BREEDING MAIZE FOR HIGHER YIELD AND QUALITY UNDER DROUGHT STRESS 1

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1 Maydica 54 (2009): BREEDING MAIZE FOR HIGHER YIELD AND QUALITY UNDER DROUGHT STRESS 1 M.J. Carena 2,*, G. Bergman 3, N. Riveland 3, E. Eriksmoen 4, M. Halvorson 5 2 Department of Plant Sciences, North Dakota State University (NDSU), Dept #7670, Fargo, ND, , USA 3 Williston Research Extension Center, NDSU, Williston, ND, USA & Sidney Research Extension Center, Montana State University, Sidney, MT, USA 4 Hettinger Research Extension Center, NDSU, Hettinger, ND, USA 5 Minot Research Extension Center, NDSU, Minot, ND, USA ABSTRACT - Maize (Zea mays L.) has a wide range of adaptation. As a consequence, the U.S. Corn Belt keeps moving north and west. However, even though the ethanol industry has expanded to western North Dakota (ND) and hectares have significantly increased in the past 10 years, maize is still limited in its extension to the west due to drought. Since 2001 the NDSU maize breeding program has incorporated drought tolerance screening as a routine in its pedigree selection process of inbred line development and hybrid identification. The use of reliable locations where stress intensity can be managed for thousands of lines and hybrids annually has made, in combination to multi-stage and multi-location testing, significant improvements for drought tolerance. Data of early and late generation hybrid trials in western ND and eastern Montana (MT) showed that hybrids including ND experimental lines had significantly better yield, lower grain moisture at harvest, higher test weight, higher extractable starch, higher fermentable starch, higher oil, and higher protein than industry check hybrids. At least 40 NDSU experimental hybrids yielded better than checks in western ND with specific hybrids exceeding 193.8% improvement. Several of these have exotic alleles in their genetic background as they belong to our long-term EarlyGEM tropical and temperate inbred line development program for short-season environments. Even though single-gene transgenic approaches are currently being proposed by industry, our breeding approach demonstrates there is no limit to genetic improvement for drought tolerance when most tolerance genes are targeted in the breeding 1 This paper is dedicated to farmers facing the challenge to produce a crop under severe drought conditions in western ND and other parts of the world. The work was done with the financial and/or cooperative direct and indirect support of the North Dakota Agricultural Products Utilization Commission (NDAPUC), the North Dakota Ethanol Producers Association (NDPA), the North Dakota Corn Council Utilization (NDCCU), and the North Dakota Board for Agricultural Research (SBARE), Monsanto, and Laboulet Semences. Special thanks to Dr. Gregory Edmeades who helped initiate this work almost 10 years ago. * For correspondence (fax: ; e.mail: marcelo.carena@ndsu.edu). Re ceived July 14, 2009 process. Heterotic effects are unique for each hybrid and sequencing efforts on only B73 may limit the identification of useful alleles for drought tolerance and other complex traits. The NDSU maize breeding program has the desire to use proposed transgenic drought tolerant industry hybrids as checks for western ND and eastern MT short season environments when they finally become available. KEY WORDS: Zea mays L.; Drought; Public maize breeding; Quality. INTRODUCTION Obtaining maximum yields in maize (Zea mays L.) often requires a reduction in groundwater supplies due to irrigation and significant costs associated with production and the environment. The major abiotic stress affecting maize production on a worldwide basis is drought. Grain yield and quality losses are maximized when drought occurs during flowering time (ROBINS and DOMINGO, 1953; CLAASEN and SHAW, 1970; SHAW, 1977). Moreover, this stress occurs randomly in timing and severity making the identification of drought tolerant cultivars challenging. In the absence of stress, drought tolerance often provides a grain yield penalty. Multi-location testing and multi-stage selection have commonly been the ways breeders used to distinguish susceptible from tolerant genotypes. However, the use of reliable locations where stress intensity can be managed has made a significant improvement for breeding purposes (BARKER et al., 2005; BANZIGER, 2006) as reliable drought locations increase the accuracy of identifying drought tolerant genotypes (BOLANOS and EDMEADES, 1996). Flowering time in maize should be targeted first while grain filling tolerance, grain number, and overall grain quality (e.g. test weight) can be con-

