Field Crops Research 116 (2010) 268277

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Field Crops Research

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Genotype environment interaction for zinc and iron concentration of wheat grain in eastern Gangetic plains of India
A.K. Joshi a,c,*, J. Crossa b, B. Arun c, R. Chand c, R. Trethowan d, M. Vargas e,f, I. Ortiz-Monasterio b
a

International Maize and Wheat Improvement Center (CIMMYT), South Asia Ofce, Kathmandu, Nepal, India International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, C.P. 06600 Mexico, D.F., Mexico c Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India d Plant Breeding Institute, The University of Sydney, PMB11, Camden, NSW 2570, Australia e noma de Chapingo, Km 38.5 Carretera Me xico-Texcoco, Chapingo, Mexico Universidad Auto f Biometrics and Statistics Unit, CIMMYT, Apdo. Postal 6-641, 06600 Mexico, D.F., Mexico
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 September 2009 Received in revised form 17 January 2010 Accepted 17 January 2010 Keywords: Micronutrient Triticum aestivum Genotype environment interaction Site regression model Factorial regression Partial least square

Zinc and iron are important micronutrients for human health for which widespread deciency occurs in many regions of the world including South Asia. Breeding efforts for enriching wheat grains with more zinc and iron are in progress in India, Pakistan and CIMMYT (International Maize and Wheat Improvement Centre). Further knowledge on genotype environment interaction of these nutrients in the grain is expected to contribute to better understand the magnitude of this interaction and the potential identication of more stable genotypes for this trait. Elite lines from CIMMYT were evaluated in a multilocation trial in the eastern Gangetic plains (EGP) of India to determine genotype environment (GE) interactions for agronomic and nutrient traits. Agronomic (yield and days to heading) data were available for 14 environments, while zinc and iron concentration of grains for 10 environments. Soil and meteorological data of each of the locations were also used. GE was signicant for all the four traits. Locations showed contrasting response to grain iron and zinc. Compared to iron, zinc showed greater variation across locations. Maximum temperature was the major determinant for the four traits. Zinc content in 3060 cm soil depth was also a signicant determinant for grain zinc as well as iron concentration. The results suggest that the GE was substantial for grain iron and zinc and established varieties of eastern Gangetic plains India are not inferior to the CIMMYT germplasm tested. Hence, greater efforts taking care of GE interactions are needed to breed iron and zinc rich wheat lines. 2010 Elsevier B.V. All rights reserved.

1. Introduction The importance of nutrient rich crops is widely accepted (Welch and Graham, 2000; Ortiz-Monasterio et al., 2007) to the extent that improving livelihoods through increased levels of micronutrients in food is considered second only to controlling HIV/AIDS (World Bank, 2006). Zinc and iron are two well known nutrients having signicant importance to human health (Cakmak et al., 1999). In developing countries, where there is high child and adult mortality, zinc and iron deciencies ranked fth and sixth, respectively, among the top 10 risk factors contributing to the burden of disease (WHO, 2002). It has been proposed that a unique opportunity exists for agriculture to invest in developing more nutrient-dense staple food crops (Underwood, 2000) that could

* Corresponding author at: International Maize and Wheat Improvement Center (CIMMYT), South Asia Ofce, Kathmandu, Nepal, India. Tel.: +91 977 4269564; fax: +91 977 4229804. E-mail addresses: a.k.joshi@cgiar.org, joshi_vns@yahoo.co.in (A.K. Joshi). 0378-4290/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2010.01.004

help reduce malnutrition (Calderini and Ortiz-Monasterio, 2003). Keeping this in mind, a global biofortication initiative (HarvestPlus) within the Consultative Group on International Agricultural Research (CGIAR) was launched to breed and disseminate crops for better nutrition (HarvestPlus Brief, 2006). The goal of HarvestPlus is to reduce micronutrient malnutrition among poor populations in different countries including Asia thereby improving food security and enhancing the quality of life. The two major micronutrients being focused by HarvestPlus in wheat are iron and zinc. Enriching wheat grains by greater micronutrient concentrations carry inherent advantage of reducing malnutrition in regions such as South Asia (Ortiz-Monasterio et al., 2007). Wheat is a major crop in the Gangetic Plains of South Asia where the major cropping sequence is ricewheat and diversication using pulses, fruits and vegetables is generally below the desired level (Chatrath et al., 2007). The ricewheat cropping areas in South Asia is practiced in around 14 million ha (Hobbs, 2001; Joshi et al., 2007a) and covers around one-third of the total rice area and two-fth of the total wheat area in India, Pakistan, Nepal, and Bangladesh, and account for some 30% of those nations rice and wheat outputs (Hobbs and

