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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Managing Reproductive Isolation in Hybrid Seed Corn Production

^a Doebler's Pennsylvania Hybrids, Inc., daleireland{at}doeblers.com
^b Landec Ag, Inc. 201 N. Michigan, Oxford, IN 47971, dwilson{at}landecag.com
^c Department of Agronomy, Iowa State University, Ames, IA 50011
^d Burris Consulting 1707 Burnett Ave, Ames, IA 50010, burrisconsulting{at}msn.com
^e Pioneer Hi-Bred International, Inc., 6900 NW 62nd Avenue, Johnston, IA 50131, Michael.Lauer{at}Pioneer.com

^* Corresponding author (westgate{at}iastate.edu)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Production of hybrid corn (Zea mays L) depends on cross-pollination between male and female inbred parents. As such, reproductive isolation of seed fields is required to ensure genetic purity of the hybrid progeny. Customer demand for improved genetic purity prompted the seed industry to examine the level of genetic purity resulting from current isolation practices. A 3-yr study was conducted to monitor purity of hybrid seed produced in 315 fields from 24 seed companies in North America. Each field was near a commercial corn field shedding pollen synchronously with the seed parent: a worse case scenario. Seed samples were collected at five locations along a 200-m transect established perpendicular to the nearest potential adventitious pollen source, and 100 seed from each location were subjected to isozyme analysis to determine percent out-crossing. Isolation distance, seed field size, field block size, number of border rows, adventitious field size, male:female row ratio, male population, and male pollen class were analyzed as continuous predictors and as variable class predictors of out-crossing at the field margins and field midpoints. Year had a significant (p 0.05) impact on observed out-crossing. Isolation distance, border rows, and male pollen class were significant predictors of out-crossing at the field margin but not at the field midpoint. A significant interaction between isolation distance and border rows was observed at both the field margin and midpoint. There also was an interaction between male:female ratio and male pollen class on out-crossing at the field margin. These results indicate that current practices used to isolate hybrid seed fields often achieve the goal of producing 99% genetically pure seed, but much higher levels of out-crossing can and do occur. Because out-crossing generally was greater and more variable at the field margins than at the field midpoint, adjustments to field management that focus on minimizing out-crossing at the field margins should lead to consistently high levels of genetic purity from hybrid seed fields.

Abbreviations: PMP, plant-made pharmaceuticals • GEE, generalized estimating equations • OLS, ordinary least squares (regression) • M:F, male:female row ratio • CI, confidence interval • dist, isolation distance • b_rows, border rows • f_size, field size • mf_ratio, male:female row ratio • cont_size, contaminant field size • mpc, male pollen class • NE, northeast • SE, southeast • NW, northwest • SW, southwest

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
PRODUCTION of genetically pure hybrid corn seed depends on cross-pollination between specific male and female inbred parents. It also depends on effective reproductive isolation of the female inbred from adventitious sources of corn pollen. Current isolation standards adopted by crop improvement organizations are intended to maintain an acceptable level of genetic purity across various environments and production conditions (Indiana Crop Improvement Association, 2002). Seed companies, however, often have their own standards for isolation that differ from public guidelines. These isolation standards are based mainly on practical experience and limited experimental investigation (Jones and Newell, 1946; Jones and Brooks, 1950; Hutchcroft, 1958; Rogers and Parkes, 1995; Indiana Crop Improvement Association, 2002).

Isolation standards typically include a minimum distance from all potential pollen sources, and a recommended number of border rows of the inbred male parent for a given isolation distance (Hutchcroft, 1958; Wych, 1988; Indiana Crop Improvement Association, 2002). Common regimes for yellow seed corn production are (isolation/border rows) 76 m (165 ft)/24 rows, 100 m (330 ft)/12 rows, and 200 m (660 ft)/6 rows (Wych, 1988; Indiana Crop Improvement Association, 2002). Greater isolation distance decreases the potential for adventitious pollen to enter the seed field, and additional border rows of male inbred parent are thought to minimize out-crossing by the adventitious pollen.

