereas it was intermediate between

the wheat cultivars and the wild donors for the two

BC1F3 and one BC2F2 derivatives analyzed (Table 8)

suggesting that the Aegilops and their introgressive

wheat derivatives had higher inorganic matter per

unit grain weight. The micronutrient content analyzed

and expressed on ash weight basis for the Ae. kot-

schyi parents was 75.96–90.27% higher for Fe and

65.11–63.75% higher for Zn than the wheat cultivar

WL711. The BC1 and BC2 derivatives had nearly

60% higher grain ash iron and zinc content than the

recurrent wheat parent suggesting that the useful

variability of Ae. kotschyi for higher ash Fe and Zn

content has been transferred to wheat.

Discussion

The low mean and range of grain iron and zinc

content in elite bread and durum wheat cultivars in

this study also strongly emphasize the need of

screening and identification of useful variability

among related wild species of wheat for an effective

biofortification program (Monasterio and Graham

2000). Cakmak et al. (2000) also reported lower iron

and zinc content among T. turgidum ssp. durum and

T. aestivum cultivars as compared to wild and

primitive Triticum species. Scientists with CIMMYT

Fig. 2 Meiotic chromosome pairing of interspecific hybrids of

‘Chinese Spring’ (PhI) 9 Ae. kotschyi 3573 (a) (5II), (b) (4II);

‘Chinese Spring’ (PhI) 9 Ae. kotschyi 396, (c) (4II), (d) (6II);

‘Chinese Spring’ (PhI) 9 Ae. kotschyi 3790, (e) (3II), and (f)(1III + 2II)

60 Genet Resour Crop Evol (2009) 56:53–64

123

0

20

40

60

80

100

120

140

160

180

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Wheat cultivars, F1 Hybrids, Donor parents

Iro

n c

on

ten

t in

fla

g l

eave

s(m

g/k

g)

Wheat cultivar F1 Hybrid Donor parent

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Wheat cultivars, F1 Hybrids, Donor parents

Zinc

con

tent

in fl

ag le

aves

(mg/

kg)

Wheat cultivar F1 Hybrid Donor parent

Fig. 3 Iron (upper)and zinc

(lower) content in flag

leaves of wheat cultivars,

their F1 hybrids and

Aegilops parents; crosses

1–3 have WL711as

recipient parent with being

(1) Ae. kotschyi 3573, (2)

Ae. kotschyi 3774, (3)

Ae. kotschyi 400804;

crosses 4–15 have ‘Chinese

Spring’ (PhI) as recipient

parent, donors being

(4) Ae. kotschyi 3573, (5)

Ae. kotschyi 3774,

(6) Ae. kotschyi 396, (7)

Ae. kotschyi 394,

(8) Ae. kotschyi 3790, (9)

Ae. kotschyi 395, (10)

Ae. kotschyi 393,

(11) Ae. kotschyi 388, (12)

Ae. kotschyi 390,

(13) Ae. peregrina 3791,

(14) Ae. peregrina 3519

and (15) Ae. peregrina13772

Table 6 Iron and zinc content in flag leaves of partially fertile and sterile BC1 plants with chromosome numbers

S.no. Crosses and pedigree Number of

chromosomes

Iron

(mg/kg)

Zinc

(mg/kg)

1 CS (PhI)/Ae. kotschyi 396//PDW 274-1 31 110.6 30.3

2 CS (PhI)/Ae. kotschyi 396//PDW 274-2 32 95.3 31.0

3 CS (PhI) Ae. kotschyi 396//UP 2425-1 44 131.2 32.8

4 CS (PhI) Ae. kotschyi 396//UP 2425-2 44 132.0 32.5

5 CS (PhI)/Ae. kotschyi 396//PBW 343-1 31 183.6 32.6

6 CS (PhI)/Ae. kotschyi 396//PBW 343-2 33 141.7 28.3

7 CS (PhI)/Ae. kotschyi 396//PBW 343-3 43 129.8 26.5

8 CS (PhI)/Ae. kotschyi 396//UP 2382-1 54 79.0 36.7

9 CS (PhI)/Ae. kotschyi 396//WL 711-1 46 108.8 36.5

10 CS (PhI)/Ae. kotschyi 396//WL 711-3 41 107.9 30.2

11 CS (PhI)/Ae. kotschyi 396//WL 711-4 56 137.6 27.1

12 CS (PhI)/Ae. kotschyi 396//WL 711-5 39 101.3 28.1

13 CS (PhI)/Ae. kotschyi 3790//UP 2338-2 39 127.9 29.4

14 CS (PhI)/Ae. kotschyi 3790//UP 2338-3 40 156.9 35.1

15 CS (PhI)/Ae. kotschyi 3790//HD 2687-1 39 172.4 38.2

16 CS (PhI)/Ae. kotschyi 3790//HD 2687-2 40 162.2 24.3

17 CS (PhI)/Ae. kotschyi 3790//PDW 274-1 39 191.0 24.9

18 T. aestivum lr. ‘Chinese Spring’ (PhI) 42 66.7 35.0

19 T. aestivum cv. WL 711 42 56.5 29.3

20 Ae. kotschyi acc. 3790 28 135.6 55.8

21 Ae. kotschyi acc. 396 28 158.6 63.9

Genet Resour Crop Evol (2009) 56:53–64 61

123

and HarvestPlus have already used Ae. tauschii and

T. dicoccoides synthetics in wheat breeding program

for biofortification of wheat for iron and zinc content

(Calderini and Monasterio 2003).