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Morris, 1996) and over half of the 24 million ha of ricewheat systems in the Asian subtropics (Ladha et al., 2000; Joshi et al., 2007b). The malnutrition can be especially much higher for rural population in the eastern Gangetic plains, who frequently have small holdings, with little land or protability (Ortiz Ferrara et al., 2007). After signicant expansion during the initial years of the Green Revolution, the wheat production area in the region appears to have stabilized in the last decade (Evenson et al., 1999; Joshi et al., 2007b). Wheat ranks rst in dietary shares in northern India represented by Gangetic plains (Joshi et al., 2007b). The importance of wheat in food and nutrition security of this region can be understood from the fact that three of the wheat producing states (Uttar Pradesh, Punjab, and Haryana) falling in this belt account for nearly 80% of the total wheat production (Joshi et al., 2007b). Because wheat is a staple food of South Asia (SA) and its consumption is pretty high (100150 g/person/day), it is an important source of nutrients for around 40% of the population. Adapted and higher-yielding genotypes will play a key role in meeting the regional demand for grain, and in addition to an increased yield potential, such genotypes will require greater nutritive value to meet the widespread problem of malnutrition. Therefore, nutrient rich high yielding wheat cultivars offer the most economical and feasible means for improving micronutrient nutrition in rural areas not only meeting food security in South Asia but also human health to a large part of population. If these cultivars can also accumulate more micronutrient in the straw this would also potentially improve animal health since wheat straw is popular cattle feed. The environments in the vast eastern Gangetic plains (EGP) of South Asia, that include parts of India, Nepal, and Bangladesh, vary greatly in moisture supply, temperature, soil type, and soil nutrient status (Chatrath et al., 2007; Joshi et al., 2007c). Under such conditions, genotype location (G L) interaction for agronomic and grain nutrient is expected to be large and may not permit differentiation of the performance of genotypes across environments. Proper characterization and understanding of locations are very important for screening superior breeding lines capable of displaying greater concentration of zinc and iron in the grain (Ortiz-Monasterio et al., 2007). The importance of using suitable sites to evaluate germplasm for particular traits is well recognized (Crossa et al., 2002). For instance, understanding environment is considered important in breeding for traits that depend on many factors (Campbell and Lafever, 1980; Ghaderi et al., 1980; Fox and Rosielle, 1982; Yau et al., 1991; Joshi et al., 2007c). Likewise, understanding environment could be more important in breeding for traits such as grain micronutrients that are dependent on soil status of nutrients, especially for data from large and heterogeneous areas where wheat is grown and tested. Many tools and techniques have been suggested for characterizing and grouping environments, with biplot analysis considered the most valuable. Interpretation of performance of a number of genotypes in a broad range of environments is generally affected by large G E interactions (Gauch and Zobel, 1996). Analysis of variance describes only the main effects; it tests the signicance of the G E interaction but provides no insight into the particular patterns of genotypes or environments that give rise to the G E interaction. Multiplicative models for multi-environment trials have been used for studying G E interaction, examining genotypic yield stability and adaptation, and developing methods for clustering sites or cultivars into groups with statistically negligible crossover G E interaction (Crossa and Cornelius, 1997; Crossa et al., 2002). Multiplicative models have an additive (linear) component (i.e., intercept, main effects of sites and/or genotypes) and a multiplicative (bilinear) component (G E interaction) and, thus, are also named linearbilinear models. A type of linear

bilinear model suitable for grouping sites and cultivars without cultivar rank change is the sites regression model (SREG). This model is also named GGE (Yan et al., 2001) because it includes the effects of genotype plus G E interaction. Biplots obtained from graphing the rst two components of the multiplicative part of SREG (genotype plus G E interaction) are useful for summarizing and approximating patterns of response that exist in the original data (Gabriel, 1971, 1978). Crossa et al. (2002) showed that, for SREG models, the biplot of the rst two multiplicative components graphs the interaction variation due to non-crossover genotype plus G E interaction versus the interaction variation due to crossover genotype plus G E interaction explainable by a second bilinear term if and only if the primary effects of sites are all of the same sign. They further demonstrated how the SREG biplots could be used for identifying subsets of sites and also for genotypes with non-crossover G E interaction. Statistical models that incorporate large number of environmental and genotypic variables into the analysis of multienvironment trials have also been used for studying and explaining G E interactions (Vargas et al., 1998, 1999). Among these, two models are the factorial regression (FR) (Denis, 1988; van Eeuwijk et al., 1996) and the partial least squares (PLS) regression method (Aastveit and Martens, 1986). It has been suggested (Vargas et al., 2001) that the multiplicative decomposition obtained from PLS can be presented graphically in the form of a biplot with treatments, environments, and covariables represented as vectors in a twodimensional space. The factorial regression models have two main advantages: (i) testing of the hypotheses related to the signicance of the effects for the available external covariables and (ii) use of standard selection procedures for variable subsets, like stepwise regression, for model construction. Therefore, when there are many covariables having unknown inuence on an important trait, partial least squares (PLS) and factorial regression (FR) analysis can be used to nd out what variables are contributing signicantly. In addition, PLS and FR are statistical models that incorporate external environmental and/or cultivar variables for studying and interpreting genotype environment interaction (GEI) (Vargas et al., 1999). Very few attempts have been made to classify and characterize eastern Gangetic plains locations of South Asia for iron and zinc concentration of wheat. In this study, we have examined three years of agronomic data following SREG, FR and PLS to (1) evaluate the magnitude and nature of genotype, location, and G L interaction effects for agronomic traits and grain zinc and iron concentration in the EGP of South Asia; (2) study the contribution of soil and meteorological factors in agronomic and nutrient concentration of genotypes; (3) evaluate the newly developed genotypes at CIMMYT for iron and zinc concentration in the grains along with their agronomic performances compared to the established varieties of South Asia. 2. Materials and methods 2.1. Experimental data A three-year (20052007) study from eastern Gangetic plains of India was done as part of a collaborative effort between the Biofortication Challenge Program (Harvest Plus), CIMMYT (International Maize and Wheat Improvement Centre, Mexico) and BHU (Banaras Hindu University, Varanasi, India) with the objective of identifying biofortied wheat varieties adapted in South Asia. 2.2. The multi-environment trial The trial consisted of the 1st South Asia Micro-Nutrient Yield Trial (SAMNYT) having 20 entries with four checks, viz., HUW 234