Until recently, these isolation standards have not been scrutinized because genetic purity of seed corn produced in the USA has met or exceeded international standards for purity (Wych, 1988; Rogers and Parkes, 1995; Organization for Economic Co-operation and Development, 2000; Indiana Crop Improvement Association, 2002). Recent concerns by U.S. international trade partners regarding transgenic seed in nontransgenic seed stocks, however, have raised significant doubts whether isolation guidelines designed for customer needs in the past are meeting current market demands (Rogers and Parkes, 1995). The European Community in particular is concerned about nontransgenic seed containing low levels of transgenic contaminants (Rogers and Parkes, 1995; Organization for Economic Co-operation and Development, 2000). Likewise, use of corn to produce plant-made pharmaceuticals (PMP) has been under intense scrutiny because of the potential for cross-pollination by airborne corn pollen. Requirements for reproductive isolation of corn fields producing PMPs is considerably more stringent than those for conventional hybrid seed production (Animal and Plant Health Inspection Service, 2003; Stevens, 2002). These developments underscore the need to examine the effectiveness of reproductive isolation in hybrid seed fields.

Reproductive isolation as it is managed in hybrid seed production has two interrelated components: biological isolation and physical isolation. Biological isolation includes elements such as flowering time, density of pollen shed, absolute amount of pollen shed, and the ratio of male rows to female rows. Inherent within biological isolation is competition for female silks by both the desirable male pollen and adventitious pollen sources. Physical isolation affects the capacity of pollen to arrive and enter the seed field. Factors include distance between the seed field and adventitious pollen source, relative orientation of the seed and source fields, seed field size, the size of the block employing the same pollinator, and the size of the adventitious source field.

Number of border rows is thought to contribute to both physical and biological isolation of the female inbred parent. Additional pollen parent plants at the seed field edge are thought to affect adventitious pollen entry into the seed field (Wych, 1988). As such, border rows of the pollen parent commonly are planted around hybrid seed fields in an attempt to saturate the field margin with pollen, and provide protection from adventitious pollen entry (Jones and Brooks, 1950; Indiana Crop Improvement Association, 2002). These additional rows also separate the seed parent from the potential adventitious pollen sources.

Production of pure hybrid seed requires that the overwhelming proportion of pollen reaching the plane of exposed silks on the seed parent comes from the male inbred. Greater pollen production supplied by the male border rows should favor pollination by the desired parent. Within most seed fields, however, only 20 to 25% of the plants are pollen parents. Additionally, inbred pollen parents typically have relatively small tassels that produce much less pollen than their hybrid progeny (Kiesselbach, 1949; Wych, 1988; Rasse et al., 2000). Seed producers often attempt to compensate for this level of low pollen production by increasing male:female row ratios and/or increasing male inbred plant density. There is a general lack of published information, however, relating the capacity for pollen production and resulting genetic purity in seed production.

There is considerable evidence that the spatial relationship between the potential adventitious pollen source and the seed field is an important determinant of genetic purity (Bateman, 1947a, 1947b; Jones and Newell, 1948; Jones and Brooks, 1950; Hutchcroft, 1958; Raynor et al., 1972; Paterniani and Stort, 1974; Garcia et al., 1998; Luna et al., 2001). The corn pollen grain is relatively large, which limits its mobility in air (Aylor, 2002; Jarosz et al., 2003). Yet information regarding corn pollen travel and environmental effects on pollen dispersal and viability is limited (Roy et al., 1995; Garcia et al., 1998; Luna et al., 2001; Ma et al., 2004). Early studies (e.g., Jones and Newell, 1946; Raynor et al., 1972) reported higher levels of pollen were deposited on the lee side of their experimental plots than on the windward side and that few grains were detected at greater distances. Paterniani and Stort (1974), in a study of four source fields, reported that pollen was "well mixed in the air" and decreased in quantity as one moved from the source. The few pollen grains that traveled greater distances remained viable, however, as Jones and Newell (1946) reported several isolated stalks of detasseled corn were pollinated from a corn source some 250 m away. The size of the adventitious pollen source probably contributes to the potential for out-crossing as well (Jones and Brooks, 1950; Di-Giovanni and Kevan, 1991). Jones and Brooks (1950) suggested that the shape of the seed field in relation to the potential adventitious source might significantly affect the level of out-crossing. To our knowledge, this information has not been applied in a systematic fashion to assess the effectiveness of isolation practices for hybrid seed production.

