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Eastern Cereal and Oilseed Research Center (ECORC), Central Experimental Farm, Research Branch, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, Canada, K1A 0C6
* Corresponding author (mab{at}agr.gc.ca).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1% out-cross. The generally recommended 200-m distance between two genotypes (inbreds, populations, hybrids, and wild relatives) appears to be appropriate for Bt or other GM maize, as well.
Abbreviations: Bt, Bacillus thuringiensis • CHU, crop heat unit • GM, genetically modified
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maize pollen grains are one of the heaviest and largest (about 90–100 µm in diameter) among the wind-dispersed pollen grains, thus limiting the distance maize pollen can travel (Raynor et al., 1972; Burris, 2001). Under natural conditions, the majority of pollen grains from a plant are normally assumed to fall within the row space. It is also suspected that a small amount of pollen can be transported over longer distances given favorable wind speeds and appropriate humidity (Kiesselbach, 1949; Garcia et al., 1998). Raynor et al. (1972) recorded only 0.2% pollen deposition per unit area at 60 m from the original source. This is in agreement with the earlier finding of Bateman (1947) that only 1% of the pollen grains at source was found at 27 m. Luna et al. (2001) reported that cross-fertilization in maize could occur at a maximum distance of 200 m from the source. Other biological factors such as pollen density, pollen radius, and sedimentation velocity are also important factors in determining the distance of the pollen drift (Luna et al., 2001). Maize pollen generally remains viable only for 1 to 2 h after dehiscence (Luna et al., 2001). However, depending on the environmental factors, mainly temperature (Goss, 1968; Schoper et al., 1987; Jemison and Vayda, 2001), humidity (Goss, 1968; Barnabas, 1984; Garcia et al., 998; Traore et al., 2000; Jemison and Vayda, 2001) and atmospheric water potential (Luna et al., 2001), it may remain viable for up to 24 h after shed. Cool temperatures and high humidity favor pollen longevity.
The issue of the degree of pollen dispersal and cross-fertilization between maize genotypes has become increasingly important with the recent and continued release of new transgenic maize hybrids. Several transgenic hybrids have been developed with herbicide tolerance or pest resistance, and are now commercially cultivated. One of the most common examples is the maize transformed with a gene from the bacterium Bacillus thuringiensis to express the insecticidal 1 epidopteran-active crystalline protein (Cry1Ab) endotoxin for the control of European corn borer [Ostrinia nubilalis (Hübner)] (Koziel et al., 1993). Such genes can be naturally transferred to conventional (non-Bt) genotypes in adjacent fields via pollen dispersal. This "loss of control" over the engineered gene is one of the most discussed environmental effects associated with the use of transgenic plants (Scriber, 2001; Saeglitz and Bartsch, 2001). For maize producers, the major issue is that contamination of conventional hybrids by pollen from neighboring transgenic hybrids will restrict the marketing of the grain harvested from the contaminated field. Grain harvested from a contaminated conventional field is essentially declared as transgenic, will not be accepted at all grain elevators and will have to be channeled to specific elevators, processors and even countries who will accept transgenic grain. Thus, there is an urgent need to understand the pollen-mediated gene flow, and the minimum distance required to isolate conventional maize hybrids from neighboring GM maize fields.
Pollen, as the carrier of the male gamete, is an important vector of gene flow from one plant to another. The maintenance of genetic purity in cross-pollinated plants is the most important issue for hybrid and breeder's seed production (Jones and Brooks, 1950; Burris, 2001). Out-crossing in maize varieties is prevented either by time isolation (temporal) or distance isolation (spatial). An isolation distance of 185 to 200 m is recommended between two maize fields for seed production (Luna et al., 2001). Garcia et al. (1998) found complete pollen control at a distance of >184 m. They observed that most of the pollen settled on the soil surface within the source field itself. We, therefore, hypothesized that production of non-GM maize is possible by removing the outside rows of non-GM maize plants adjacent to the GM maize field. For seed production, the recommended distance of 200 m between two genotypes (inbreds, populations, hybrids, and wild relatives) was also appropriate for Bt or other GM maize, as well.
