Characterization of Drought-Tolerant Maize MON ...

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Characterization of Drought-Tolerant Maize MON 87460 for Use in Environmental Risk Assessment Bernard Sammons,* Joy Whitsel, LeAnna G. Stork, William Reeves, and Michael Horak

Abstract MON 87460 is a maize (Zea mays L.) product that expresses cold shock protein B to impart drought tolerance. Here we describe our evaluation of MON 87460 for agronomic and phenotypic parameters, ecological interactions, reactions to abiotic stressors, ability to persist in uncultivated areas, root growth, and acrossseason water use compared with those of a conventional control hybrid with a similar genetic background. These data were used for the environmental risk assessment of MON 87460, a process required for commercialization of any new genetically modified crop. Any statistically significant differences were considered in the context of the genetic variation known to occur in maize and were assessed for their potential impact on plant pest (weed) potential and their potential environmental impact. The environmental risk assessment included several product-specific studies to assess the trait for unintended effects such as tolerance to stresses other than drought. The results of these studies revealed no effects of the genetic modification that would result in increased pest potential or adverse environmental impact of MON 87460 compared with a conventional control and no evidence for pleiotropic effects. The results of the plant characterization studies and the subsequent environmental risk assessment support the conclusion that the environmental risks associated with cultivation or import of MON 87460 are no different from the risks associated with conventional maize.

Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63141. Received 10 July 2013. *Corresponding author (bernard.sammons@ monsanto.com). Abbreviations: CSPB, cold shock protein B; DAT, days after treatment; GM, genetically modified; NPTII, Neomycin phosphotransferase II.

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rought stress is a major cause of yield reduction in maize (Zea mays L.), and its effects have far-reaching global socioeconomic implications. In both temperate and tropical regions suitable for commercial maize production globally, the average annual maize yield loss attributable to moderate water deficits is approximately 15% (Barker et al., 2005). During periods of severe drought, these losses can be much higher and can potentially result in complete crop failure. Climate change is expected to increase the frequency of adverse growing conditions. In a 2008 report, the U.S. Climate Change Science Program concluded that a combination of increased temperature variability, altered rainfall patterns, and the resulting increases in drought frequency will magnify yield variability (CCSP, 2008; Hatfield et al., 2008). Consequently, improving drought tolerance in maize is a major goal of plant-breeding efforts. Advances in conventional plant breeding and agronomic practices have made contributions to improving drought tolerance and yield potential of maize. Nearly all conventional maize hybrids currently on the market have been bred to exhibit some degree of drought tolerance. Drought tolerance traits introduced via biotechnology and used in combination with conventional breeding and appropriate agronomic practices are anticipated to further improve maize yield stability under water-limited conditions.

Published in Crop Sci. 54:719–729 (2014). doi: 10.2135/cropsci2013.07.0452 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

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Monsanto has developed MON 87460, a maize product that can exhibit reduced yield loss under water-limited conditions (Monsanto Company, 2009; USDA-APHIS, 2011). MON 87460 is the first abiotic stress–tolerant crop plant produced through biotechnology to achieve commercial introduction. Like conventional maize, MON 87460 is still subject to yield loss under water-limited conditions, but the impacts can be less severe on MON 87460 than on conventional maize. Under severe water deficit, grain yields for both MON 87460 and conventional maize can be reduced to zero. MON 87460 expresses cold shock protein B (CSPB) from Bacillus subtilis and neomycin phosphotransferase II (NPTII) from Escherichia coli, which served as the selectable marker to accelerate transformation and event selection. In bacteria, cold shock proteins are believed to help preserve normal cellular functions during certain stresses by binding cellular RNA and maintaining RNA stability and translation. As in bacteria, the CSPB protein in MON 87460 binds RNA and appears to help maintain plant cellular functions under stress conditions (Monsanto Company, 2009).