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(an improved long term check from EGP of India), HUW 510 and PBW 343 (an improved check from India), and Inqalab 91 (an improved check from Pakistan). The other lines used were developed at CIMMYT using primary hexaploid T. dicoccon-based synthetics, backcrossed twice to the adapted recurrent parent, selected for yield and iron and zinc status in Mexico (OrtizMonasterio et al., 2007; Trethowan, 2007). The trial was grown at 14 locations during Rabi season in three years 20042005, 2005 2006, 20062007 in the eastern Gangetic plains of India. Rabi season is the winter season in northern India where crop is sown in NovemberDecember and harvested in AprilMay of the subsequent year. The locations used in the three years of testing were 20042005: village Bhurkura (Mirzpuar), Ghurahoopur (Mirzapur), Bhadawal (Mirzapur), Bharooiya (Mirzapur), Mauparasi (Azamgarh), and Research Farm Banaras Hindu University; year 20052006: village Bhurkura (Mirzapur), Mauparasi (Azamgarh), Pidkhir (Mirzapur), Research Farm Banaras Hindu University; and 20062007: village Bhurkura (Mirzapur), Ghurahoopur (Mirzapur), Pidkhir (Mirzapur) and Research Farm Banaras Hindu University. All locations have ricewheat as the major cropping system and were characterized by high temperatures and relative humidity (RH). For each line, 75 g of seed was used to plant six rows 5 m long with row spacing of 25 cm. Each line was hand sown with two replications. Identical fertilization practices (120 kg N:60 kg P2O5:40 kg K2O ha1) were followed at all locations in all years. Zinc was not applied in the trials. Full doses of K2O and P2O5 were applied at sowing; nitrogen was supplied in split applications, with 60 kg N ha1 at sowing, 30 kg N ha1 at the rst irrigation (21 days after sowing), and 30 kg N ha1 at the second irrigation (45 days after sowing). Trial design was a 10 2 alpha lattice with two replications. 2.3. Agronomic and nutrient traits Observations were recorded for days to heading, grain yield and grain iron and zinc concentrations. For days to heading, an entry was considered to have achieved heading when 50% of the spikes emerged out of the ag leaf. The grain yield per plots was converted to kilograms per hectare adjusted at 12% moisture content. Zinc and iron analysis was performed at Waite Analytical Services, Adelaide, Australia. All samples were analyzed on an oven dried basis. Samples for Inductively Coupled Plasma analysis were digested using nitric/perchloric acid in open glass tubes. Sample solutions were analyzed using Inductively Coupled Plasma Atomic Emission Spectrometry (ICPAES) following standard procedure and quality management. Grain samples were collected taking full care to avoid soil and equipment contaminations. Around 50 spikes from each of the plot were hand harvested and kept in clean paper envelopes. Care was taken that spikes were taken from plants that were fully standing and were in no way in contact with soil. Threshing was done by hand. After threshing, air cleaner was used to remove all foreign material. Any further visible impurity was manually cleaned. Twenty grams subsample was collected from each sample. Samples were placed in new, clean paper bags and then dried in a clean oven at 75 8C for 48 h to destroy any pathogen spores. Soil available zinc and iron were measured using the methods of DTPA extraction. For each genotype three samples were used. Soil samples from each location were collected. Samples represented top (upper half 030 cm) and deep soil (between 30 and 60 cm). Using a soil auger, soil samples were taken on parallel paths in each of the plot of the trial in a location. Thus, a total of 40 samples cores of around 1 kg were taken from each location for each of the two soil depths. Soil was mixed from all the cores from a single location and 500 g sample was derived following standard

procedure (Pandey et al., 2005). Samples were placed in new, clean paper bags and then dried in a clean oven at 75 8C for 48 h to destroy any pathogen spores. 2.4. Statistical analyses All analyses were performed using SAS software (SAS, 2003). For 20 genotypes, ANOVA was performed using combined data for all environments. For agronomic traits, data of 14 sites was used while for grain zinc and iron concentrations, 10 sites data was used. The combination of sites and years were named by the rst three to four letters of the site. Thus, the names combining the location and year of testing were, for example, Bhur5, site Bhurkura for year 5; Ghur5, Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6; Bhur7, site Bhurkura for year 7; Ghur7, site Ghurahoopur for year 7; Pidk7, site Pidkhir for year 7; BHU7, site Banaras Hindu University for year 7. Heritability (h2) was estimated from the analysis of variance following Nyquist (1991). The formula used was (h2 = s2g/s2p; where, h2 = heritability estimate, s2g is genotypic variance and s2p is phenotypic variance; s2p = s2g + s2ge/y + s2e/ry where s2ge is the genotypeenvironment interaction variance and s2e is the residual error variance; y = number of years and r = number of replications). 2.4.1. Site regression model (SREG) The SREG is given by yi j m j
t X k1

lk aik g jk ei j

where yi j is the mean of the ith cultivar in the jth environment for g cultivars and e sites (i = 1, 2, . . ., g and j = 1, 2. . ., e); m j is the site mean; lk (l1 ! l2 ! . . . ! lt ) are scaling constants (singular values) that allow the imposition of orthonormality constraints on the singular vectors for cultivars, ak a1k ; . . . ; agk 0 and P 2P 2 sites, g k g 1k ; . . . ; g ek 0 , such that and i aik j g jk 1 P P 0 i aik aik0 j g jk g jk0 0 for k 6 k ; aik and g jk , for k = 1, 2, 3, . . ., are called primary, secondary, tertiary, . . ., effects of ith cultivar and the jth site, respectively; ei j is the residual error assumed to be normally and independent distributed with 0 means and variance s2/r (where s2 is the pooled error variance and r is the number of replicates). The number of bilinear terms is t min (g, e). Estimates of the multiplicative parameters in the kth bilinear term are obtained as the kth component of the deviations from the additive part of the model. In the SREG model, only the main effects of cultivars plus the G E interaction are absorbed into the bilinear terms. The biplots of the SREG had the primary and secondary effects of genotypes and locations plotted. Useful conclusions can be drawn from the biplot about relationships among locations, genotypes and G L interaction. For example, locations located in the same direction of the biplot equally discriminate genotypes, whereas locations in the opposite direction ranked the genotypes differently. Crossa et al. (2002) pointed out that if the primary effects of sites were all of the same sign, the rst component in biplots of SREG would be related to non-crossover genotype plus G E interaction variability, whereas the second component accounted for crossover genotype plus G E interaction variability, such that the ideal test location or the genotype should have a large rst primary effect and a near-zero secondary effect (Yan et al., 2001).