The endosperm of maize kernels can be yellow or white. These colors are easily observable and can be used as markers in genetic studies or tools for evaluating cross-fertilization. It is possible to determine the degree of out-cross between genotypes, by planting a white-kernel hybrid next to a yellow hybrid (Wicks and Mack, 1996). The objective of this study was to determine the extent of cross-fertilization of a maize genotype by foreign pollen from neighboring maize fields. Specifically, by growing white-kernel, non-Bt maize adjacent to yellow kernel Bt maize hybrids sided by side as a model system, we determined the effects of synchronization of the receptive silking period of a maize genotype with the pollination of the other maize hybrids, weather conditions during the flowering period, and distance between the hybrids on the rate of cross-fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Field Experiment
At all sites, soil samples (0–30 cm) were taken before planting each year to determine soil nutrient level and general properties to ensure adequate fertilizer applications. Adequate phosphorus (P) and potassium (K) were applied during the land preparation according to the soil test recommendations. Fertilizer urea at 200 kg N ha–1 and herbicide [Fieldstar (flumetsulam/clopyralid) 216 g ha–1 + Primextra Lite (s-metolachlor/atrazine) 3.3 L ha–1] mixtures were applied and incorporated into the soil before planting at each site in all years. The maize was planted at a density of 73000 plants ha–1 in 76.2-cm row spacing in a north–south row orientation. In each field, the yellow Bt maize was planted in the center (36 rows of 27 by 27 m) while a white maize hybrid was planted in the surroundings to fill a total area of 1 ha (100 by 100 m) for Site #1 and 0.68 ha (82.3 by 82.3 m) for the other two sites (Fig. 1)
. In this region, the prevailing wind in July and August is generally assumed from the northwest direction. Therefore, the white maize planted in the east and south direction of the yellow Bt maize was assumed to be in the "downwind" direction and designated as downwind, while the white maize in the west and north direction was upwind. Hybrids and planting dates for each site-year are listed in Table 1. In 2000, for Site #1, the hybrids for which the flowering dates of yellow and white maize were synchronized better than for the other sites. In the other years, more synchronised hybrid pairs were chosen. In Sites #2 and #3, the pairs of white and yellow kernel hybrids were planted on the same day as they had similar silking dates. In addition, to provide white maize with an extended period of pollen availability of yellow kernel maize, mixtures of two yellow hybrids with differences up to 60 Crop Heat Units (CHU; Brown and Bootsma, 1993) were used in 2002 in Sites #2 and #3 (Table 1). Phenological progressions of both hybrids at each site were recorded. A plant was considered to be at pollination stage if at least one anther was releasing pollen as checked daily before 1000 h. Similarly, date of silking stage was recorded if at least one silk was emerged from the sheath. Fields were monitored daily from the beginning of tasseling and silking, and times taken to pollen-shed and silking in 50% plants were recorded.
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At maturity, a systematic sampling of the white maize was conducted to determine the pattern and extent of cross-fertilization of white maize by pollen from neighboring yellow Bt hybrid. In Site #1, in both downwind and upwind directions, ears of white maize were sampled from rows No. 1 (the first row of white kernel maize adjacent to the yellow kernel maize), 7, 13, 19, 25, 31, 37, 43, and 48 (37 m) bordering the yellow Bt maize. These rows were chosen on the basis of the fact that the dominant maize header in Ontario is six rows wide, thus contamination after potentially removing 0, 1, 2 ... 6 header widths could be evaluated. In addition, 36 rows represent three passes of a 12-row maize planter and perhaps the widest isolation buffer maize growers would adopt. In the north and south ends of yellow Bt maize rows, white maize ear samples were taken from rows 1, 7, 13, 19, 25, and 33 (row number was arbitrarily defined, but plant number was counted always starting from the white kernel plant bordering the yellow kernel maize). Sampling scheme was the same in Sites #2 and #3 except that there were only 24 rows of white maize in the assumed upwind directions. In all designated rows, ears from every 10th plant of white maize (i.e., 1st, 11th, 21st, and so on) were collected for a total of 47 to 57 ears per row, marked and stored in onion bags for air-drying before counting the kernels. The relative distance to the yellow maize determined the position of all sampled plants in the field. A plant was considered as 0% cross-fertilization if there were no yellow-colored kernels in the sampled ear as well as in the ears of two adjacent plants (e.g., if the target was an 11th plant, plants 10th, 11th, and 12th were also field-checked to ensure no yellow kernels were present). In the straight south and north sides of the yellow maize rows, every 10th plant of white maize from Rows 1, 7, 13, 19, 25, and 31 was sampled starting from the 1st plant in each sampling row. In this way, a total of about 2800 ears were collected each year except in year 2002 when Site #1 was abandoned due to severe drought and rootworm damage. Within a single ear, the total number of rows, number of kernels per row and the total number of yellow kernels per ear were counted. Percent out-cross was calculated as the number of yellow kernels divided by the total number of kernels (white+yellow) per ear.