Environmental Risk Assessment of MON 87460 A scientifically sound environmental risk assessment is required for all genetically modified (GM) crops before unrestricted release into the environment (Nickson, 2008). The scientific principles underlying the environmental risk assessments completed for herbicide-tolerant and insect-protected GM crops commercialized to date are now being applied to crops that are modified for improved tolerance to abiotic stress (Nickson, 2008). For MON 87460, the assessment included the standard plant characterization studies conducted for any new biotechnology-derived maize product, that is, phenotypic and agronomic field evaluations and volunteer-potential studies (reported here), as well as studies of pollen viability and morphology, seed dormancy, and seed germination (reported in a petition for deregulation to USDA [Monsanto Company, 2009]). In addition, the environmental risk assessment for MON 87460 included six studies that were developed or modified on the basis of the nature of the trait. These additional studies included assessments for persistence outside cultivation; root growth and development; and drought, cold, heat, and salt tolerance. The endpoints considered in these studies were established to determine whether MON 87460 exhibited any differences from conventional corn that would indicate the trait posed a hazard requiring further evaluation. These studies were not intended to investigate differences that could describe how CSPB functions in MON 87460. Monsanto’s petition for deregulation to the USDA (Monsanto Company, 2009), Monsanto’s submission to Japan’s Ministry of Agriculture, Forestry and Fisheries/Ministry 720

of the Environment (Monsanto Japan Limited, 2012), and Castiglioni et al. (2008) present data developed to describe the function of bacterial CSPs expressed in plants. Here we describe the regulatory strategy used to support the environmental risk assessment of MON 87460, and we discuss our approach to problem formulation used to design the assessment of this product. We then present the studies and plant characterization data needed to support the environmental risk assessment for MON 87460. This report describes the first application of previously defined environmental risk strategies applied in the commercial development of drought-tolerant maize.

Problem Formulation The initial steps in an environmental risk assessment are focused on problem formulation, which involves defining assessment endpoints and developing a conceptual model and an analysis plan (USEPA, 1998). Assessment endpoints are designed on the basis of protection goals, and for MON 87460, these goals include protecting farmlands by preventing the introduction and dissemination of plant pests (weedy or invasive pests) in the United States as described in the Plant Protection Act (USDA, 2000). The assessment endpoints used in the environmental risk assessment for MON 87460 were summarized in comparative measured endpoints (plant characterization studies) conducted in the field, greenhouse, or growth chamber. Consistent with the environmental risk assessments conducted for previously approved agronomic traits, the measured endpoints were used to assess for altered plant pest (weed) potential and for adverse environmental impacts of the drought tolerance trait. Because MON 87460 expresses CSPB, which has been shown to improve stress adaptation in plants (Castiglioni et al., 2008), studies to assess for altered stress tolerance were also conducted to identify possible pleiotropic effects in maize that expresses CSPB. The product concept developed for MON 87460 is that maize expressing this trait can tolerate water-limited conditions and can provide a yield benefit compared with control plants. Other responses not consistent with the mode of action could be considered as unintended effects and would be assessed in terms of potential adverse environmental impacts. The current model for assessing risk is that risk is a function of defined harm (hazard) and its likelihood of occurrence (exposure; Conner et al., 2003; USEPA, 1998). With deregulated status granted for MON 87460 by USDA–APHIS, the likelihood of occurrence (exposure) of the trait is high because it could be cultivated on a wide scale in maize-producing regions. Thus, the risk assessment for MON 87460 focused on the potential harm (hazard) of the MON 87460 trait.

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Table 1. Characteristics measured for phenotypic, agronomic, and environmental interactions assessment of MON 87460. Category Plant phenotypic and agronomic characteristics

Plant environmental interactions

†

Evaluation description (measurement endpoints)

Characteristics measured

Evaluation timing

Seedling vigor

Stage V2–V4

Rated on 0–9 scale, where 0 = dead and 9 = above-average vigor

Early stand count

Stage V2–V4

Number of emerged plants per plot

Final stand count

Preharvest

Number of plants per plot

Stay green

Maturity

Rated on a 0–9 scale, where 0 = entire plant is dried and 9 = entire plant is green