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To understand the contribution of different covariables (meteorological and soil nutrient variables), partial least squares analysis and factorial regression was done. The meteorological variables used were temperature maximum before owering (TMXBF); temperature maximum after owering (TMXAF); temperature minimum before owering (TMNBF); temperature minimum after owering (TMNAF); relative humidity before owering (RHBF); relative humidity after owering (RHAF); rainfall before owering (RBF); rainfall after owering (RAF). The soil nutrient variables were zinc concentration in 030 cm soil depth (Zn_30); zinc concentration in 3060 cm soil depth (Zn_60); iron concentration in 030 cm soil depth (Fe_30); iron concentration in 3060 cm soil depth (Fe_60). The proportion of the variation accounted for by each of the signicant covariables for each factor of the PLS was estimated. To understand the contribution of different covariables (meteorological and soil nutrient variables), partial least squares analysis and factorial regression was done. The meteorological variables used were temperature maximum before owering (TMXBF); temperature maximum after owering (TMXAF); temperature minimum before owering (TMNBF); temperature minimum after owering (TMNAF); relative humidity before owering (RHBF); relative humidity after owering (RHAF); rainfall before owering (RBF); rainfall after owering (RAF). The soil nutrient variables were zinc concentration in 030 cm soil depth (Zn_30); zinc concentration in 3060 cm soil depth (Zn_60); iron concentration in 030 cm soil depth (Fe_30); iron concentration in 3060 cm soil depth (Fe_60). The proportion of the variation accounted for by each of the signicant covariables for each factor of the PLS was estimated. 2.4.2. Partial least square analysis The PLS extracts the main variation patterns from one data table X that have relevance also to another data table Y from the same individuals. The PLS can be seen as an extension of principal component analysis because it allows extraction of the main variation patterns within X and Y permitting study of the structure between X and Y. Thus, the latent variables extracted are the essence contained in the variables of X that are also relevant to Y so that both matrices are modeled simultaneously. Thus the bilinear representation of X and Y is as follows: X t1 p01 t2 p0 . . . E T p0 E; Y t1q01 t2q02 . . . F Tq0 F where the matrix T contains the Z scores, matrix P contains the Z loadings, matrix q contains the Y loadings and F and E are the residual matrices. The basic idea is that the relationship between Y and X is transmitted through the latent variables t. The choice of what is X and what is Y does not have to follow the traditional: X = cause and Y = effect like in the classical regression where X are called independent (or regressor) variables and Y are called dependent variables (Martens and Martens, 2001). In the context of multi-location trials, the Y matrix consists of variables (grain yield, days to maturity, etc.) measured on genotypes in different environments and the X matrix comprises covariables that have been measured in either the genotypes (days to anthesis, physiological maturity, etc.) or in the environments (minimum temperatures, maximum temperatures, etc.). In this context, the covariables measured in X can explain some of the variability existing in Y. In other words, genotypic or environmental covariables can help us to explain G E interaction. The results of the PLS can be graphically displayed in the form of biplots (Gabriel, 1971) in which coordinates for environments, genotypes and environmental covariables corresponding to the rst two PLS components are simultaneously depicted by vectors

in a space with starting points at the origin (0, 0) and end points determined by the value of the coordinate. Genotypes and environments having the same directions have positive interactions and those having opposite directions have negative interaction. Details of the univariate and multivariate PLS regression algorithms are given in Vargas et al. (1998, 1999). 2.4.3. Factorial regression model The treatment environment is modeled in relation to environmental covariables (with the regression coefcient depending on the treatment) or in relation to treatment covariables (with the regression coefcient depending on the environment). A FR model for the mean of the ith treatment in the jth environment, for which the interaction includes G (centred) treatment covariables xi1 to xiG, can be written in matrix notation as follows: EY m1I 1J 0 t 1J 0 1I b0 XG 0 where the fourth term on the right side of the equation (T E) consists of the product of the known treatment covariables, xi1 to xiG (G I 1), represented by the I G matrix X = (xig) and multiplied by the unknown environmental effects (or environmental potentialities), gj1gjG, denoted by the J G matrix G = (gjg) (Vargas et al., 2001). Convenient constraints on the parameters are sum to zero over i for the parameters ti and over j for bj and gjg. The treatment covariables are known, but the environmental potentialities should be estimated. A FR model in which the T E term contains H (centred) environmental covariables, zj1 to zjH, can be written as follows: EY m1I 1J 0 t 1J 0 1I b0 zZ0 where the fourth term on the right side of the equation (T E) consists of the product of treatments having differential effects (sensitivity), zi1 to ziH (H J 1), collected in the I H matrix z = (zih) and multiplied by the values of the environmental covariables that are collected in the J H matrix Z = (zjh). The values of the environmental variables are known, but the treatment sensitivities need to be estimated. 3. Results Genotypic effect was signicant for grain yield and days to maturity across locations and years, but not for grain zinc and iron (Table 1). However, GE interactions were signicant for grain yield, days to maturity, grain iron and zinc concentrations when genotypes were used as xed effects and environment as random. The means of grain yield of 20 genotypes varied from 2177 to 3147 kg/ha whereas,

Table 1 Analysis of variance for yield, days to heading, grain iron and zinc concentration of 1st SAMNYT wheat lines in three years (20052007) of testing in 14 locations of the eastern Gangetic plains of India. Source DF Means sum of squares of traits Yield Env Gen Env Gen Error Source 13 19 247 280 7,023,725.3 1,242,646.6** 231,651.2** 108,826.1 DF
**

Days to heading 356.20** 23.40** 28.50** 10.34 Means sum of squares of traits Grain zinc Grain iron 509.66** 21.56 23.52 16.625 0.37

Env Gen Env Gen Error Heritability

**

9 19 171 200

3961.19** 10.70 14.11 17.1625 0.25

Signicant at P = 0.01.

