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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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