Ear height

Maturity

Distance from the soil surface at the base of the plant to the ear attachment node

Plant height

Maturity

Distance from the soil surface at the base of the plant to the flag leaf collar

Stalk-lodged plants

Preharvest

Number of plants per plot broken below the ear

Root-lodged plants

Preharvest

Number of plants per plot leaning at the soil surface at >30° from the vertical

Days to 50% pollen shed

Pollen shed

Days from planting until 50% of the plants have begun to shed pollen

Days to 50% silking

Silking

Days from planting until 50% of the plants have silks exposed

Root growth and development

VT–R2

Root biomass, root:shoot biomass ratio, water use efficiency

Grain moisture

Harvest

Moisture percentage of harvested shelled grain

Test weight (lb/bu)

Harvest

Test weight of harvested shelled grain

Yield (bu/ac or Mg/ha)

Harvest

Harvested weight of shelled grain, adjusted to 15.5% moisture

Dropped ears

Preharvest

Number of mature ears dropped from plants

Insect, disease, and abiotic stressors from field

Variable, from planting to harvest†

Qualitative assessment of each plot, with rating on a 0–9 scale‡

Abiotic stress tolerance to drought, cold, heat, and salt from greenhouse or growth chamber

Stage V2–V6

Measurements included plant height, growth stage, vigor, chlorophyll content, and biomass; leaf rolling rated on a 1–5 scale where 1 = no rolling and 5 = severe

Soil water relations

Across the season

Soil moisture depletion in mm water

Persistence within cultivation (volunteer potential)

After fall planting and the following spring

Number of plants present as volunteer maize in plots

Persistence outside cultivation

Variable, from planting to harvest

Variable, phenotypic assessments from planting to harvest that included early and final stand counts, vigor ratings, plant height, and number of ears and seed per plot

Plant response to arthropod damage, disease damage, and abiotic stressors was evaluated at four growth stages: V2–V4, V10–V15, VT–R5, and R6. For arthropod and abiotic stressor damage, an observational rating scale was used where 0 = none (no symptoms observed), 1–3 = slight (symptoms not damaging to plant development), 4–6 = moderate (intermediate between slight and severe), and 7–9 = severe (symptoms damaging to plant development). For disease damage, plants were visually assessed before harvest for ear and kernel rot and for stalk rot.

‡.

Hazard Assessment Four potential hazards were considered in this assessment: (i) the potential for MON 87460 to become a weed of agriculture by persisting in areas managed for cultivation, (ii) the potential for MON 87460 to become a weed of nature, (iii) the potential for adverse environmental impact on arthropods and other nontarget organisms, and (iv) the potential for the drought tolerance trait to cause increased use of soil water and limit future crop choices. Table 1 presents a list of characteristics measured as part of the phenotypic, agronomic, and environmental interactions assessment of MON 87460. Included among these characteristics is a set of assessment endpoints (USEPA, 1998) that allowed a determination of whether MON 87460 is crop science, vol. 54, march– april 2014

likely to pose an environmental risk different from that of conventional maize with respect to the four potential hazards being assessed. The number of stalk-lodged and root-lodged plants and the number of dropped ears allowed a determination of whether MON 87460 is more or less likely to return grain to the soil that could serve as the source of future volunteer populations. Observations collected during studies of persistence within and outside cultivation provided a direct assessment of whether the drought tolerance trait alters the potential for unharvested maize grain to germinate and compete with subsequent crops or nonagricultural plant populations. Qualitative assessments of plant interactions with insects, disease, and abiotic stressors in the field provided an indication