2 288 M.J. CARENA, G. BERGMAN, N. RIVELAND, E. ERIKSMOEN, M. HALVORSON Germplasm Improved by Intra & Inter Population Recurrent Selection (RS) (Advanced Cycles, ~10%) Germplasm Adapted from Stratified Mass Selection (~5%) Elite x Elite (within Heterotic Group, ~40%) Elite Industry Lines under MTAs (~5%) Elite x Elite (across Heterotic Group, ~5%) Top Progenies from RS (full-sibs, half-sibs, S1s, and S2s, all) S0 NDSU Maize Populations Top Backross Progenies from the NDSU EarlyGEM Adapted Breeding Crosses (~35%) Inbred Line Development (see Table 2) FIGURE 1 - Germplasm pre-breeding sources for NDSU maize inbred line development. sidered secondary targets (EDMEADES, personal communication). A significant increase in the anthesissilking interval (ASI), the difference between pollen shed and ear silking of the same genotype, is the typical response of maize under drought stress (DOW et al., 1984; EDMEADES et al., 2000). ASI is an indicator of ear growth rate since delayed silk emergence is associated with reduced ear growth under stress. There is statistically significant variation for ASI among maize genotypes. Recurrent selection including reduced ASI has made possible a good partitioning of assimilates to the developing ear under drought stress by increasing yield and reducing ASI as well as barrenness (EDMEADES et al., 2000). Early grain filling has been suggested as another opportunity for breeding (GRANT et al., 1989; NESMITH and RITCHIE, 1992) against reduced number of kernels and their test weight. In addition, kernel number was observed to be highly correlated with grain yield under water stress meaning that physiological mechanisms confer the ability to maximize kernel number under water deficit (O NEILL et al., 2004). These responses, however, vary considerably among maize inbred lines and hybrids (BRUCE et al., 2002; O NEILL et al., 2004). The correlation between inbred lines and hybrids for drought tolerance ranges from 0.35 to 4.0 (BANZIGER, 2006) indicating the need for testing not only inbred lines per se but also hybrids for heterosis exploitation and, ultimately, cultivar development. Moreover, the genetic correlation between grain yield and ASI across a diverse array of genotypes grown under drought at flowering time was shown to be -0.6, suggesting that ASI is a visual indicator of underlying processes affecting reproductive success under drought stress (EDMEADES et al., 2000). Corn hybrid changes over 30 (TOLLENAAR and WU, 1999), 50 (CASTLEBERRY et al., 1984), and 70 (DU- VICK et al., 2004) years have shown that successful maize hybrids have adapted better to abiotic stresses through increased stress tolerance. Differences among hybrids suggest it is feasible to combine stress tolerance with high yield in future elite germplasm (O NEILL et al., 2004). The same authors suggested the ability to maintain kernel number under water stress should be a priority of maize improvement programs. Drought tolerance has complex and polygenic tolerance mechanisms associated with epistatic effects and large genotype by environment interactions. Therefore, breeding approaches exploiting polygenic effects are desirable. The recent announcement from Monsanto and BASF regarding the discovery of the drought resistance gene cspb gives us hope to utilize transgenic hybrids as indus-

3 BREEDING MAIZE UNDER DROUGHT 289 try checks by the year The industry transgenic approaches are limited to one or few genes that might give a limited genetic improvement, but a good business advantage. Drought-tolerance efforts on polygenic effects do not have a limit on genetic improvement and quantitative traits are better explained by polygenes rather than quantitative trait loci or QTL (CARENA and WICKS III, 2006). TABLE 1 Linking drought tolerance to a public breeding program for hybrid maize. NDSU Maize Inbred Line Development and Hybrid Identification Season Generation Activity Winter 1 P 1 X P 2 Elite by elite combinations within heterotic groups Winter 2 F 1 Self-pollination of hybrids developed from lines that in most cases belong to the same heterotic group (some exceptions) Summer 1 F 2 = S 0 (a) S 0 populations grown at high plant density (b) Pre-breeding from long-term programs provide additional segregating populations/progenies. Stratified mass selection (CARENA et al., 2008), intra/inter-population recurrent selection and BC Early GEM programs (CARENA et al., 2009) S 0 plants are discarded based on phenotype. We have initiated first year of testing with > 1,000 S 0 testcrosses produced in the winter Winter 3 and 4 F 3 = S 1 (a) S 0s and S 1 lines are screened under drought-controlled conditions (b) Selected S 1 plants are selfed and testcrossed to inbred and sister-line industry testers. Some lines are advanced (winter 4) (c) Specific DT hybrids are tested and produced (winter 4) Summer 2 F 4 = S 2 (a) S 2 lines are grown in breeding, disease and testcross nursery rows across locations. Emergence/vigor data collected (b) Few S 0 populations discarded based on parent hybrid data (c) S 1 testcrosses grown in 1-2 rep regional trials (First year) (d) S 2 top lines kept (TC data and phenotype across locations) Winter 5 and 6 F 5 = S 3 (a) S 3 lines are re-screened under