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A.K. Joshi et al. / Field Crops Research 116 (2010) 268277 Table 3 Mean zinc and iron concentration in the soil at two depths (30 cm and 3060 cm) in 6 locations of the eastern Gangetic plains of India. Locations Iron and zinc concentration (mg/kg dry basis) at two soil depths Fe (30 cm) Bhurkura Mauparasi Ghurahoopur BHU Bhadawal Bharooiya LSD 5% 43.89 36.63 31.30 26.51 34.72 43.30 5.21 Fe (3060 cm) 14.15 16.93 15.70 14.99 15.58 20.30 1.56 Zn (30 cm) 0.97 0.92 0.95 1.16 1.14 0.80 0.11 Zn (3060 cm) 0.37 0.26 0.40 0.77 0.50 0.40 0.12

the days to heading ranged from 73 to 84 days (Table 2). Three lines (Line Nos. 10, 11 and 12) showed signicant superiority for grain yield over the best check Inqalab 91. These lines were around 5 days later in days to heading than Inqalab 91 but were 2 days earlier than the long duration check (PBW 343). The range for mean values of iron and zinc concentration in the grain of 20 lines was 4650 and 32 34 mg/kg dry basis, respectively (Table 2). Broad sense heritability for grain zinc and iron concentrations were 0.25 and 0.37, respectively (Table 1). The correlation between iron and zinc concentration of grains was 0.18 where as the correlations of grain yield with iron and zinc concentrations of grain were 0.04 and 0.19, respectively. The soil iron and zinc concentrations varied across locations, however, compared to zinc iron concentration appeared to be better (Table 3). 3.1. Site regression model (SREG) The SREG biplot of the 20 genotype for grain yield (in 14 locations across three years) and for grain zinc and iron concentration (in 10 locations across three years) is depicted in Fig. 1ac, respectively. For grain yield and days to maturity, in almost all the three years all locations are located towards the right side of the biplot. However, for grain zinc and iron concentrations, the locations are spread to all sides. For grain yield, the rst SREG component explained 37% of the genotype plus G E interaction, whereas the second component accounted for 15% of the

variability. Likewise the contribution of rst and second component for days to maturity, grain zinc and grain iron was 84% and 7%; 36% and 26% and, 32% and 15%, respectively. Results indicated that most of the sites were good for days to maturity because they had large values for primary effects and low values for secondary effects. However, for grain yield and nutrient concentration in the grains, the locations were substantially variable. The locations appeared highly unstable for zinc and iron concentrations. A stable location demonstrates a large rst primary effect (non-crossover G E variability) and near-zero secondary effect (crossover G E variability) in the biplot (Crossa et al., 2002).

Table 2 Mean performance and rank of 1st SAMNYT lines for agronomic traits and zinc and iron content (mg/kg dry basis) in the grains in the three years (20052007) of testing in the eastern Gangetic plains of India. 1st SAMNYT No. 401 402 403 404 405 406 407 408 409 410
*

Pedigree

Days to heading 75.29 81.07 74.96 73.64 80.64 79.46 81.89 81.21 80.18 79.68 79.36 79.18 80.11 78.75 79.61 79.46 79.75 80.21 81.29 77.75

Rank

Grain yield (kg/ha) 2635.04 2663.13 2785.82 2732.68 2756.86 2764.94 2451.54 2618.93 2790.34 3147.02 2956.82 3082.23 2793.13 2177.39 2757.13 2810.55 2817.87 2780.44 2538.18 2594.91

Rank

Iron conc. in grains 46.96 46.79 46.95 47.60 48.39 48.56 50.20 47.73 47.58 47.82 48.92 48.04 46.63 47.02 47.48 49.40 48.86 47.53 49.48 49.54

Rank

Zinc conc. in grains 33.82 33.38 34.52 34.47 34.19 32.60 34.79 34.38 33.71 33.95 34.50 33.68 32.69 32.70 33.19 33.15 34.12 32.58 32.96 33.24

Rank

411

412* 413 414 415 416 417 418 419 420

HUW 510 PBW343 INQALAB 91 HUW 234 REBECA F2000 UP 262 T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-26M-010Y-2ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-35M-010Y-5ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-11M-010Y-4ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-11M-010Y-5ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-70M-010Y-2ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-70M-010Y-3ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//TUI/. . .CMSA01M00382T-040Y-42M-010Y-3ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//TUI/. . .CMSA01M00382T-040Y-42M-010Y-5ZLB-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-73M-010Y-5ZLM-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-73M-010Y-4ZLM-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-73M-010Y-3ZLM-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-59M-010Y-2ZLM-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00371T-040Y-59M-010Y-6ZLM-OSY-OB T.DICOCCON PI94625/AE.SQUARROSA (372)//. . .CMSA01M00370T-040Y-15M-010Y-6ZLM-OSY-OB Mean Min Max LSD 5%

3 17 2 1 16 8 20 18 14 11 7 6 13 5 10 8 12 15 19 4

15 14 8 13 12 10 19 16 7 1 3 2 6 20 11 5 4 9 18 17

17 19 18 12 8 7 1 11 13 10 5 9 20 16 15 4 6 14 3 2

9 12 2 4 6 19 1 5 10 8 3 11 18 17 14 15 7 20 16 13

79.17 73.64 81.89 3.59

2732.74 2177.39 3147.02 751.33

48.07 46.63 50.2 2.2

33.63 32.58 34.79 3.13

Resistant to new virulence (78S84) of Yr27 race of yellow rust of wheat.

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Table 4 Proportion of variation accounted from factorial regression analysis for each of the signicant covariables for grain yield, grain iron and zinc concentration. Variable Grain yield RHAF TMXAF TMNBF RHBF Fe_30 Zn_30 % contribution of 6 variables Iron concentration in the grain TMXBF RFAF Zn_60 RHAF % contribution of 4 variables Zinc concentration in the grain TMNAF Zn_60 RFAF TMNBF Zn_30 % contribution of 5 variables SC variable % variation explained 13.01 12.42 11.94 9.13 7.67 6.09 60.26

7,671,294 7,323,023 7,038,254 5,386,681 4,522,150 3,591,001

1045.64 644.96 644.89 463.93

22.21 13.69 13.69 9.85 59.46

1369.06 751.67 653.90 604.58 495.65

29.11 15.98 13.90 12.85 10.54 82.41

TMXBF, temperature maximum before owering; TMXAF, temperature maximum after owering; TMNBF, temperature minimum before owering; TMNAF, temperature minimum after owering; RHBF, relative humidity before owering; RHAF, relative humidity after owering; RBF, rainfall before owering; RAF, rainfall after owering.