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of whether the drought tolerance trait alters the relationship between maize and its biotic and abiotic environment. Assessing the tolerance of young plants to drought, cold, heat, and salt stresses in greenhouse or growth chamber conditions allowed a determination of whether the drought tolerance trait confers a fitness advantage that could make MON 87460 more competitive under adverse conditions. Studies of root growth and development and soil water relations provided an understanding of whether the drought tolerance trait is likely to cause maize plants to extract more water from the soil and affect future crop choices. The remaining endpoints in Table 1 are measurement endpoints that provide an agronomic comparison between MON 87460 and conventional maize. The unique nature of the drought tolerance trait necessitated data collection under both well-watered and water-limited conditions. Data collected under wellwatered conditions provided an analysis of MON 87460 in the absence of trait bias, where the hypothesis was that no differences between MON 87460 and the conventional control were expected. Data collected under water-limited conditions provided an analysis of MON 87460 under conditions likely to produce trait bias, where the hypothesis was that a yield advantage for MON 87460 compared with the conventional control could be expected. Analysis under this paradigm provided an opportunity to assess whether the interaction between the drought tolerance trait and the environment could result in an increased environmental risk compared with conventional maize.

MATERIALS AND METHODS Plant Materials

Each study included MON 87460, a conventional isogenic control hybrid, and reference hybrids, as described in the Codex Alimentarius guidelines (Codex Alimentarius, 2003). The reference hybrids were commercially available and varied by study, thereby providing a range of background values common to commercial maize for the assessed characteristics. MON 87460 was produced by Agrobacterium-mediated transformation. The genetic insert in MON 87460 contains the rice (Oryza sativa L.) actin1 promoter (McElroy et al., 1990), linked to a rice actin1 intron (McElroy et al., 1991). The native coding region for cspB (GenBank accession number U58859) was introduced, with the exception that for cloning convenience the codon for the second amino acid was modified, changing the encoded amino acid from leucine to valine (Monsanto Company, 2009).

Ecological Assessments Eight different types of studies reported here were conducted in the field, greenhouse, or growth chamber to assess the environmental risk of MON 87460: agronomic and phenotypic field trials with ecological interaction data; persistence within cultivation (volunteer potential); persistence outside cultivation; root growth and development; and drought, cold, heat, and salt-stress tolerance.

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Well-Watered and Water-Limited Phenotypic and Agronomic Field Trial with Assessment of Ecological Interactions From December 2006 through June 2007, a field study was established under well-watered and water-limited conditions at four sites in Chile: Calera de Tango, Colina, Lumbreras, and Quillota (Supplemental Methods; Supplemental Figs. S1– S5). The sites are in the major crop-growing region of central Chile, which is characterized by a Mediterranean (dry-summer subtropical) climate (Vera, 2006), and irrigation is needed to maintain crops. The experiment was established at each site in a strip-plot design with three replications. Irrigation treatment was the whole plot, and hybrid (MON 87460, control, and four references) was the subplot, which was randomized in strips across the treatments. Plots were six rows, 4.5 m × 4.7 m in area, and were planted at a density of approximately 79,000 plants/ha. The whole plots (irrigation treatment) were separated by a 3-m buffer of conventional maize to minimize lateral flow between treatments. Phenotypic and ecological interactions data were collected from Rows 4 and 5 of each plot. The phenotypic, agronomic, and ecological interaction characteristics that were evaluated at each site are shown in Table 1. No precipitation occurred at any site during the crop-growing cycle. The well-watered (nonstress) treatment was managed to achieve optimal grain yield, and plants received approximately 2.5 cm of irrigation water every 3 d from planting through R6. The water-limited (stress) treatment was managed to impose water restrictions during the late vegetative to early grain-fill period, when maize is most susceptible to yield loss from drought stress (Campos et al., 2006). Plants received approximately 2.5 cm of irrigation water every 3 d from planting until approximately V10. From approximately V10 through approximately R2, less than 2.5 cm of water was applied. Irrigation for the water-limited treatment resumed at R2 and continued through R6 (Supplemental Figs. S2– S5). Successful water management was required for this study. To determine if a differential water treatment was achieved between the well-watered and water-limited irrigation treatments, each site was evaluated in a stepwise method using predetermined criteria for site inclusion on the basis of successful water management (Supplemental Methods; Supplemental Fig. S1). Confirmation of successful water management was based on site water records, water monitoring records, and the response of the conventional reference hybrids that did not express the CSPB trait. Indicators of water limitation in the reference hybrids included reduced plant height, reduced ear height, delayed silking, and a yield reduction of ³15% compared with the reference hybrids that received the wellwatered treatment. Only sites that met these criteria were included in the combined-site plant characterization analysis for MON 87460 (Supplemental Fig. S1 and Supplemental Table S1). Statistical comparisons between MON 87460 and the conventional control were conducted within each irrigation treatment.