Current guidelines for seed isolation dictate uniform isolation distance 360 degrees surrounding the hybrid seed field (Indiana Crop Improvement Association, 2002). Little regard is currently given to how seed fields and potential adventitious sources are spatially arranged as long as the seed field meets minimum seed isolation standards in all directions.

The U.S. hybrid corn industry's current reputation for producing seed of high genetic purity relies on its capacity to isolate hybrid seed fields. Transgene-free products may require <0.5% transgene content in seed for some markets, a standard that is increasingly difficult to meet as commercial transgenic production continues to gain popularity. Identifying factors of biological and physical isolation that will contribute to greater genetic purity clearly is important to the U.S. hybrid corn industry. Therefore, the objectives of this study were (i) to evaluate the capacity of current industry isolation practices to produce hybrid seed that meets higher levels of genetic purity and (ii) to identify practices that will improve reproductive isolation in hybrid seed fields. Resolving the interactions among variables that contribute to the biological and physical isolation of the hybrid seed field forms the basis of our analysis. Because this study was conducted on actual seed production fields, the results necessarily reflect the practical constraints on hybrid seed production—focus on saleable seed, variation in local agronomic practice, control and cost of land area, and uncontrollable sources of adventitious pollen. These variables limit the resolving power of our analysis but do provide direct relevance to the hybrid seed industry.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
This study is based on a survey of genetic purity in selected seed production fields currently in use by hybrid seed companies in the U.S. Corn Belt. The analysis of these data focuses on identifying aspects of reproductive isolation most closely correlated with the successful production of genetically pure seeds.

Field Selection
Participating seed production plants from all major hybrid seed corn companies in North America were asked to identify 20 hybrid production fields as candidate fields for this study. Candidate fields were representative of normal seed production but had at least one commercial corn production field in the vicinity that was considered a potential source of adventitious pollen. Flowering notes taken on each of these fields were the date of anthesis (50% of plants shedding pollen) for the pollen parent, date of silking (50% plants with silks visible) for the seed parent, as well as anthesis dates of potential adventitious pollen sources within 200 m of the seed field. In addition to flowering data, each location collected information on the previous crop, pollen parent border row number, pollen parent plant population, pollen to seed parent row ratio, pollen parent flowering classification (Poor, Fair, or Good), spatial orientation of any potential adventitious source relative to the seed field, size of the adventitious pollen source, size of the hybrid seed field, distance to the nearest potential adventitious pollen source, and the size of the contiguous area using the same pollen parent (commonly referred to as "block size").

Flowering notes were collected on at least 1020 candidate fields. Of these, 315 fields from 24 seed companies in eight U.S. states (Illinois, Indiana, Iowa, Michigan, Minnesota, Nebraska, and South Dakota) and Ontario, Canada, were selected for further study because they met two criteria: (i) synchronous flowering between the inbred pollen and seed parents and (ii) flowering date of the potential adventitious source coincided with that of the seed field. The application of these criteria was meant to represent a worse case scenario for seed production. The entire set of fields included a broader range of flowering scenarios than could have been included for analysis. Fields that had synchronous flowering of inbred parents with no adventitious presence, for example, likely produced extremely pure seed. About 75% of the candidate fields considered for study met this criterion. Likewise, fields with asynchronous flowering between the inbred parents and an adventitious pollen source would likely produce impure and nonrepresentative seed. These fields were excluded in favor of those with synchronous flowering of the hybrid parents and the adventitious source because the latter were more likely to provide a true test of isolation practices with moderate (and potentially manageable) levels of out-crossing.

