Concentration of cadmium and other elements in the grain of near-isogenic durum lines

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1 Concentration of cadmium and other elements in the grain of near-isogenic durum lines J. M. Clarke 1, W. A. Norvell 2, F. R. Clarke 1, and W. T. Buckley 3 1 Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, Saskatchewan, Canada S9H 3X2 ( clarkej@em.agr.ca); 2 U.S. Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, NY, USA 14850; 3 Brandon Research Centre, Agriculture and Agri-Food Canada, P.O. Box 1000A, R.R. #3, Brandon, Manitoba, Canada, R7A 5Y3. Received 26 April 2001, accepted 11 September Clarke, J. M., Norvell, W. A., Clarke, F. R. and Buckley, W. T Concentration of cadmium and other elements in the grain of near-isogenic durum lines. Can. J. Anim. Sci. 82: Durum wheat (Triticum turgidum L. var durum) shows genetic variation for concentration of the heavy metal cadmium in the grain. This variation is being exploited to develop cultivars with low cadmium concentration, but there is no information on the effect of incorporation of the trait on uptake of other elements or on economic traits such as yield. Five pairs of near-isogenic high/low cadmium durum wheat lines and their parents were grown in a randomized complete block trial with three replications. Trials were grown at Swift Current, Saskatchewan in 1994, at Swift Current, Stewart Valley and Regina, Saskatchewan in 1995 and 1996, at Langdon and Fargo, North Dakota in 1995, and at Casselton and Langdon, North Dakota in Grain yield, test weight, kernel weight and protein concentration were determined. Grain cadmium, calcium, copper, iron, magnesium, manganese, nickel and zinc were measured by atomic absorption or inductively coupled plasma emission spectroscopy. The low cadmium trait had no significant effect on average yield, grain protein concentration, test weight, or kernel weight as indicated by comparison of the high and low cadmium isolines. Average grain cadmium concentration differed among years within locations, among locations within years, and among genotypes. The average grain cadmium concentration of the high cadmium isolines was approximately double that of the low cadmium isolines. There were significant genotypic differences in grain concentration of the other elements, but the differences were not associated with the high and low cadmium isogenic lines. The low cadmium allele seems to be specific for cadmium, lowering cadmium without altering concentrations of other elements or affecting economic traits. Key words: Triticum turgidum L. var durum; grain cadmium concentration; grain yield Clarke, J. M., Norvell, W. A., Clarke, F. R. et Buckley, W. T Concentration de cadmium et d autres éléments dans le grain des lignées quasi isoéniques de blé dur. Can. J. Plant Sci. 82: La concentration de cadmium, un métal lourd, dans le blé dur (Triticum turgidum L. var. durum) varie avec le génome. On se sert de cette variation pour créer des cultivars à faible teneur en cadmium, mais on ne sait pas grand-chose au sujet des répercussions d une telle modification génétique sur l absorption des autres éléments ni sur les caractères d importance économique comme le rendement. Les auteurs ont cultivé cinq paires de lignées de blé dur quasi isogéniques à haute/faible teneur en cadmium et leurs lignées parentales dans le cadre d un essai en blocs randomisés, répété trois fois. Les essais se sont déroulés à Swift Current (Saskatchewan) en 1994, à Swift Current, Stewart Valley et Regina (Saskatchewan) en 1995 et 1996, à Langdon et Fargo (Dakota Nord) en 1995, et à Casselton et Langdon (Dakota Nord) en On a mesuré le rendement grainier, le poids spécifique, le poids de l amande et la concentration de protéines du blé cultivé. La concentration de cadmium, de calcium, de cuivre, de fer, de magnésium, de manganèse, de nickel et de zinc dans le grain a été déterminée par spectroscopie d absorption atomique ou par spectroscopie d émission avec plasma induit par haute fréquence. Le gène responsable de la réduction de la teneur en cadmium n a pas d incidence sensible sur le rendement moyen, la concentration de protéines dans le grain, le poids spécifique ni le poids de l amande lorsqu on compare les lignes isogéniques à teneur élevée et faible en cadmium. La concentration moyenne de cadmium dans le grain varie d une année à l autre au même endroit, d un endroit à l autre la même année et d un génotype à l autre. La concentration