The cross-fertilization data with distance to the pollen source of yellow Bt maize were fitted to an exponential equation:
 | [1] |
And a modified exponential equation:
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Means and standard deviations (STD) of cross-fertilization of individual rows were calculated and presented. Synchronization of pollen donor and silking receptor, and patterns of average kernel number, barrenness and percent cross-fertilization were assessed against wind conditions during the flowering period.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crop Phenology
Pollen shedding and silking dates, time taken to reach the stages and CHU accumulated for both yellow and white kernel maize hybrids are presented in Table 1. The Cargill hybrid 4521Bt (yellow) in Site #2 (year 2000), V414W (white) in Site #3 (year 2001) and both hybrids at Sites #2 and #3 in 2002 had non-uniform plant growth (uneven plant size) and development (phenological stages) resulting in a longer periods to complete their flowering, and probably asynchronous pollination within the population.
Environmental Effects
Seasonal climatic conditions affected the level of cross-fertilization. Overall maximum cross-fertilization occurred in the first row of the white maize adjacent to the yellow-kernel Bt hybrid. The observed maximum out-cross was over 82% in all years (Table 2). The average level of out-cross in the first adjacent row was greater in 2000 (18.2%) than in 2001 (12.3%) or 2002 (13.3%). A consistent pattern was also observed in subsequent rows (Table 3). However, the effect of wind direction was different among the three years. Although the assumed wind direction was northwesterly, this was not always the case, particularly in Site #2 in 2000 and 2001 (Fig. 3). Non-prevailing wind evidenced in all the site-years (Fig. 3) may have had a large impact on cross-fertilization, which cannot be pinpointed from the current study. In 2000, cross-fertilization in the first adjacent row was on average 27.6% downwind and only 8.7% upwind (Table 3). A similar pattern of out-cross was observed in 2002 (24.9% downwind and only 4.9% upwind). In 2001 this pattern was reversed: downwind had only 10.6% out-crossing compared to 14.0% in upwind direction, indicating the impact of variable wind directions during flowering (Fig. 3). In general, in 2000 and 2002, all sites had greater out-cross in downwind than in upwind directions, but in 2001, Sites #2 and #3 had lower level of out-cross in downwind than upwind while, Site #1 had greater out-cross in downwind than upwind (Table 3). Apparently, instantaneous wind direction changes at the time of silking of the white maize had much larger effect than the overall prevailing wind direction.
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Site Effect
Cross-fertilization of white maize by yellow kernel Bt hybrid varied in all sites. Irrespective of year and wind direction, the mean percentage of cross-fertilizations recorded in the first adjoining row of white maize was 17% in Site #1, 14.2% in Site #2 and 14.5% in Site #3. However, the sites did not follow the same pattern over the three years. In 2000, Site #1 had the highest out-crossing (27.4%) in the first adjacent row of white maize followed by Site #2 (14.3%) and Site #3 (12.7%). In 2001, Site #2 had the highest out-crossing (19.1%) followed by Site #3 (10.3%) and Site #1 (6.6%). In 2002, Site #2 had higher cross-fertilizations (20.7%) than Site #3 (9.1%), while Site #1 was abandoned. The lower percentage of cross-fertilization in Site #1 in 2001 was mainly due to uneven plant growth as affected by the drought, which also resulted in more barren plants. The difference between the sites was however, mainly within the 13 rows (10 m) adjacent to the pollen source; thereafter, cross-fertilization of white maize by pollen from neighboring yellow Bt maize exponentially declined in all site-years (Table 3).