drought and crossed to 2-3 industry testers from each heterotic group (b) Top families are re-sampled and all lines are advanced A range of S 4 -S 6 lines are obtained Summer 3 F 6 = S 4 (and beyond) (a) Second-year evaluation of S 2 testcrosses in 5-10 regional locations (e.g. western ND for drought, northeastern ND for cold, etc) in addition to several traits including drydown, earliness, test weight, lodging resistance, grain quality, etc) Two replications at each location (incomplete block designs) (b) S 5 seed of selected S 4 families (based on S 2 TC data) is bulked in breeding nursery for single-crosses. (c) Select top lines (more competitive than check hybrids) for each state region. Top ears from each line are advanced Winter 7 and 8 S 5 and beyond (a) Lines advanced and crossed to 8-10 elite industry testers (b) Lines and hybrids are re-tested for drought tolerance Summer 4 S 6 and beyond (a) Experimental NDSU hybrids are tested in locations (Third-year testing) and multi-state regional trials (b) Lines are grown in the potential releases section of nursery Winter 9 and 10 S 7 and beyond (a) Lines advanced and crossed to new elite industry testers (b) Lines and hybrids are re-tested for drought tolerance Summer 5 S 8 and beyond (a) Fourth-year test at 30+ locations in cooperation with Industry and Foundation Seed Companies Winter 11 RELEASE of NDSU inbred lines. After 3-5 years evaluation, NDSU lines have up to approximately 70 environments of data for release consideration

4 290 M.J. CARENA, G. BERGMAN, N. RIVELAND, E. ERIKSMOEN, M. HALVORSON Production of maize in ND continues to grow significantly (USDA, 2009). The ethanol industry has significantly expanded over to U.S. northern and western areas where maize is still limited in its extension due to significant environmental challenges (mainly drought) and the availability of elite early maturing hybrids. Therefore, local ethanol plants need early maturing hybrids with above average drought tolerance and quality for an ideal maizeethanol relationship in the region. The objective of this long-term project is to develop lines and populations for improved grain yield and quality under drought stress. The approach is complementary to the one being performed by industry since it is a non-transgenic approach with no limits of genetic improvement and impact considering drought tolerance is a very complex trait with several genes controlling its expression. FIGURE 2 - S 1 and S 2 line production under drought stress managed environment (Salta, Argentina). MATERIALS AND METHODS A subset of 25 segregating populations with approximately 100,000 F 2 individuals was selected from the NDSU maize breeding program. Over 9,000 early and late generation lines derived from this subset were evaluated in Buin, Chile and Salta, Argentina for drought tolerance. This sample represents a large variation of tropical and temperate germplasm adapted and improved for ND environmental conditions (Fig. 1). Tropical and late temperate germplasm included over 3,000 lines from the EarlyGEM project (CARENA et al., 2009a; HALLAUER and CARENA, 2009) already adapted through a modified backcross methodology. A strong drought tolerance screening was incorporated in the NDSU maize breeding program during early generation inbred line development in 2001 (Table 1). The target was the MonDak region including western ND and eastern Montana (MT). Winter nursery screening of genotypes (both inbreds and hybrids) was carried out with essentially no rain during the growing season (Fig. 2). Irrigation was supplied at a rate approximate to crop water use rate up to the V7 stage (BANZINGER, 2006), essentially for stand establishment when irrigation was completely withdrawn. Water treatments consisted of a well-watered control and a treatment of drought imposed at the V7 corn growth stage. We recorded the dates when 50% of the plants within a plot or nursery row reached anthesis and silking. Specifically male flowering was measured by counting the number of days until half of the plants in the row had produced pollen on at least half of the tassel. Female flowering was measured by counting the number of days until half of the plants had silks. The ASI was determined by taking the absolute number of the difference in days between male and female flowering. Before harvesting nursery rows, husks from self-pollinated ears were removed (Fig. 3) for visual screening among and within genotypes (Fig. 4) for a second screening stage for grain filling. The following winter season hybrids were produced between NDSU drought tolerant lines (Fig. 5) and selected industry testers from foundation seed companies. These hybrids were evaluated under controlled stress conditions (winter nursery) and across drought-prone northern U.S. FIGURE 3 - Husk removal of S 2 lines screened under drought stress managed environment. FIGURE 4 - Grain filling screening among and within maize inbred progeny lines.