moyenne de cadmium dans le grain des lignées isogéniques à teneur élevée en cadmium est à peu près le double de celle observée chez les lignées à faible teneur. La concentration des autres éléments dans le grain présente de fortes variations génotypiques, mais celles-ci n ont aucun lien avec les lignées isogéniques à forte ou à faible teneur en cadmium. L allèle qui code la faible teneur en calcium semble n agir que sur cet élément et en réduit la concentration sans modifier celle des autres éléments ni d autres caractères économiques. Mots clés: Triticum turgidum L. var. durum, concentration de cadmium dans le grain, rendement grainier Excessive concentrations of essential micronutrients or nonessential trace elements, such as heavy metals, can be toxic to humans and animals (McLaughlin et al. 1999). Among the many elements found in plants, McLaughlin et al. (1999) concluded that cadmium and selenium are of greatest concern in terms of food contamination. High levels of cadmium in food crops are a concern in human diets, particularly 27 in staple foods such as the cereal grains, which represent a large portion of the diet (Wagner 1993). Genetic variation in mineral uptake by plants has been recognized for many years (Saric 1983), and there appears to be potential to manipulate the accumulation of essential or non-essential elements through plant breeding (Graham et al.1992). Genetic studies and manipulation of cadmium

2 28 CANADIAN JOURNAL OF PLANT SCIENCE concentration by breeding have been reported in sunflower (Helianthus annuus L.) (Li et al. 1995a,b) and durum wheat (Triticum turgidum L. var durum) (Clarke et al. 1997b). Cultivars with genetically determined low levels of cadmium can provide an economical and permanent means of reducing cadmium concentration in these crops to ensure continued acceptability in countries which have statutory limits for cadmium in grain and food products. Such cultivars would give farmers an effective alternative to the restriction of production of certain crops to only those soils with low available cadmium (Oliver et al. 1995), or to utilization of uneconomical crop rotations or other management techniques intended to reduce cadmium to acceptable levels (Oliver et al. 1993). Cadmium concentration of plants is influenced by environmental conditions. Factors such as high precipitation (Andersson and Pettersson 1981), low soil ph (Oliver et al. 1998), soil salinity (Norvell et al. 2000; Weggler-Beaton et al. 2000) and low availability in the soil of chemically similar metals such as zinc (Oliver et al. 1994) tend to increase cadmium uptake. Further information on the effects of environment on cadmium concentration would facilitate the choice of screening environments for breeding low cadmium cultivars and provide an indication of the efficacy of low cadmium cultivars in reduction of grain cadmium concentration in different environments. Decisions to deploy the low cadmium trait into new durum cultivars will depend on interactions of uptake of cadmium with uptake of other essential micronutrients. Various micronutrients such as copper, iron, magnesium and manganese are essential in animal and plant nutrition and disease resistance. Manganese, for example, affects reaction to the disease take-all of cereals (McCay-Buis et al. 1995). The objective of this study was to determine the effect of the allele for low grain cadmium concentration (Clarke et al. 1997b) on grain cadmium concentration of durum wheat grown in different environments, and to assess the effects of the allele on uptake of other elements and on economic traits such as grain yield. MATERIALS AND METHODS Five pairs of near-isogenic high/low cadmium durum wheat genetic stocks (Clarke et al. 1997a) and their parents were grown in a randomized complete block design with three replications. The two 8982-pairs derive from the cross Kyle/Nile, the W9260-pair from the cross DT617/DT471, the W9261-pair from the cross DT630/DT471 and the W9262-pair from the cross Kyle*2/Biodur. Kyle (PI537310; McLeod et al. 1991) is from the Swift Current breeding program, Nile was obtained from ICARDA (Aleppo, Syria), Biodur is from France, and the other parents are breeding lines from the Agriculture and Agri-Food Canada durum program. Trials were grown in Canada near Swift Current in 1994, near Swift Current, Stewart Valley and Regina, Saskatchewan, in 1995 and 1996, and in the United States, near Langdon and Fargo, North Dakota, in 