Persistence within Cultivation (Volunteer Potential) In November 2006 the volunteer potential of MON 87460, the conventional control, and six reference hybrids was evaluated at three locations in the United States: Jefferson County, IA; Guthrie County, IA; and Parke County, IN. These sites are in the US Corn Belt and had been used for testing during the 2006 summer

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season. At each site, plots were established in a randomized complete block design with three replications. Approximately 200 F2 seed of each entry were scattered on the surface of a 6.1-m × 1.5-m plot and incorporated into the soil with a disc or field cultivator. The number of plants per plot was counted in fall 2006 until soil temperatures fell below 10°C and resumed again in spring 2007 when environmental conditions became favorable for germination and emergence. Monitoring ended in mid-June 2007.

Persistence Outside Cultivation To assess the ability of MON 87460 to persist and establish populations in areas not cultivated for maize production, phenotypic and agronomic characteristics encompassing plant growth, development, and yield were assessed for MON 87460, the control, and the references in unmanaged environments. Four sites were established in 2007 (Effingham County, IL; Shelby County, MO; York County, NE; and Carson County, TX) and in 2008 (Effingham County, IL; Boone County, IN; Rapides Parish, LA; and York County, NE). These sites were chosen to represent a diverse set of noncultivated land areas (e.g., set-aside ground or pasture) found near areas of agricultural production. Each site was unmanaged and received no agricultural inputs (i.e., no fertilizers, tillage, or herbicides), allowing MON 87460, the conventional control, and the three reference maize hybrids to compete with existing vegetation and be subject to abiotic and biotic stressors present in each environment. The experiment was established at each site in a randomized complete block design with three replications. One hundred F2 seeds of each entry were planted in each four-row, 3-m × 3-m plot using methods appropriate to the site (e.g., no-till planting equipment or by hand). Early and final stand count, number of ears produced per plot, number of ears produced per plant, and seed produced per plot were determined. Additionally, replacement values (Rissler and Mellon, 1996) were calculated for each environment as the ratio of the number of seeds produced to the number of seeds sown.

Soil Water Relations Field trials were conducted in 2007 and 2008 in Yolo County, CA, which is in the Central Valley and is characterized as a Mediterranean climate (Norton, 2001), where irrigation in the summer months is necessary to produce a viable crop. Capacitance soil moisture probes (EnviroSMART, Sentek) were used to characterize the depletion of moisture from the soil profile during the growing season. In both 2007 and 2008, plots were established in either a water-limited or well-watered treatment using a separate randomized complete block design. The wellwatered treatment was managed to provide optimal grain yield by targeting replacement of 100% crop evapotranspiration (ET) from planting through physiological maturity. The water-limited treatment was managed to impose water-limited conditions during the late vegetative through early grain-fill growth stages (~V10–R3). Rescue irrigations were provided as needed in the water-limited treatment to prevent crop failure. The goal of the water-limited treatments was to reduce plant growth and yield by up to 50% compared with plants in the well-watered treatment. Throughout the remainder of the growing season (i.e., before stress and after stress), irrigation targeted 100% ET replacement. No precipitation occurred at any site after treatment initiation, which is defined at each site as approximately crop science, vol. 54, march– april 2014