The physical relationship between a typical seed field and an adventitious pollen source is shown in Fig. 1. The selected fields had at least 50% of the seed field and potential adventitious pollen source overlapping to the East, North, South, or West. Fields with diagonal exposure (NE, SE, NW, SW) were excluded from the field orientation analysis. Although this is not a random sampling of all field orientations, it provided increased resolving power in the field orientation analysis since at least 50 fields were included in each cardinal direction.

View larger version (62K): [in this window] [in a new window]   Fig. 1. Diagram of sampling transect within a seed production field relative to an adventitious pollen source. Selected seed fields were adjacent to an adventitious pollen source that overlapped at least 50% of the seed field to the North, East, South, or West. Sampling transects were located on the side of the field adjacent to the adventitious pollen source. Ears were collected along the transect at 1.8 m (6 ft), 9.4 m (31 ft), 20.6 m (68 ft), 35.8 m (118 ft), and 200 m (660 ft) from the field edge nearest to the adventitious pollen source. Blocked area surrounding the seed field indicates border rows, which varied in number with distance between seed field and adventitious pollen source.

At each sampling location along a transect, 20 ears were collected from the center two female rows. A primary ear was selected at random near the transect, and then an ear was collected from every tenth plant along the row for a total of 10 ears. Collection continued in the same manner from an adjacent row to provide a total of 20 ears for the location sample. These 20 ears were dried, shelled, and pooled to produce the source of kernels for genetic analysis. Kernels were sized to pass between 26/64 and 16/64 mesh screens to approximate seeds suitable for commercial purposes (Wych, 1988).

Genetic Purity Testing
One hundred seeds from each pooled sample of 20 ears were subjected individually to isozyme analysis by starch gel electrophoresis (Stuber et al., 1988). This analysis was performed at each seed company's quality assurance lab. If a participating company did not possess an electrophoresis lab, private labs were employed for this purpose. Seeds showing two or more unexpected alleles were considered out-crosses in accordance with standard commercial practice (Smith and Register, 1998). Data were reported as the percentage of out-crossed seeds in each 100-seed subsample.

Predictor Variables
Table 1 lists the predictor variables for managing genetic purity considered in this study. YEAR defines the seasons this study was conducted. DIRECTION describes the orientation of the seed field relative to the potential adventitious pollen source. SAMPLE LOCATION is the position along each transect from which a sample was collected. Margin represents values from samples collected at the four locations within 35.8 m of the field edge. The MALE PARENT CLASS is a description of the relative efficiency of the male inbred parent for affecting seed set. Classifications of "Poor," "Fair," or "Good" were provided by the seed companies on the basis of their assessment of absolute pollen production, pollen shed dynamics, or yield performance in seed production fields. This study did not attempt to distinguish between these criteria. ISOLATION DISTANCE is the physical separation between adjacent edges of the seed field and the potential adventitious pollen source. SEED FIELD SIZE is the area of the hybrid seed field containing the seed and pollen parents. BORDER ROWS is the number of pollen parent rows that surround the seed field. ADVENTITIOUS SOURCE SIZE is the area of the potential adventitious source field. The number of male inbred plants per acre is reported as the MALE POPULATION. FIELD BLOCK SIZE is the number of contiguous acres containing the same pollen parent, regardless of seed parent. The field block size was usually the same size as a seed field, but in some cases the block of male parent encompassed several fields. The MALE:FEMALE ratio is the percentage of the field area allocated to the male seed parent. A field planted in a 1:4 male:female row ratio, for example, allocates 20% of the field area to the male pollen parent.