3.2. Factorial regression (FR) and partial least squares (PLS) analysis The results of factorial regression and partial least square analysis depicting the contribution of covariables are given in Tables 4 and 5, respectively. For grain yield, six covariables were signicant and contributed around 60% of variation. Among these, RH and temperature after owering contributed the most. However, RH and temperature before owering were also signicant. In addition, iron and zinc in the soil (030 cm depth) also contributed signicantly. The biplot (Fig. 2a) showed that all these covariables were located towards the left side of the biplot. The genotypes 7 and 15 were the most stable. For iron concentration in the grain, four covariables (maximum temperature before owering, rainfall after owering, zinc in 30 60 soil depth and RH after owering) were signicant and accounted for around 59% of variation. The covariables, RH after owering and maximum temperature before owering were located towards the right side of the biplot where as rainfall after owering and zinc in 3060 soil depth towards the left side (Fig. 2b). The genotypes 19 and 6 were the most stable but carried relatively lesser iron concentration than genotypes 3 and 13 that also showed good stability.
Ghur7, site Ghurahoopur for year 7; Pidk7, site Pidkhir for year 7; BHU7, site Banaras Hindu University for year 7. b: SREG biplot of number of locations and years on the performance of iron concentration in the grains of twenty wheat lines in 10 environments in eastern Gangetic plains of South Asia. Bhur5, site Bhurkura for year 5; Ghur 5, Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6. c: SREG biplot of number of locations and years on the performance of zinc concentration of grains of twenty wheat lines in 10 environments in eastern Gangetic plains of South Asia. Bhur5, site Bhurkura for year 5; Ghur 5, site Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6.

Fig. 1. a: SREG biplot of number of locations and years on the grain yield performance of twenty wheat lines in 14 environments in eastern Gangetic plains of South Asia. Bhur5, site Bhurkura for year 5; Ghur 5, site Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6; Bhur7, site Bhurkura for year 7;

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A.K. Joshi et al. / Field Crops Research 116 (2010) 268277 Table 5 Results of partial least square analysis for grain yield, grain iron and zinc concentration for a set of twenty wheat lines tested in eastern Gangetic plains of India. Factor Grain yield Factor 1 Factor 2 Total Grain iron concentration Factor 1 Factor 2 Total Grain zinc concentration Factor 1 Factor 2 Total % explained for X 34.46 23.44 57.90 % explained for Y 12.85 11.27 24.12

27.03 16.28 43.31

16.17 18.52 34.69

29.99 18.16 48.15

16.29 21.53 37.82

For grain zinc concentration, ve covariables explained around 82% of variation. These covariables in descending order of their contribution wereminimum temperature after owering, zinc in 3060 cm soil depth, rainfall after owering, minimum temperature before owering and zinc in 030 soil depth. In the biplot (Fig. 2c), two covariablesthe minimum temperature before and after owering were on the right side of the graph, where as the other three variables (zinc in 030 and 3060 cm soil depth, and rainfall after owering) on the right side.
Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6; Bhur7, site Bhurkura for year 7; Ghur7, site Ghurahoopur for year 7; Pidk7, site Pidkhir for year 7; BHU7, site Banaras Hindu University for year 7. TMXBF, temperature maximum before owering; TMXAF, temperature maximum after owering; TMNBF, temperature minimum before owering; TMNAF, temperature minimum after owering; RHBF, relative humidity before owering; RHAF, relative humidity after owering; RBF, rainfall before owering (RBF); RAF, rainfall after owering (RAF). Zn_30, zinc concentration in 030 cm soil depth; Zn_60, zinc concentration in 3060 cm soil depth; Fe_30, iron concentration in 030 cm soil depth; Fe_60, iron concentration in 3060 cm soil depth. b: PLS biplot of number of locations and years with environmental and soil covariables on the performance of iron concentration in the grains of twenty wheat lines in 10 environments in eastern Gangetic plains of South Asia. Signicant variables are given in bold letters. Bhur5, site Bhurkura for year 5; Ghur 5, site Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6; Bhur7, site Bhurkura for year 7; Ghur7, site Ghurahoopur for year 7; Pidk7, site Pidkhir for year 7; BHU7, site Banaras Hindu University for year 7. TMXBF, temperature maximum before owering; TMXAF, temperature maximum after owering; TMNBF, temperature minimum before owering; TMNAF, temperature minimum after owering; RHBF, relative humidity before owering; RHAF, relative humidity after owering; RBF, rainfall before owering (RBF); RAF, rainfall after owering (RAF). Zn_30, zinc concentration in 030 cm soil depth; Zn_60, zinc concentration in 3060 cm soil depth; Fe_30, iron concentration in 030 cm soil depth; Fe_60, iron concentration in 3060 cm soil depth. c: PLS biplot of number of locations and years with environmental and soil covariables on the performance of zinc concentration in the grains of twenty wheat lines in 10 environments in eastern Gangetic plains of South Asia. Signicant variables are given in bold letters. Bhur5, site Bhurkura for year 5; Ghur 5, site Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site Mauparasi for year 5; BHU5, site Banaras Hindu University for year 5; Bhur6, site Bhurkura for year 6; Mau6, site Mauparasi for year 6; BHU6, site Banaras Hindu University for year 6; Pidk6, site Pidkhir for year 6; Bhur7, site Bhurkura for year 7; Ghur7, site Ghurahoopur for year 7; Pidk7, site Pidkhir for year 7; BHU7, site Banaras Hindu University for year 7. TMXBF, temperature maximum before owering; TMXAF, temperature maximum after owering; TMNBF, temperature minimum before owering; TMNAF, temperature minimum after owering; RHBF, relative humidity before owering; RHAF, relative humidity after owering; RBF, rainfall before owering (RBF); RAF, rainfall after owering (RAF). Zn_30, zinc concentration in 030 cm soil depth; Zn_60, zinc concentration in 3060 cm soil depth; Fe_30, iron concentration in 030 cm soil depth; Fe_60, iron concentration in 3060 cm soil depth.