the V8 to V10 growth stage (when water was withheld from the water-limited treatment) up to and including approximately the R3 growth stage. In the 2007 study, 10 replicates each of MON 87460 and the control were grown under water-limited conditions from the V7 to R2 growth stages. In addition, five replicates each of MON 87460 and the control were grown under well-watered conditions. In the 2008 study, 20 replicates each of MON 87460 and the control were grown under water-limited conditions from the V10 to V14 growth stages, and five replicates each of MON 87460 and the control were grown under well-watered conditions. Each plot consisted of eight 6.5-m rows planted with 50 kernels per row, with a planting density of approximately 88,000 plants/ha in both years. In both the 2007 and 2008 studies, border rows were grown between treatments to ensure that water did not migrate from the well-watered plots to the water-limited plots. In both years, the soil moisture probes were installed within the plots just after stand establishment. The probes had five sensors at soil depths of 20, 30, 50, 70, and 100 cm that monitored the available soil moisture within a 10-cm radius from each sensor. Probes were installed in 10 replicates each of MON 87460 and the control in the water-limited treatment block, and in 5 replicates each for MON 87460 and the control in the well-watered treatment block. Cumulative soil moisture depletion calculations were made by summation of the daily soil moisture depletion values over the course of the growing season. Grain was harvested using a combine, and the weight of grain on a plot basis was used to calculate yield.

Root Growth and Development The effect of water-limited conditions on water use efficiency and the growth and development of roots of MON 87460 compared with a conventional control was assessed in a greenhouse. MON 87460 and control plants were established in pots 1-m tall × 20-cm diameter filled with Turface Field and Fairway artificial soil medium. The greenhouse was maintained at approximately 30°C day temperature and 20°C night temperature, with supplemental lighting provided as needed. The experimental design was a complete factorial treatment structure (2 × 2) in a randomized complete block with 12 replications. The factors were irrigation treatment (well watered or water limited) and hybrid type (MON 87460 or control). Irrigation treatments were managed by volumetric water content (VWC), where well-watered pots were maintained at 56% VWC for the entirety of the study and waterlimited pots were maintained at 56% VWC before stress imposition and 30 to 40% VWC on stress imposition. Volumetric water content was the percentage of the pot volume occupied with water and was maintained by weight, where the target weight at each irrigation event = [(pot weight) + (dry planting medium weight) + (mass of target volume of water)]. Stress was imposed on the water-limited treatment from the V7 growth stage for 25 d after treatment (DAT), when the plants in the well-watered treatment reached approximately the VT to R1 growth stage. Plant height and growth stage were evaluated before treatment, 13 DAT, and at harvest (22 DAT). Dry weights of shoot, root, and ear biomass were measured 25 DAT, and whole-plant water use efficiency (defined as total biomass produced divided by water consumed), root:shoot ratio, and total water used were calculated.

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Table 2. Treatments for each abiotic stress tolerance assay. Cold tolerance (growth chamber) Treatment level Optimal Mild Moderate Severe

Heat tolerance (growth chamber)

Drought Salt tolerance tolerance (greenhouse) (greenhouse)

Day, night† Day, night† AWHC target temptempfor each erature erature irrigation‡ 30, 22°C 20, 15°C 15, 10°C 4, 4°C

30, 22°C 40, 35°C 43, 35°C 47, 35°C

98–100% 73–81% 56–60% 45–50%

Soil EC target dS/m§ 0.05) were detected between MON 87460 and the control for any of the assessed phenotypic characteristics in the well-watered treatment (Table 3). In the combined-site analysis of the water-limited treatment, no significant differences (p > 0.05) were detected between MON 87460 and the control for any of the assessed phenotypic characteristics, with the exception of yield (Table 3). MON 87460 exhibited higher yield (p £ 0.05) than the conventional control (7.2 vs. 5.4 Mg/ha) as expected owing to efficacy of the drought tolerance trait under water-limited conditions. A total of 87 ecological interaction qualitative comparisons were made in each of the water treatments. No differences in any ecological stressor were observed between MON 87460 and the control in the well-watered or water-limited treatments (Table 3). Observations included assessments of abiotic stressors (cold, hail, heat, and nitrogen deficiency), disease stressors (ear rot, Fusarium, gray leaf spot, leaf blights, root rot, rust, seedling blight, smut, and stalk rot), and arthropod stressors (aphids, seedcorn maggots, thrips, and wireworms) (Monsanto Company, 2009).