Ae. longissima (S), Ae. kotschyi (US), and Ae.

peregrina (US) all having S genome gave consistently

higher levels and range of both iron and zinc content.

Exhaustive screening of a number of accessions of

diploid and tetraploid wild Triticum and Aegilops

species has shown that the non-progenitor S and U

genomes may be the useful sources for transferring

genes controlling high iron and zinc content to wheat.

The S genome being closely related to the B genome of

polyploid wheat (Faris et al. 2002; Dvorak and Zhang

Table 7 Iron and zinc content of seeds of some fertile BC2 and BC1F2 plants from interspecific crosses between wheat and

Ae. kotschy i accessions

S.no. Crosses and pedigree Iron (mg/kg) Zinc (mg/kg)

1 BC2F2 CS (PhI)/Ae. kotschyi 396//PBW 343-3///WL711-8 60.3 41.3

2 BC2F2 CS (PhI)/Ae. kotschyi 396//PBW 343-3///WL711-25 30.1 27.5

3 BC2F2 CS (PhI)/Ae. kotschyi 396//PBW 343-3///WL711-42 70.3 49.5

4 BC2F2 CS (PhI)/Ae. kotschyi 396//PBW 343-3///WL711-44 35.3 36.3

5 BC2F2 CS (PhI)/Ae. kotschyi 396//PBW 343-3///WL711-65 67.7 48.3

6 BC2F2 CS (PhI)/Ae. kotschyi 3790//PDW 274-1///PBW 373-5 74.4 37.1

7 BC2F2 CS (PhI)/Ae. kotschyi 3790//PDW 274-1///PBW 373-14 79.8 38.4

8 BC2F2 CS (PhI)/Ae. kotschyi 3790//PDW 274-1///PBW 373-17 26.8 23.3

9 BC2F2 CS (PhI)/Ae. kotschyi 3790//PDW 274-1///PBW 373-18 68.8 50.2

10 BC1F3 CS (PhI)/Ae. kotschyi 396//UP 2382-1-1 61.0 41.5

11 BC1F3 CS (PhI)/Ae. kotschyi 396//PBW 343-3-1 65.9 49.8

12 BC1F3 CS (PhI)/Ae. kotschyi 3790//UP 2338-2-2 69.3 42.3

13 BC1F3 CS (PhI)/Ae. kotschyi 3790//UP 2338-2-5 36.3 22.1

14 BC1F3 CS (PhI)/Ae. kotschyi 3790//UP 2338-2-16 36.5 26.1

15 BC1F3 CS (PhI)/Ae. kotschyi 3790//UP 2338-2-23 69.3 37.9

16 BC1F3 CS (PhI)/Ae. kotschyi 3790//UP 2338-2-38 76.8 39.7

17 Ae. kotschyi 396 79.6 45.1

18 Ae. kotschyi 3790 76.5 40.7

19 T. aestivum cv. PBW 343 27.9 20.4

20 T. aestivum cv. WL711 25.3 19.5

Table 8 Ash content in wheat cultivars, Ae. kotschyi accessions and wheat, Ae. kotschyi BC1F2 and BC2 derivatives and their grain

ash iron and zinc content

S.no. Grain material Ash (%) Fe (lg/g)

of ash

% Change in ash Fe

content over WL711

Zn (lg/g)

of ash

% Change in ash Zn

content over WL711

1 WL711 1.58 1,607 – 1,342 –

2 CS (PhI) 1.65 1,702 5.94 1,181 –11.43

3 Ae. kotschyi 3790 2.21 2,828 75.96 2,215 65.11

4 Ae. kotschyi 396 2.07 3,058 90.27 2,197 63.75

5 BC1F3 CS (PhI)/Ae. kotschyi3790//UP2338-2-1-8

1.81 2,525 57.15 1,932 43.95

6 BC1F3 CS (PhI)/Ae. kotschyi3790//UP2338-2-1-19

1.77 2,541 58.13 2,224 65.66

7 BC2F2 CS (PhI)/Ae. kotschyi396//PBW343-3///WL711-1-45

2.02 2,755 71.42 2,248 67.55

62 Genet Resour Crop Evol (2009) 56:53–64

123

1990; Daud and Gustafson 1996) can be effectively

used for transferring useful variability for high iron and

zinc content into wheat. T. boeoticum and T.

monococcum with Am genome most closely related

to that of A genome of polyploid wheat, Ae. tauschii

with D genome and T. dicoccoides having AB

genomes have already been reported to have high

grain iron and zinc content (Monasterio and Graham

2000; Cakmak et al. 2000). The useful variability from

S genome can be transferred to wheat through induced

homoeologous chromosome pairing using PhI (Chen

et al. 1994; Aghaee-Sarbarzeh et al. 2002). In addition

to the S genome, some other non-progenitor genomes

(U, M) also controlling high iron and zinc can be

exploited for biofortification using PhI mediated

induced homeologous chromosome pairing.