Fig. 2. a: PLS biplot of number of locations and years with environmental and soil covariables on the performance of grain yield in twenty wheat lines in 14 environments in eastern Gangetic plains of South Asia. Signicant variables are given in bold letters. Bhur5, site Bhurkura for year 5; Ghur 5, site Ghurahoopur for year 5; Bhad5, site Bhadawal for year 5; Bharooiya5, site Bharooiya for year 5; Mau5, site

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PLS analysis of genotypic variable and GE interaction for grain yield performance displayed various sites for year 20042005 in the left quadrangles while for the years 20052006 and 20062007 in the right quadrangles or in the opposite quadrangles indicating that the performance of genotypes was entirely different in these years (Fig. 2a). Minimum temperature after owering and RH after owering were placed opposite to each other. Likewise, minimum temperature before owering and RH before owering were also placed opposite to each other indicating that rainfall before or after owering had increasing effect on the minimum temperature. In the same way the variables, rainfalls before or lower owering were in the opposite quadrangle to maximum temperature before or after owering, respectively (Fig. 2a). This indicated that rainfall before or after owering led to lower maximum temperature. The variables, temperature minimum or maximum before and after owering were present in different quadrangles indicating contrasting expression among them in the three years of testing. For iron and zinc concentrations in the wheat grains, genotypic response widely varied across the environments as indicated by vectors that radiated in all the directions in PLS biplot that depicted genotypic variables, environments and genotypes (Fig. 2b and c). Variations in the pattern of response within the location was highly evident from the fact that most of the locations in the year 2005 were independent of their position in the year 2006 and were often placed opposite to each other. The biplot clearly differentiated the 2005 and 2006 sown environments at all locations (Fig. 2b and c) showing the complexity of evaluation in grain iron and zinc concentrations. However, for both nutrients maximum temperature before owering appeared to play an important role. This could be owing to the reason that temperature before owering also contributes to proper development of the embryo where a large proportion of the micro-elements reside. PLS analysis of genotypic variable and G E for grain iron concentration placed variables such as Fe_30 and Zn_30 in the right upper most quadrangle while genotypic variables Fe_60 and Zn_60 were in the left uppermost (Fig. 2b). In general, different locations in the year 2005 were in different quadrangle than the 2006 indicating contrasting performance in the locations across the two years of testing. PLS analysis of genotypic variable and G E for grain zinc concentration placed variables such as minimum temperatures before and after owering in the right upper most quadrangle (Fig. 2c) indicating their similar role. On the other hand, genotypic variables maximum temperatures before and after owering were in the opposite quadrangles indicating their opposite role in grain zinc concentration. We hypothesize that maximum temperature is often correlated with higher solar radiation which in turn is correlated with more grains per meter square and lower micronutrient concentration. While maximum temperature during the grain lling period tends to be associated with poor grain lling (smaller grains), and this in turn tends to results in a higher micronutrient concentration. The Zn_30 and Zn_60 were also in the opposite quadrangles indicating that their contribution in grain zinc concentration is quite different. The variable Zn_60 appeared to play greater role in the grain zinc concentration. The genotypes 2, 5 and 6 were on the same quadrangle as the Zn_30 indicating their capacity to have greater zinc in the grain when zinc was higher in top soil. On the other hand, genotypes 1, 11, 13, 14, 15, 16 were on the quadrangle where Zn_60 was present indicating their capacity to exploit zinc from the deeper soil. 4. Discussion This study attempted to address three specic questions: rst, whether iron and zinc concentration in the wheat grains display stable performance? Second, what is the contribution of soil and

meteorological factors in agronomic traits and nutrient concentration in the grain of genotypes, and third, whether newly developed genotypes at CIMMYT could be suitable for higher iron and zinc concentrations along with superior agronomic performance compared to the established varieties of South Asia? The iron and zinc concentrations in the wheat grains were highly unstable. Their performance varied across locations and years. This was reected in quite low values for broad sense heritability of grain zinc and iron concentration. The location with the highest value of iron concentration in the grain was 51.8 mg/kg dry basis and the lowest 42.1 showing only a difference of 9.7 mg/ kg dry basis across locations. For zinc concentration in grains, the location with the highest value was 53.2 mg/kg dry basis and the lowest 22.3 showing a wide difference of 31.1 mg/kg dry basis across locations. The critical deciency level of zinc for wheat was reported to be 0.60 mg/kg in the soil (Bansal et al., 1990). Therefore, the soil concentration of zinc appeared to be moderate and relatively more variable than iron which was noted to display good soil concentration. Since micronutrient of grains essentially depends on micronutrient content of the soil, the grain concentration of zinc was expected to be moderate and variable. The iron and zinc values across locations indicated that available iron in the soil is much more stable than zinc across the region (eastern Uttar Pradesh, India). The higher variability of grain zinc values suggest that in some locations genetic biofortication may need to be complemented with agronomic biofortication, meaning the use of zinc fertilizers. Among various meteorological factors, both temperature and relative humidity before and after owering displayed signicant inuence on the grain yield. Likewise, maximum temperature before owering and relative humidity and rainfall after owering inuenced iron concentration in the grain. For zinc concentration in the grain, rainfall after owering and minimum temperature before and after owering appeared to play signicant role. The negative effect of higher temperature on grain yield of wheat is well known for long (Howard, 1924; Fischer, 1985; Reynolds et al., 1994; Joshi et al., 2007b). High temperature is an important problem in South Asia and is believed to increase in future due to global warming (Schneider, 1989; IPCC, 2007; Battisti and Naylor, 2009). In this study, grain iron and zinc concentration was also found to be inuenced by higher temperature. Therefore, future breeding program should not only focus on breeding for heat tolerance but also on breeding early maturing varieties that are able to escape terminal heat stress. Zinc level in the soil (depths 030 and 3060 cm) appeared to play a signicant role in grain nutrients. The iron and zinc concentration in the wheat grain depends largely on environmental conditions, particularly soil availability (Fiel et al., 2005). Therefore, the iron and zinc concentrations in grain show variation in soil or medium availability. For example, the same variety C 306 was found to display a wide variation in grain iron and zinc concentration depending on the soil/medium where it was grown130 mg/g of zinc and 220 mg/g of iron in nutrient solution (Welch et al., 2005) compared to 31 and 33 mg/g of zinc and iron, respectively under eld conditions in high pH soil (Ortiz-Monasterio et al., 2007). Another reason for greater GE interaction for iron and zinc concentration could be their quantitative inheritance as reported in maize and rice (Gregorio, 2002; Long et al., 2004). Some reports in wheat also suggest quantitative control in wheat (Trethowan, 2007; Trethowan et al., 2005). The complexity of the inheritance of grain iron and zinc concentrations in wheat, plus the associated low heritability and the large environment and GE interaction effects, progress in the genetic analysis of these traits is expected to be slower than many traits. However, in spite of these challenges there is evidence that breeding for increased levels of micronutrients is feasible (Ortiz-Monasterio et al., 2007).