Persistence Within and Outside Cultivation In some crops, seed remaining in the field after harvest has the potential to overwinter and volunteer in the subsequent cropping season. The purpose of the persistence within cultivation study was to assess for differences between MON 87460 and the conventional control to overwinter and establish as volunteers. Second, although it is known that modernday maize cannot survive outside cultivation (OECD, 2003), studies of persistence outside cultivation were conducted to crop science, vol. 54, march– april 2014

assess whether the ability of plants of MON 87460 to survive in unmanaged, competitive environments had changed compared with a conventional maize control. No volunteer plants were observed at any site or observation time during the fall or spring, indicating that MON 87460 seed did not exhibit changes in potential to volunteer (Table 3). In the persistence outside cultivation studies conducted in 2007 and 2008, there were two statistically significant differences detected in the 2007 study (Tables 3 and 4). At one of the four sites, early and final stand counts were higher for MON 87460 compared with the control. However, the critical assessment for the persistence outside cultivation study is the calculated replacement value and associated interpretation regarding persistence of maize in an environment that was not managed for cultivation. Grain was produced at a single site in each year for both MON 87460 and the control, and replacement values were calculated. Replacement value is the ratio of seeds produced to seeds planted; thus, a value of 1 indicates that a population is neither increasing nor declining. In both cases, the values were much less than 1.0 (Tables 4 and 5), indicating that the populations in both years were declining and did not persist. For grain generated in the 2008 study, zero kernels germinated in a subsequent germination assay (data not shown), indicating that no viable kernels were produced in the study.

Soil Water Relations and Root Growth and Development To assess for differences in season-long water use, a comparative assessment of moisture depletion was conducted for MON 87460 compared with a conventional control

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Table 4. Persistence outside cultivation—2007. Early stand count (plants/plot)

Final stand count (plants/plot)

No. ears produced (ears/plot)

Mean ±SE

Mean ±SE

Mean ±SE

†

Site‡

MON 87460

Control

Reference range§

IL MO NE TX

53.0 ±2.08 41.3* ±1.76 2.3 ±2.33 –

57.3 ±5.24 24.0 ±3.06 0.3 ±0.33 –

30.7–48.3 21.3–30.3 0.0–1.0 –

MON 87460 16.7 ±1.45 38.3* ±1.67 1.0 ±1.00 –

Control

Reference range

MON 87460

Control

Reference range

22.0 ±1.15 21.0 ±2.31 0.0 ±0.00 –

9.3–19.3 19.3–28.0 0.0–0.3 –

– 8.7 ±3.18 – –

– 3.7 ±1.45 – –

– 3.0–10.7 – –

No. seed produced (seed/plot)

Average no. ears (ears/plant)

Mean ±SE

Mean ±SE

Replacement value¶

Site

MON 87460

Control

Reference range

MON 87460

Control

Reference range

MON 87460

Control

Reference range

IL MO NE TX

– 7.7 ±1.86 – –

– 1.3 ±0.88 – –

– 2.3–28.3 – –

– 0.2 ±0.08 – –

– 0.2 ±0.05 – –

– 0.2–0.4 – –

0 0.15 0 0

0 0.03 0 0

0.0–0.0 0.05–0.57 0.0–0.0 0.0–0.0

*Indicates significant differences detected between MON 87460 and the control (p £ 0.05). †

SE, standard error; n = 3 for MON 87460 and control at each site. Dash (–) indicates no data available.

‡

Site codes: IL = Effingham County, IL; MO = Shelby County, MO; NE = York County, NE; TX = Carson County, TX.

§

Reference range was calculated from the minimum and maximum mean values from among the reference hybrids.

¶

Because of seed counting errors, the exact number of seed sown in each plot varied. Therefore, replacement values at the MO site were conservatively based on 50 seeds sown per plot by using the formula Replacement Value = (Number of seeds produced)/50. Replacement values were not statistically analyzed. Replacement values