1995, and near Casselton and Langdon, North Dakota, in The 1994 trial did not include the W9262-pair or Biodur. The Saskatchewan trials consisted of four-row plots 3 m long, with a row spacing of 0.23 m and a seeding rate of 290 seeds m 2. Plots were separated by a row of spring-planted winter wheat. In North Dakota, the trials in 1995 and 1996 consisted of four-row plots, 3.2 m long with 30-cm row spacing and a seeding rate of approximately 230 seeds m 2. Planting, soil fertility, pesticide application, and harvest were typical of local production conditions. Grain yield was determined at all locations except Fargo in 1995 and Swift Current in Test weight and kernel weight were determined at all sites in 1996, and grain protein and yellow pigment concentration were measured at the Canadian sites in Grain samples from the 1994 and 1995 Canadian sites were analyzed for cadmium, zinc, iron, and the 1995 samples were also analyzed for manganese. The 1996 Canadian samples were analyzed for cadmium only. Oven-dry grain or plant samples (5 g) were ground with a laboratory mill with stainless steel blades, and 0.5 g was digested in 6 ml trace-metal grade HNO 3 /HClO 4 (2:1 vol/vol) for 16 h at room temperature. The samples were then digested in a block digester for 60 min at 100 C, 10 min at 120 C, and 45 min at 220 C. A reagent blank and certified NIST (Nat. Inst. of Standards and Technology, Gaithersburg, MD) reference sample was processed with each set of 40 samples. Cadmium was determined on a 20 µl aliquot with a Hitachi Z8200 (Hitachi Scientific Instruments, Nissel Sagyo Canada Inc., Rexdale, ON) flame/furnace atomic absorption spectrophotometer with polarized Zeeman background correction, using graphite tubes. Grain samples from the 1995 and 1996 sites in North Dakota were analyzed for cadmium, zinc, iron, manganese, copper, nickel, calcium, magnesium, potassium and sodium. Well-mixed sub-samples of 1 g whole grain were digested in pyrex 1 tubes in concentrated HNO 3 /HClO 4 acids and then on a temperature-regulated digestion block. The residues were dissolved in 10 ml of 0.75 M HNO 3 for analysis as described below. The acid digests were analyzed by inductively-coupled argon plasma emission spectrometry on a Thermo Jarrell-Ash model Trace Analyzer 1 (TJA Solutions, Franklin, MA) with axial plasma. Generalized shifts in background emission were determined at off-peak wave length positions, and deducted from simultaneous measurements at on-peak wavelengths for each element. Sample and standard solution matrices were matched. Replicate samples and NIST standard grain samples were included routinely, and standard samples of grain were analyzed occasionally. The coefficient of variation (CV) among duplicates was <5%, and recovery of method-of-addition increases in concentrations were between 95 and 105%. Analyses on five occasions of NIST wheat sample 1567a yielded Cd concentrations of (± 0.001) µg kg 1 in comparison to the certified value of (± 0.002) µg kg 1. 1 Mention of proprietary product or vendor does not imply approval or recommendation by the U.S. Department of Agriculture.

3 CLARKE ET AL. CADMIUM AND OTHER ELEMENTS IN DURUM GRAIN 29 Table 1. Grain cadmium concentration (µg g 1 ) of durum isogenic pairs and parents grown in 11 environments in 1994 to Swift Swift Stewart Swift Stewart Genotype z Current Fargo Langdon Regina Current Valley Casselton Langdon Regina Current Valley 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED y Contrast ** ** ** ** ** ** ** ** ** ** ** H vs. L x z Isogenic line designations ending L indicate low cadmium accumulator, and H indicates high accumulator. y Standard error of a difference. x Significance of single degree of freedom contrast of all high vs. all low isolines: * P < 0.05, ** P < PROC MIXED of SAS (Littell et al. 1996) was used for the mixed models analysis with lines fixed and replications random. Bartlett s test for homogeneity of errors (Steel et al. 1997) over environments was performed on each trait. The errors were heterogeneous, so separate analyses were performed for each trait in each environment within each year. Comparisons between each pair of isogenics, and a comparison between all high and all low isogenics, were performed with the ESTIMATE statement. RESULTS AND DISCUSSION Average grain cadmium concentration was approximately 2.5 times greater for the high than for the low isolines within each environment (Table 1). The differences between the high and low