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Higher GE interaction and lower stability of genotypes for iron and zinc concentration in the grain suggest that an important step in breeding for enhanced concentrations of iron and zinc should be the testing stability of iron and zinc accumulation across the target environment. The correlation between grain iron and zinc (0.18) was not strong as reported in some of the studies (Morgounov et al., 2007; Ortiz-Monasterio et al., 2007). This is probably due to a very low range of zinc values among genotypes in these studies. Morgounov et al. (2007) also found low correlation when all the spring and winter genotypes were pooled for analysis. Likewise the correlation between grain yield and iron and zinc concentrations in the grain was also low. This weaker correlation between grain yield and nutrient concentrations is probably associated with low variation in both zinc and iron among the genotypes studied. The genotypes tested in the present study showed absence of signicant variation for iron and zinc concentrations in the grains of the advanced CIMMYT wheat lines tested. If the genotypes in this study had shown a relatively wide range of zinc grain concentrations the GE studies would have been more robust and meaningful. Their performance was at part to the three most popular cultivars of South Asia in the last decadePBW 343 and HUW 234 in India and Inqalab 91 in Pakistan. The cultivar HUW 234 is the most popular cultivar of eastern Gangetic plains under late sown conditions and in mid-1990s occupied around 4 5 million ha. It is still grown in about 2 million ha. PBW 343 is grown in more than 7 million ha in India while Inqalab 91 in around 6 million ha in Pakistan (Ortiz Ferrara et al., 2007). However, three lines (Table 2) proved signicantly superior in yield over the best check (Inqalab 91). These lines are also resistant to the new virulence of yellow rust resistance gene Yr27. The lines obtained from CIMMYT were initially tested for their iron and zinc concentrations at their research station at Cd. Obregon, Sonora, Mexico. The soil and grain analysis of systematic checks at the CIMMYT research station at Cd. Obregon showed that soil zinc and to a lesser degree iron concentrations were heterogeneous (Ortiz-Monasterio et al., 2007) and therefore could have confounded or masked genetic differences among lines, therefore preventing the identication of lines with genetically superior concentrations of grain iron and zinc. It has been suggested that given the generally strong GE interaction for iron and zinc concentration, screening for iron and zinc concentration could be highly unreliable in breeding for enhanced micronutrient concentration (Oury et al., 2006). Therefore, special strategies need to be followed to avoid this problem. Ortiz-Monasterio et al. (2007) suggested two strategies to overcome this problem(i) using a systematic check, alpha lattice designs, and spatial analyses of segregating and advanced populations and (ii) use of zinccontaining fertilizer (foliar or soil applied) to try to homogenize soil zinc concentration. There are reports suggesting there is a wide range in the iron and zinc concentrations in the wheat grains (Ortiz-Monasterio and Graham, 2000; Ortiz-Monasterio et al., 2007). Another study evaluating a set of high yielding lines under eld conditions, the zinc values were found between 15 and 35 mg/g but increased to 43 mg/g in some genotypes, while the iron concentrations ranged from 20 to 60 mg/g (Oury et al., 2006). In contrast, the range in mean iron and zinc concentration of grains in the present study was only 4650 and 3234 mg/g. It has been reported that the breeding target for the Gangetic plains of South Asia is to increase iron and zinc levels by 25 and 10 mg/g above a baseline value represented by the mean of the genotypes grown around different locations in the in the region. The present study suggests that the base line for the eastern Gangetic plains (using PBW343 and HUW 234 as checks) should be 47 and 33 mg/g for iron and zinc, respectively. Increased iron and zinc grain concentrations are invisible traits; agronomic advantages are most appealing for the

farmers (Ortiz-Monasterio et al., 2007). This requires using limited back cross approach (Singh and Huerta-Espino, 2004; Singh et al., 2000) crossing agronomically superior lines with iron and zinc rich donors and taking a large population in the early segregating generations. There is good evidence to breed superior yielding varieties for eastern Gangetic plains following this approach (Singh et al., 2007). In addition, availability of a good molecular marker for such genes (Ortiz-Monasterio et al., 2007) or a linkage between a gene regulating senescence (GpcB1 6BS) and high levels of protein, iron, and zinc in the grain as suggested by Uauy et al. (2006) could also be extremely useful. Acknowledgments We are grateful to the farmers who carried out the various trials reported in this study. Financial support for this work was provided by HarvestPlus (www.HarvestPlus.org), a global alliance of agriculture and nutrition research institutions working to increase the micronutrient density of staple food crops through biofortication. References
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