5 BREEDING MAIZE UNDER DROUGHT FIGURE 5 - NDSU experimental drought tolerant EarlyGEM line included in winter testcross isolations. 291 FIGURE 7 - Hybrid trials including drought tolerant maize genotypes with a range of relative maturities conducted in Williston, ND. FIGURE 6 - Hybrid trials including a range of drought tolerant maize genotypes conducted in Williston, ND.

6 292 M.J. CARENA, G. BERGMAN, N. RIVELAND, E. ERIKSMOEN, M. HALVORSON FIGURE 8 Annual harvested acreage (Y1 axis, yellow dots in thousands) and productivity (Y2 axis, red dots, bu/a) of ND maize. Midwest environments (Williston, Hettinger, and Minot in ND, and Sidney, MT). Both transgenic and non-transgenic industry testers were utilized for making NDSU x industry hybrids including NDSU drought tolerant lines. ND Research Extension Centers and farmers provided land space and management each year. Corn hybrid experiments (Fig. 6) were carried out across 15 drought-prone and irrigated environments in 2006, 2007, and In addition to, male and female flowering (e.g. ASI), emergence percentage and seedling vigor were taken for screening lines in summer nurseries through a rank summation index. The first stage of phenotypic selection was also based on maturity, stalk and root lodging, ear quality, drought tolerance, and seed set on lines per se. A range of relative maturities (RM) from 75 to 90 RM was utilized (Fig. 7). At harvest time, plants per plot were counted and harvested. Grain yield at harvest (Mg ha-1) was based on the total weight of the seeds adjusted on a 155 g H2O kg-1 grain moisture basis after ears in the plot had been shelled. Grain quality data were generated by the FOSS 1241 grain analyzer on 500 g kernels samples from every plot in most environments. The heritability index was utilized to estimate the repeatability of hybrids across locations within years. Augmented, partially balanced lattice, and randomized complete block (RCBD) designs were utilized to measure the variation present in the drought managed location. Partially-balanced lattice designs with two replications per location were also utilized across dry land and irrigated ND and MT locations across years and testers. Individual analyses of variance were performed for all traits at each location, as well as across locations using the SAS Lattice and GLM procedures (SAS, 1990). Efficiency of the lattices relative to a RCBD were calculated, if needed, following Fisher s procedure (STEEL et al., 1997). For the combined analyses of variance adjusted means were utilized when the relative efficiency of lattices was higher than 105% compared with a RCBD for each trait. Expected mean squares were based on a mixed linear model that considers environments and replications as random effects and entries as fixed effects. Mean comparisons were assessed by the least significant difference (LSD) since it has been shown to be an adequate test for detection of differences (CARMER and SWANSON, 1971). The effective error was utilized to calculate the LSD values within environments for traits showing high relative efficiency. FIGURE 9 - Drought tolerant (left) and drought susceptible (right) hybrids in a partially-balanced lattice 10x10 experimental design in Williston, ND. FIGURE 10 - Drought tolerant hybrids with 182% and 200% grain yield response compared to top industry check in Williston, ND. FIGURE 11 - Drought susceptible hybrid with 59% grain yield response compared to top industry check in Williston, ND.