Although data for many of these predictors were collected as continuous variables, analyses also were conducted on predictor classes generated from these data. In some cases, this was done because the limited number of seed fields precluded meaningful analysis. In most cases, however, creating predictor classes simplified data analysis without sacrificing the ability to determine the significance of a response variable in determining the observed level of out-crossing. Natural breaks in predictor variables (defined by predominant management practices) were used to define classes in most cases. When predictors were more normally distributed, the median value was used as a boundary between classes (see Fig. 2).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Distribution of out-crossing values and management variables for the 315 seed production fields analyzed from 1998 to 2000. A: out-crossing values for all sampling locations. B: isolation distances between seed fields and nearest adventitious pollen source. C: seed field sizes. D: adventitious pollen source field sizes. E: number of border rows. F: male/female row ratios. Distributions of the management variables (B–F) were used to define predictor classes for comparison of out-crossing percentages at the field margins and field midpoints.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Figure 2B through F shows the distribution of physical characteristics of isolation associated with the 315 seed fields examined in this study. These include isolation distance (Fig. 2B), seed field size (Fig. 2C), adventitious field size (Fig. 2D), number of border rows (Fig. 2E), and male:female row ratio (Fig. 2F). Although the values for each characteristic vary widely, predominant management options are evident in nearly every case: 50 or 100 m isolation distance, 40 ha field size, 30 or 60 ha adventitious field size, 6 border rows, and 1:4 male:female row ratio. As such, the fields chosen for study provide a fair representation of the management options currently used by hybrid seed field managers.

Management Variables as Predictors of Out-Crossing
Initially, we considered each field management option as an independent continuous variable that could be used to predict the observed levels of out-crossing. Table 2 shows the correlations between these predictor variables and percent out-crossing, as well as correlations among predictor variables. Among the eight predictor variables measured, only Isolation Distance and Field Block Size were correlated significantly (p << 0.05) with percent out-crossing. In both cases, the correlation was negative, indicating the level of out-crossing decreased with greater distance and larger block size. Nonetheless, the correlations were fairly weak (r –0.14) for both predictor variables. Seed field size, male population, and male parent class also were weakly correlated with out-crossing (r = ± 0.10), but the significance of these correlations was not considered compelling, given the size and variability of the data set.

View this table: [in this window] [in a new window]   Table 2. Correlations among continuous predictor variables of out-crossing (r) measured in 315 seed fields sampled in 1998, 1999, and 2000. Correlations significant at p 0.05 are underscored. Values in parentheses: number of transects included in each analysis (n).

The correlation between the Border Rows and Isolation Distance was quite high (r = –0.65) and highly significant (p < 0.0001). Indeed, Fig. 3 shows that field managers tend to use fewer border rows when seed fields are isolated by more than 100 m. But there is considerable variation in both predictor variables at shorter isolation distances. Note that the number of border rows varied from 0 to 24 around seed fields isolated by 100 m, and that 24 border rows were reported around fields isolated by 20 to 100 m.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Relationship between Number of Border Rows and Isolation Distance for 315 seed production fields included in this study. Many points are obscured because of common values in the data.

View this table: [in this window] [in a new window]   Table 3. Logistic regression coefficients and significance tests for predictors of out-crossing at the Field Margin. Year and direction coefficients different from zero, comparisons between coefficients different from zero, predictor main effects and selected interactions significant at p 0.05 are underscored. The model was a marginal logistic regression of the form logit(pij) = b0 + btreat(xij), where Var(Yij) = pij (1 – pij), and Corr(Yij,Yik) = for each variable.

View this table: [in this window] [in a new window]   Table 4. Logistic regression coefficients and significance tests for predictors of out-crossing at the Field Midpoint. Year and direction coefficients different from zero, comparisons between coefficients different from zero, predictor main effects and selected interactions significant at p 0.05 are underscored. The model was a marginal logistic regression of the form logit(pij) = b0 + btreat(xij), where Var(Yij) = pij (1 – pij), and Corr(Yij,Yik) = for each variable.