isolines were all highly significant and consistent. The consistency of these differences will permit selection for genotypic differences in grain Cd in environments where average grain cadmium concentrations are modestly elevated. It would be practical to avoid environments where very low concentration of grain Cd (e.g. < 0.01 µg g 1 ) makes comparisons of lines difficult because of analytical uncertainty at concentrations near detection limits and errors contributed by even minor sources of contamination. Average grain cadmium concentration differed among years within locations and among location within years (Table 1). Large differences among years were observed for Regina, Swift Current, and Langdon. The year differences can not be separated from differences due to soil conditions associated with differing placement of the trials within sites each year. The variation of grain cadmium concentration over environments was not positively associated with grain yield. Indeed, there was a moderate negative correlation (r = 0.73*, n = 9), although this was due almost entirely to the Langdon site (r = 0.14 if Langdon removed), where yields were relatively high and Cd concentrations were relatively low in Positive relationships between cadmium concentration and grain yield are sometimes observed when a yield increase results from fertilization which improves growth and root exploration (Grant and Bailey 1998). On the other hand, elevated levels of grain cadmium may be associated with suppressions in growth caused by stresses such as salinity (Norvell et. al. 2000). However, in the present experiment, there was little effect of yield on cadmium concentrations, and thus, the differences among locations were most likely due to undetermined environmental factors. There was a significant difference between the low and high cadmium isogenic lines for grain yield in one of the nine environments in which yield was measured (Table 2). The yield of the high cadmium isogenic lines was slightly higher than that of the low isogenic lines at Swift Current in There were no significant differences between the low and high cadmium isogenic lines for grain protein concentration, test weight, kernel weight, days to maturity or lodging score in any of the environments (not shown). These results agree with observations in our breeding populations involving diverse low cadmium parents (unpublished). Incorporation of the low cadmium allele into durum cultivars is unlikely to have any effect on agronomic or other economic traits. Although there were many significant genotypic differences in grain concentration of elements other than Cd, these differences were generally not associated with the high and low cadmium isogenic lines (Tables 3 7).

4 30 CANADIAN JOURNAL OF PLANT SCIENCE Table 2. Grain yield (kg ha 1 ) of durum isogenic pairs and parents grown in nine environments in 1994 to Swift Swift Stewart Stewart Genotype z Current Langdon Regina Current Valley Casselton Langdon Regina Valley 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED y Contrast H vs. L x * NS NS NS NS NS NS NS NS z Isogenic line designations ending L indicate low cadmium accumulator, and H indicates high accumulator. y Standard error of a difference. x Significance of single degree of freedom contrast of all high vs. all low isolines: * P < 0.05 and NS, non-significant. Table 3. Grain zinc concentration (µg g 1 ) for near isogenic high/low cadmium durum lines grown in different environments in 1994 to Genotype Swift Current Fargo Langdon Regina Swift Current Stewart Valley Casselton Langdon 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED z Contrast H vs.l y NS NS * NS NS NS NS NS Significant (P < 0.05) genotypic differences in grain zinc concentration were found in four of the seven environments in which zinc was measured. Among the parent lines, DT618 tended to have high grain zinc concentration. Differences in zinc concentration among isolines were significant at only Langdon (P < 0.05) in 1995 (Table 3), and marginally so at Regina (P < 0.10), suggesting that Cd and Zn accumulation is controlled by independent mechanisms. Hinesly et al. (1982) found that grain zinc concentration was not significantly different between low and high cadmiumaccumulating maize hybrids, although leaf zinc concentration was greater for the high accumulator in 2 of 3 yr. They also concluded that cadmium and zinc accumulation are genetically independent traits. Zinc deficiency is a widespread problem in cereal crop production, limiting grain yields and causing deficiency problems in cereal-based human diets. Durum appears to be more sensitive than common wheat and other cereals to zinc deficiency (Cakmak et al. 1998). Graham et al. (1992) reported genotypic variation in zinc uptake efficiency of