7 BREEDING MAIZE UNDER DROUGHT 293 RESULTS AND DISCUSSION Commercial hybrids available in ND are mostly bred and developed elsewhere. As a consequence, adaptation is challenging. Hybrids are often late maturing and, therefore, low test weight and starch content, especially under drought conditions, are a continuous challenge. In addition, poor grain quality is often present in market hybrids due to state environmental challenges affecting the effective utilization of our state maize into ethanol. These environmental challenges are mainly the short period between killing frosts, the limited heat supply, and the limited rainfall, especially in western ND. Therefore, drought tolerance, cold tolerance, grain quality, test weight, early seedling vigor, uniform emergence in cold soils, fast dry down, and early maturity are very important characteristics (as important as grain yield) essential to a hybrid grown in ND. In spite of these environmental challenges ND farmers planted over 2.5 million acres of maize in the past two years (Fig. 8). Breeding for early maturity, drought tolerance, and grain quality are some of the reasons maize is becoming more adapted to these once considered marginal areas. ND is an excellent place to test what is screened under managed conditions. Research cooperators as well as current maize breeding locations are located TABLE 2 - NDSU experimental top maize hybrid mean comparisons across western ND environments in Hybrid Pedigrees Grain Yield Grain Test Weight Starch Protein Oil Mg ha -1 Moisture Kg hl -1 % % % IRRIGATED TRIALS ND x II N/A 66.0 N/A N/A N/A Pioneer 39D N/A 61.3 N/A N/A N/A Pioneer 39D N/A 65.0 N/A N/A N/A Wensman 6084BtRR 8.9 N/A 62.5 N/A N/A N/A Mycogen 2J N/A 61.3 N/A N/A N/A LSD(0.05) 1.9 N/A 4.3 N/A N/A N/A DRY LAND TRIALS TR2040 x ND NP2123Bt x ND TR1017Bt x ND ND x NP2123Bt ND X II TR2040 x ND TR1017Bt x ND TR1017Bt x ND TR1017Bt x ND TR2040 x ND LH176 x ND NP2123Bt x ND TR2040 x ND LH176 x ND TR1017Bt x ND TR1017Bt x ND Wensman 6084BtRR Mycogen 2J DKC LSD(0.05) Early and late generation hybrid testing has four stages and utilizes up to eight industry testers per heterotic group upgrated yearly, sometimes less than eight due to the lack of yearly maturing industry testers (<85RM).

8 294 M.J. CARENA, G. BERGMAN, N. RIVELAND, E. ERIKSMOEN, M. HALVORSON TABLE 3 - NDSU experimental top maize hybrid mean comparisons across western ND environments in Hybrid Pedigrees Grain Yield Grain Test Weight Starch Protein Oil Mg ha -1 Moisture Kg hl -1 % % % IRRIGATED TRIALS LH176 x ND Pioneer 39D Pioneer 39K LSD(0.05) DRY LAND TRIALS TR1017Bt x ND N/A N/A N/A TR1017Bt x ND N/A N/A N/A TR1017Bt x ND N/A N/A N/A TR1017Bt x ND N/A N/A N/A TR1017Bt x ND N/A N/A N/A TR2040 x ND N/A N/A N/A TR2040 x ND N/A N/A N/A Pioneer 39D N/A N/A N/A LSD(0.05) N/A N/A N/A TR4615 x ND N/A N/A N/A LH176 x ND N/A N/A N/A TR4615 x ND N/A N/A N/A LH176 x ND N/A N/A N/A NP2123Bt x ND N/A N/A N/A LH176 x ND N/A N/A N/A LH176 x ND N/A N/A N/A Pioneer 39K N/A N/A N/A Pioneer 39D N/A N/A N/A LSD(0.05) N/A N/A N/A NP2123Bt x ND N/A N/A N/A NP2123Bt x ND N/A N/A N/A NP2123Bt x ND N/A N/A N/A NP2123Bt x ND N/A N/A N/A Pioneer 39D N/A N/A N/A Mycogen 2J N/A N/A N/A LSD(0.05) N/A N/A N/A in strategic places for drought screening where ethanol plants have been developed and sustained drought is typical of this region. Therefore, the need for early maturing drought tolerant genotypes is priority before utilizing maize for ethanol. NDSU maize breeding is one of the few if not the only breeding program present in the MonDak region and winter nurseries manage the stress besides the advantage of having one or two extra generations. The key to breeding for drought tolerance is to manage the stress. In both winter nursery places we know the fields are dry, it never rains, and on site cooperators are set up with an irrigation system that allows us to decide when to stop irrigating, collect data, select the best lines and hybrids, and test them in the target U.S. areas the following season. In maize breeding the purpose of both early and late generation hybrid trials is to discard thousands of genotypes utilizing different experiments and testers (Table 1). Drought was severe (as expected) in all years of evaluation. State hybrid maize performance trials including industry hybrids across western ND Research/Extension Centers had to be discarded in most cases as industry hybrids often showed significant barrenness. However, breeding trials including pre-screened lines and hybrids for