Table 3 also presents the results of the logistic regression analyses for predictors of out-crossing at the field margin. These data were generated using all samples collected within 38.5 m from the edge of the seed fields. Isolation Distance, Number of Border Rows, and Male Parent Class all were significant (p < 0.05) predictors of out-crossing at the field margin. Two interactions among these predictors also were significant at p < 0.05; Border Rows x Isolation Distance, and Male:Female Ratio x Male Parent Class.

When a similar analysis was conducted for out-crossing data collected at the field midpoint (200 m from the edge of the seed field), none of the management variables tested was a significant predictor of out-crossing (Table 4). A significant (p = 0.011) interaction between Isolation Distance x Border Rows, however, was detected. No other interactions tested were significant (p < 0.05).

In summary, the analysis of eight field management practices as continuous predictor variables of out-crossing revealed that Isolation Distance, Border Rows, and Male Parent Class were significant predictors of out-crossing at the field margin but not at the field midpoint. There was a significant interaction between Isolation Distance and the number of Border Rows for out-crossing at both the field margin and field midpoint. And there was an interaction between Male:Female Row Ratio x Male Pollen Class for out-crossing at the field margin. The greater number of management factors exposed as predictors of out-crossing at the field margins by this analysis likely reflects the greater and more variable levels of out-crossing in these samples.

The interaction between Border Rows and Isolation Distance was examined further by comparing the out-crossing levels among predictor classes generated from the distribution of Border Rows and Isolation Distances within the data set. These classes were created primarily on the basis of natural breaks in the predictor values, but relevance to current field management practices also was considered (Fig. 2). Border Row Classes included fields with fewer than 12 border rows (dominated by those with 6 rows), fields with 12 to 18 border rows, and fields with more than 18 rows. Isolation Distance Classes includes fields less than 100 m from the adventitious pollen source, at 100 m from the source, and those greater than 100 m from the adventitious pollen source. These classes were dominated by two conditions: fields with 12 to 18 border rows and less than 100 m from an adventitious pollen source, and those with less than 12 border rows and at 100 m from an adventitious pollen source (Table 5). Parsing the fields into these Border Row Classes revealed a major impact of border row number on percent out-crossing at the field margin but not at the field midpoint. Fields isolated by less than 100 m and with >18 border rows had lower out-crossing at the field margin than those with fewer border rows (Table 5). At the field midpoint, increasing border rows had little impact on out-crossing in fields isolated by less than 100 m. Apparently, out-crossing was greater in fields with more than 18 border rows and isolated by 100 m. But this result is not reliable, since only 2 of 315 fields met these criteria (Table 5). Taken together with the results of logistic regression analysis (Tables 3 and 4), these data show that border rows have a greater impact on out-crossing at the field margin, which tends to have higher and more variable levels of out-crossing. These data do not support the notion, however, that a large number of border rows will overcome the risk of out-crossing associated with shorter isolation distance from the field midpoint.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Mean out-crossing levels observed at each field sampling location in 1998, 1999, and 2000. Samples were collected at 1.8 m (6 ft), 9.4 m (31 ft), 20.6 m (68 ft), 35.8 m (118 ft), and 200 m (660 ft) from the field edge nearest to the adventitious pollen source. Data are the mean for 60 field transects in 1998, 93 transects in 1999, and 162 transects in 2000. The Y-axis error bars represent the 90% CI. Plotted distances are offset each year for clarity.