5 CLARKE ET AL. CADMIUM AND OTHER ELEMENTS IN DURUM GRAIN 31 Table 4. Grain iron concentration (µg g 1 ) for near isogenic high/low cadmium durum lines grown in different environments in 1994 to Genotype Swift Current Fargo Langdon Regina Swift Current Stewart Valley Casselton Langdon 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED z Contrast H vs. L y NS NS * NS NS NS NS * Table 5. Grain manganese concentration (µg g 1 ) for near isogenic high/low cadmium durum lines grown in different environments in 1995 and Genotype Fargo Langdon Regina Swift Current Stewart Valley Casselton Langdon 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED z Contrast H vs. L y NS NS NS NS NS NS NS durum, and concluded that grain zinc concentration could be improved by breeding. The apparent genetic independence of uptake of cadmium and zinc is thus positive for efforts to breed for increased efficiency of zinc uptake. The difference in average grain iron concentration for the high and low isogenic lines was not consistent (Table 4), with the high lines greater than the low lines at Langdon (P < 0.05) and nearly so in Regina (P < 0.10) in 1995, and the reverse at Langdon in 1996 (P < 0.05). Overall there were significant (P < 0.05) genotypic differences in grain iron concentration at all locations except Swift Current in DT630 showed consistently low iron concentration. Average grain manganese concentration did not differ for the high and low isogenic lines (Table 5), but there were significant (P < 0.05) genotypic differences at all locations. DT618 showed consistently low manganese concentration, and Nile was consistently high. Grain copper concentration of the low cadmium isogenic lines was significantly greater (P < 0.05) than the high cadmium lines at Langdon in 1996 (Table 6), but not at the other locations. There were significant (P < 0.05) genotypic differences at all locations. Trends in genotypic differences were not strong, although DT630 tended to have high copper concentration. Grain calcium concentration tended to be higher for the high than for the low isogenic lines, but approached significance only at Langdon in 1995 (P < 0.10). There were significant (P < 0.01) genotypic differences at all locations except Langdon Average

6 32 CANADIAN JOURNAL OF PLANT SCIENCE Table 6. Grain copper, magnesium and calcium concentration (µg g 1 ) for near isogenic high/low cadmium durum lines grown at Fargo and Langdon, ND in 1995 and at Casselton and Langdon, ND 1996 Copper Magnesium Calcium Genotype Fargo Langdon Casselton Langdon Fargo Langdon Casselton Langdon Fargo Langdon Casselton Langdon 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED z Contrast H vs. L y NS NS NS * ** * NS * NS NS NS NS Table 7. Grain potassium, sodium and nickel concentration (µg g 1 ) for near isogenic high/low cadmium durum lines grown at Fargo and Langdon, ND in 1995 and at Casselton and Langdon, ND 1996 Potassium Sodium Nickel Genotype Fargo Langdon Casselton Langdon Fargo Langdon Casselton Langdon Fargo Langdon Casselton Langdon 8982-SF-L SF-H TL-L TL-H W9260-BC-L W9260-BC-H W9261-BG-L W9261-BG-H W A-L W A-H Biodur DT DT DT Kyle Nile Mean SED z Contrast H vs. L y ** * NS NS NS NS NS NS NS ** NS NS grain magnesium concentration was greater (P < 0.05) for the high than for the low isogenic lines at Fargo and Langdon in 1995, but the reverse for Langdon in There were significant (P < 0.05) genotypic differences at all locations, but genotype ranking was not consistent over environments. Average grain potassium concentration was greater (P < 0.05) for the high than for the low isogenic lines at Fargo and Langdon in 1995, and there were significant (P < 0.05) genotypic differences at all locations except Langdon in 1996 (Table 7). Nile tended to have high potassium concentration. Average grain sodium concentration did not differ for the high and low isogenic lines, but there were significant (P < 0.01) genotypic differences within locations, except at Langdon in Nile had high sodium concentration. Average grain nickel concentration was