Among the screened wild relatives in general and

selected donors in particular, accessions with high

grain iron content were also found to have high grain

zinc content, which strongly suggests similar mech-

anism of uptake, translocation and deposition of the

two micronutrients. Welch and Graham (2004) also

reported high correlation between grain iron and

grain zinc concentrations in wheat cultivars and

related species. Phytosiderophores like mugineic

acids are known to facilitate uptake of iron, zinc

and other micronutrients (Takagi et al. 1998; Marsch-

ner and Romheld 1994). Some of the accessions of

Aegilops species with high grain and leaf iron and zinc

also possess high level of phytosiderophores (our

unpublished results).

Analysis of variance for iron and zinc content in

wild accessions and wheat cultivars revealed highly

significant variation among them and high heritability

indicating suitability of the set of donor parents for

biofortification of wheat cultivars.

Leaves of all the Aegilops species had higher

micronutrient content than T. aestivum cv. WL711

with 52.16 mg/kg iron and 31.72 mg/kg zinc (Fig. 1).

Significantly high positive correlation between grain

and flag leaf content of both iron and zinc in the

selected donors strongly suggests the presence of a

genetic system(s) in the donors for more efficient

uptake/transport of the micronutrients as compared to

that of the bread and durum wheat cultivars. This

high positive correlation between flag leaf and grain

iron and zinc suggests the possibility of using flag

leaves for an early selection of plants with potentially

high iron and zinc in the segregating generations of

interspecific crosses rather than waiting till harvest

for grain analysis. This could facilitate selection of

high yielding and disease resistant plants among

those plants with high iron and zinc in leaves similar

to that of marker assisted selection. Garnett and

Graham (2005) reported nearly 77%, 62% and 42%

remobilization of wheat shoot iron, copper and zinc,

respectively in wheat into grains during anthesis to

maturity under controlled experimental conditions.

This supports our observations of higher content of

leaf and grain iron than their zinc content. It appears

that remobilization of zinc follows the same pattern

as that of iron but in lower proportion (Garnett and

Graham 2005). The ancestral wild allele of transcrip-

tion factor NAM-B1 responsible for accelerated

senescence during grain filling period of wheat (Uauy

et al. 2006) can be fine tuned and used for high level

of translocation of iron and zinc from the biofortified

leaves to grains without any appreciable loss of grain

yield.

Higher iron and zinc content in flag leaves of most

of the interspecific hybrids than their parents provides

an excellent ‘proof of the concept’ that the screened

and selected Aegilops accessions have the requisite

superior genetic system(s) for higher uptake/transport

of micronutrients which expresses in the wheat

background. Higher leaf iron content in most of the

BC1 plants with approximately the expected 75% of

the wheat complement further confirms the effec-

tiveness of their superior genetic system.

Chemical analysis of the selfed seeds of fertile

derivatives in advanced backcross generations was

done for further confirmation of the ‘proof of

concept’ (Table 7). The iron and zinc content of

grains of fertile BC2 and BC1F2 plants showed

variation ranging from that of the wheat parent to

their wild donors. The variation could be attributed to

the presence/transfer of one or more chromosomes/

chromosome segments of the wild donors controlling

the efficient uptake and translocation of the micro-

nutrients. As the grain size of the fertile derivatives

was almost similar or even greater than that of the

wheat parent, the higher iron and zinc found in their

seeds is not due to reduced harvest index unlike the

synthetic hexaploids where Calderini and Monasterio

(2003) found lower grain yield per plant to be a major

contributing factor to their higher micronutrient

contents. The recovery of fertile derivatives with

seeds as bold as that of the wheat cultivars and

Genet Resour Crop Evol (2009) 56:53–64 63

123

micronutrient content nearly as high as that of the

wild donors not only on the grain weight basis but

also on the grain ash basis unequivocally confirms

that Ae. kotschyi possesses efficient genetic system

for uptake/translocation of the micronutrients which

could be effectively used for biofortification of wheat

cultivars.

Acknowledgements The help of Department of

Biotechnology, Govt. of India for supporting the work

through a project, ‘Biofortification of wheat for enhanced

iron and zinc content by conventional and molecular breeding’

is gratefully acknowledged. The authors are highly thankful to

the Head, Institute Instrumentation Centre, I.I.T. Roorkee and

Mr. R. Juyal for their help in chemical analyses.

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