Male Parent Class
Male Parent Class (Good, Fair, or Poor) assigned to each male inbred by seed companies typically involves several practical considerations including total pollen production, pollen shed duration, and silk coverage. Averaged across Pollen Parent Classes, Border Rows, and Years, Male Parent Class had a significant effect on the level of out-crossing at both the field margin and field midpoint (Table 8). Seed fields with Poor pollen parents consistently had higher levels of out-crossing than those with male parents classified as Fair or Good. Out-crossing at the field margin was greater than at the field midpoint for all male pollen classes. And nearly twice as much out-crossing was reported at the field midpoint in fields with Poor pollen parents, compared with those with Good pollen parents. Thus, fields with Poor pollen parents had higher levels of out-crossing throughout the field, indicating pollen production was not sufficient to protect the interior of these fields from adventitious pollen sources.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The analyses of out-crossing in 315 seed production fields that flowered synchronously with a nearby adventitious pollen source reinforced the significance of Isolation Distance for maintaining genetic purity of harvested seed. The greater the Isolation Distance between the seed field and the potential adventitious source, the greater the probability of meeting the standard of 99% genetic purity in the harvested seed. The number of Border Rows and Male Parent Class, however, also surfaced as important management factors influencing the production of genetically pure seed at the field margins. There was a significant interaction between Isolation Distance and number of Border Rows as predictors of out-crossing percentage. Border Rows had a large impact on out-crossing at the field margin, the region of the field which tended to have higher and more variable levels of out-crossing. There was little evidence, however, to support the commonly held notion that a large number of border rows will overcome the risk of out-crossing associated with shorter isolation distance for the bulk of the field (i.e., field midpoint).

While these results indicate that current isolation practices often achieve the goal of producing 99% genetically pure seed, higher levels of out-crossing can and do occur, particularly at the field margins (i.e., within 36 m of the field edge). Because out-crossing in this area of the seed field generally is greater and more variable than that observed in the bulk of the field, adjustments to field management that focus on minimizing out-crossing at the field margins should lead to consistently higher levels of genetic purity in hybrid seed production. Currently, the decision to segregate all or a portion of a seed field suspected of having an unacceptable level of out-crossing must be delayed until the genetic purity can be assessed after harvest—as was done in this study. The capability to predict the level of out-crossing on the basis of flowering characteristics of the inbreds and potential adventitious pollen entry would enable this decision to be made soon after pollination. Modeling approaches are now available to help seed field managers make this decision in a timely manner (Lizaso et al., 2003; Westgate et al., 2003, 2005; Fonseca et al., 2004).

	ACKNOWLEDGMENTS

 
The authors are especially grateful to Pioneer—A Dupont Company and their cooperating seed plant personnel for 3 yr of seed field data collection. Special thanks to Dave Shoultz and to Dave Langer for initiating and supporting this project. Thanks is extended to the American Association of Seed Certifying Agencies, American Seed Trade Association, and the U.S. EPA for their support and funding of the inter-industry portion of this study. Thanks to the following companies for 1 yr of seed field data collection: Burrus Brothers & Associated Growers, Cargill Inc., Curry Seed Co. Inc., Dairyland Seed Co., Inc., Fontanelle Hybrids, Garst Seed Co., Inc., Golden Harvest, J.C. Robinson, Inc., Great Lakes Hybrids, Inc., Kaltenberg Seed Farms, LG Seeds, Monsanto Seed Co., Inc., Mycogen Seeds, Inc., Syngenta Seeds, Inc. and Wyffels Hybrids, Inc. Special recognition is also due Dave Shoultz for his suggestion and support of this inter-industry project from its inception. Additionally, we would be remiss not to thank the following project team members who provided guidance and supported these efforts: Ace Catlett, Gary Lawrance, Dan Smith, Jerry Tank, Bob Wych, and Bob York. Thank you also to Greg Mangold and the Beal Seed Testing laboratory at Pioneer Hi-Bred International, Inc. for their significant contribution in electrophoresis testing. And finally, thank you to Kerry Mounsey, Scott Koenigsfeld, and Kent Mowrer for their support and assistance throughout this endeavor. The authors also gratefully acknowledge the assistance of Bruce Craig, Department of Statistics at Purdue University, and Jodi Edwards, USDA-ARS at Ames, IA, for their guidance regarding data analysis.

Received for publication January 7, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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