7 CLARKE ET AL. CADMIUM AND OTHER ELEMENTS IN DURUM GRAIN 33 greater (P < 0.01) for the low than for the high isogenics at Langdon in 1995, and there were significant (P < 0.01) genotypic differences at all locations. Biodur had consistently high grain nickel concentration and DT471 was relatively low. Graham et al. (1999), Farnham et al. (1999) and Bouis (2000) suggest there is the genetic potential within most crops to breed for enhanced nutritional characteristics. This objective is urgent in the case of staple foods consumed in less-developed nations where nutritional needs for several essential mineral elements are not being met (Graham et al. 1999; Bouis 2000). Breeding for improved nutritional quality of durum wheat should be possible given the genetic variation for the essential trace elements iron, manganese and zinc in this small sample of genotypes, and this conclusion is supported by other evidence of genetic variation for micronutrient elements presented by Khabaz-Saberi et al. (1999) and Graham et al. (1999). In conclusion, the allele for low cadmium concentration does not negatively affect major economic traits, and should not cause problems when incorporated into durum cultivars. Further, the allele seems to be specific for cadmium and has generally no effect or small and inconsistent effects on concentrations of other elements. This should permit breeding of durum wheat genotypes with lower levels of grain cadmium, while maintaining or raising the levels of essential nutrients. ACKNOWLEDGMENTS We gratefully acknowledge Drs. E. Elias and B. L. Johnson (North Dakota State University, Fargo) for growing and harvesting the North Dakota trials, G. E. Winkleman (Semiarid Prairie Agricultural Research Centre), M. A. Rutkze (USDA Plant, Soil and Nutrition Lab.), and I. S. Murchison and C. R. Dooley (Brandon Research Centre) for analyses of grain, and J. Ross, L. Thiessen, M. Olfert, P. Taylor and C. Knockert (Semiarid Prairie Agricultural Research Centre) for technical assistance. Andersson, A. and Pettersson, O Cadmium in Swedish winter wheat: Regional differences and their origin. Swed. J. Agric. Res. 11: Bouis, H. E Enrichment of food staples through plant breeding: A new strategy for fighting micronutrient malnutrition. Nutrition 16: Cakmak, I., Torun, B., Erenoglu, B., Ozturk, L., Marschner, H., Kalayci, M., Ekiz, H. and Yilmaz, A Morphological and physiological differences in the response of cereals to zinc deficiency. Euphytica 100: Clarke, J. M., Leisle, D., DePauw, R. M. and Thiessen, L. L. 1997a. Registration of durum wheat genetic stocks near-isogenic for cadmium concentration. Crop Sci. 37: 297 Clarke, J. M., Leisle, D. and Kopytko, G. L. 1997b. Inheritance of cadmium concentration in five durum wheat crosses. Crop Sci. 37: Farnham, M. W., Simon, P. W. and Stommel J. R Improved phytonutrient content through plant genetic improvement. Nutr. Rev. 57(9II): S19 S26. Graham, R. D., Ascher, J. S. and Hynes, S. C Selecting zinc-efficient cereal genotypes for soils of low zinc status. Plant Soil 146: Graham, R., Senadhira, D., Beebec, S., Iglesiasc, C. and Monasteriod, I Breeding for micronutrient density in edible portions of staple food crops: conventional approaches. Field Crops Res. 60: Grant, C. A. and Bailey, L. D Nitrogen, phosphorus and zinc management effects on grain yield and cadmium concentration in two cultivars of durum wheat. Can. J. Plant Sci. 78: Hinesly, T. D., Alexander, D. E., Redborg, K. E. and Ziegler, E. 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G., Townley-Smith, T. F., DePauw, R. M., Clarke, J. M., Lendrum, C. W. B. and McCrystal, G. E Registration of Kyle durum wheat. Crop Sci. 31: 236. Norvell, W. A., Wu, J., Hopkins, D. G. and Welch, R. M Association of cadmium in durum wheat grain with soil chloride and chelate-extractable soil cadmium. Soil Sci. Soc. Am. J. 64: Oliver, D. P., Schultz, J. E., Tiller, K. G. and Merry, R. H The effect of crop rotations and tillage practices on cadmium concentration in wheat grain. Aust. J. of Agric. Res. 44: Oliver, D. P., Hannam, R., Tiller, K. G., Wilhelm, N. S., Merry, R. H. and Cozens, G. D The effects of zinc fertilization on cadmium concentration in wheat grain. J. Environ. Qual. 23: Oliver, D. P., Gartrell, J. W., Tiller, K. G., Correll, R., Cozens, G. D. and Youngberg, B. L Differential responses of Australian wheat cultivars to cadmium concentration in wheat grain. Aust. J. Agric. Res. 46: Oliver, D. P., Tiller, K. G., Alston, A. M., Cozens, G. D. and Merry, R. 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