9 BREEDING MAIZE UNDER DROUGHT 295 TABLE 4 - NDSU experimental top maize hybrid mean comparisons across western ND environments in Hybrid Pedigrees Grain Yield Grain Test Weight Starch Protein Oil Mg ha -1 Moisture Kg hl -1 % % % IRRIGATED TRIALS ND2004 x TR N/A 72.5 N/A N/A N/A ND2002 x TR N/A 68.8 N/A N/A N/A ND2001 x LH N/A 66.9 N/A N/A N/A ND2003 x TR3621Bt 11.5 N/A 75.0 N/A N/A N/A Pioneer 39D N/A 72.8 N/A N/A N/A Pioneer 39D N/A 70.0 N/A N/A N/A LSD(0.05) 1.9 N/A 2.1 N/A N/A N/A DRY LAND TRIALS NP2123Bt x ND N/A 11.3 N/A TR3621Bt x ND N/A 11.8 N/A TR1017Bt x ND N/A 12.9 N/A TR3621Bt x ND N/A 11.3 N/A TR1017Bt x ND97-6W N/A 10.9 N/A ND2001 x LH N/A 11.3 N/A ND2004 x TR1017Bt N/A 11.5 N/A ND2002 x NP2123Bt N/A 11.0 N/A ND2003 x LH N/A 10.9 N/A Pioneer 39D N/A 11.1 N/A Pioneer 39D N/A 11.6 N/A LSD(0.05) N/A 0.9 N/A LH176 x ND04-67W N/A N/A N/A LH176 x ND N/A N/A N/A LH176 x ND N/A N/A N/A LH176 x ND N/A N/A N/A Pioneer 39D N/A N/A N/A LSD(0.05) N/A N/A N/A drought tolerance showed significant differences among genotypes for economically important traits in those cases which can visually be perceived in Fig. 9, 10, and 11. Data within and among years showed NDSU experimental drought tolerant hybrids had significantly better grain yield performance under drought. Grain yields of industry hybrids available in the market ranged from 51.6% to 79.3% of the top NDSU experimental hybrids (Tables 2-4). On the other hand, only one of the three years showed significant better grain yield under irrigation for one NDSU experimental hybrid as well as similar ranks. Therefore, industry hybrids were not stable and suffered a significant yield penalty. These are environments that had basically no rain during late July, August, and September when grain yield and quality losses are maximized (ROBINS and DOMINGO, 1953; CLAASEN and SHAW, 1970; SHAW, 1977). At least 40 hybrids including ND experimental lines screened for drought tolerance yielded better than the top industry check in western dry land environments. These results confirm that screening for both ASI and grain filling is desirable for any maize breeding program developing cultivars for droughtprone environments (EDMEADES et al., 2000; O NEILL et al., 2004) as this breeding approach has no limits to genetic improvement for drought tolerance. Drought tolerant hybrids (e.g. those maintaining significant larger yields) had significantly lower grain moisture at harvest in 2006 and 2007 (Tables 3, 4). In four of these hybrids EarlyGEM lines were utilized, confirming their adaptation to very early maturing regions and that it is essential to broaden the germplasm base that the breeder works with (CARE- NA, 2008). In addition, several EarlyGEM derived lines with statistically similar yields to industry hy-

10 296 M.J. CARENA, G. BERGMAN, N. RIVELAND, E. ERIKSMOEN, M. HALVORSON brids were also lower in grain moisture at harvest showing that only one backcross generation was sufficient to adapt tropical and late temperate lines to ND (CARENA et al., 2009a). These are germplasm sources unique to NDSU. Seventeen and eight ND- SU experimental hybrids, including EarlyGEM lines and recently released lines (CARENA et al., 2009b), showed significantly better test weight values as well as better overall grain quality than commercial hybrids across years (Tables 2-4). Most of these hybrids also showed higher significant values for extractable and fermentable starch under drought conditions when compared to industry hybrids (data not shown). As with heterosis not much is known regarding the underlying genetic mechanisms of drought tolerant genotypic changes and its consequences to trait expression, crop improvement, and crop stability. What we know is that definitively drought tolerance is very complex genetically and ultimately, a combination of breeding techniques on unique germplasm screened under reliable drought environments will continue to help achieve desirable drought tolerance levels in maize. Investment in this type of research will benefit farmers that need to sustain maize production under low cost and very challenging environments. REFERENCES BARKER T.C., H. CAMPOS, M. COOPER, D. DOLAN, G.O. EDMEADES, J. HABBEN, J. SCHUSSLER, D. WRIGHT, C. ZINSELMEIER, 2005 Improving drought tolerance in maize. Plant Breed. Rev. 25: BANZINGER M., 2006 Breeding for highly variable abiotic stress environments. Intl. Plant Breed. Symp Aug. Mexico City, Mexico. BOLANOS J.S., G.O. EDMEADES, 1996 The importance of the anthesis-silking interval in breeding for drought tolerance in tropical maize Field Crops Res. 48: BRUCE W.B., G.O. EDMEADES, T.C. BARKER, 2002 Molecular and physiological approaches to maize improvement for drought tolerance. J. Exp. Bot. 53: CARENA M.J., 2008 Increasing the genetic diversity of northern U.S. maize hybrids: Integrating pre-breeding with cultivar development. In: Conventional and molecular breeding of field and vegetable crops. Novi Sad, Serbia. CARENA M.J., Z.W. WICKS, 2006 Maize population hybrids: An exploitation of US temperate public genetic diversity in reserve. Maydica 51: CARENA M.J., L. POLLAK, W. SALHUANA, M. DENUC, 2009a Development of unique and novel lines for early-maturing hybrids: moving GEM germplasm northward and westward. Euphytica (in press). CARENA M.J., D.W. WANNER, J. YANG, 2009b Linking pre-breeding for local germplasm improvement with cultivar development in maize breeding for short-season (85-95RM) hybrids. J. Plant Reg. (in press). CARMER S.G., M.R. SWANSO, 1971 Detection of differences between means: A Montecarlo study of five pairwise multi ple comparison procedures. Agron. J. 63: CASTLEBERRY R.M., C.W. CRUM, C.F. KRULL, 1984 Genetic improvements of U.S. maize cultivars under varying fertility and climatic environments. Crop Sci. 24: CLAASEN M.M., R.H. SHAW, 1970 Water deficit effects on corn. II. Grain components Agron. J. 62: DOW E.W., T.B. DAYNARD, J.F. MULDOON, D.J. MAJOR, G.W. THURTELL, 1984 Resistance to drought and density stress in Canadian and European maize hybrids. Can. J. Plant Sci. 64: DUVICK D.N., J.S.C. SMITH, M. COOPER, 2004 Long-term selection in a commercial hybrid maize breeding program Plant Breed. Rev. 24: EDMEADES G.O., J. BOLANOS, A. ELINGS, J.-M. RIBAUT, M. BANZINGER, M.E. WESTGATE, 2000 The role and regulation of the anthesis-silking interval in maize. pp In: M.E. Westgate, K.J. Boote (Eds.), Physiology and modeling kernel set in maize. CSSA Special Publication No. 29. CSSA, Madison, WI. GRANT R.F., B.S. JACKSON, J.B. KINIRY, G.F. ARKIN, 1989 Water deficit timing effects on yield components in maize. Agron. J. 81: HALLAUER A.R., M.J. CARENA, 2008 Maize breeding. pp In: M.J. Carena (Ed.), Handbook of Plant Breeding: Cereals. Springer, NY. NESMITH D.S., J.T. RITCHIE, 1992 Maize response to a severe soil water-deficit during grain filling. Field Crops Res. 29: O NEILL P.M., J.F. SHANAHAN, J.S. SHEEPPERS, R. CALDWELL, 2004 Agronomic responses of corn hybrids from different eras to deficit and adequate levels of water and nitrogen. Agron. J. 96: ROBINS J.S., C.E. DOMINGO, 1953 Some effects of severe soil moisture deficits at specific growth stages in corn. Agron. J. 45: SAS, 1990 SAS user s guide: Statistics. 4th ed. SAS Inst. Inc., Cary, NC. SHAW R.H., 1977 Water use and requirements of maize - a review. pp In: Agrometeorology of the maize crop. World Meteorology Organization Publication 480. TOLLENAAR M., J. WU, 1999 Yield improvement in temperate maize is attributable to stress tolerance Crop Sci. 39: USDA-NASS, 2009 North Dakota agricultural statistics 2009 [Online]. North Dakota Agricultural Statistics Service, North Dakota field office. U.S. Department of Agriculture, National Agricultural Statistics Service. Updated: June 2008.