Distance from the Pollen Source
The level of cross-fertilizations across site-years fluctuated greatly because of the wind directions, but as a rule, the farther away from the yellow Bt pollen source, the lesser was the percent out-cross (Table 3, Fig. 4)
. Consequently, the first row of white maize adjacent to the yellow Bt hybrid always had the highest cross-fertilizations. The extent of cross-fertilization in the subsequent rows declined exponentially to 0 or near 0% toward the edge of the field. Less than 1% cross-fertilization was found after the 37th border row (28 m) downwind from the prevailing wind direction or the 13th row (10 m) in the upwind direction in all site-years.
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Rate of cross-fertilization with distance to the pollen source was well represented by both exponential and modified exponential decline functions (P < 0.01) with R2 = 0.64 for downwind and 0.58 for upwind (Table 3). Data of cross-fertilization in the north and south sides of yellow Bt maize represented by the Eq. [1] better than Eq. [2] on the basis of the R2 values (Fig. 4). According to Eq. [1], the estimated zero (or 0.0001%) cross-fertilization would have occurred in the white maize population at about 30 m downwind or 23 m upwind from the pollen source (Table 3). Estimated zero (or 0.0001%) of white maize in the south and north sides of the yellow Bt hybrid had a short distance (11–19 m) from the pollen, suggesting that pollen traveled shorter distances, or cross-fertilization declined more quickly along the same row direction than cross row directions. In general, although the models fit the data, the R2 values in all cases were not very large (Table 3, Fig. 4), indicating factors other than distance also played important roles in the extent of cross-fertilization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The extent of pollen transfer and out-cross fertilization also depended upon the synchronization of pollen shedding of the yellow maize with the silking of the white maize, and the amount of pollen available from the yellow maize. Greater level of out-cross was expected in Sites #2 and #3 in 2002 as two yellow hybrids with different maturities were used in each site, and there was a longer period of pollen shed of the yellow kernel maize hybrids (Table 1). However, poor plant growth in size and uneven phenological progression (different silking dates) of the white maize in Site #3 of 2002 (and also 2001) was associated with the extended period of drought, which has caused asynchronous pollination between early and late appearing silks (Table 1), and thus resulted in poor kernel set and a large number of partial or even over 50% of the ear barrenness. Even if wind direction and speed are favorable for pollen dispersal, if silks are not receptive or if the air is too dry, pollen viability will be quickly lost. Therefore, synchronization of pollen dispersal and silking is very crucial in determining the extent of cross-fertilization in maize. In breeding programs, isolation in time is sometimes used to prevent cross-fertilization in seed production fields between materials concerned. For production of non-GM maize, or specialty maize, this method can also be considered.
Hybrid seed production requires close synchrony between receptive silks on the female parent and pollen shed by male parent (Westgate et al., 2003). The variation in the extent of cross-fertilization among site-years indicates that seasonal conditions influenced the level of synchrony and thus out-crossing. In 2001, hot and dry weather conditions in July and August substantially shortened the viability of pollen after shed and maturation of silking may also have been accelerated. When plants are exposed to any stress before anthesis, the time gap between male and female flowers usually lengthens (Cárcova and Otegui, 2001). Drought delayed tassel emergence, silking and grain filling (NeSmith and Ritchie, 1992), and resulted in a water deficit during the tassel and silk emergence period, which can increase the interval from silking to tasseling (Traore et al., 2000) and pollen shed (Herrero and Johnson, 1981). Prolonged drought in 2001 and 2002 seasons may have been associated with two events: (i) drought caused uneven plant growth in size and uneven phenological progress and thus led to unsynchronized flowering and reduced cross-fertilization within and among populations and kernel set and (ii) probably more importantly, drought (low moisture in the atmosphere) reduced pollen longevity. Maize pollen is susceptible to desiccation (Luna et al., 2001) and water loss in pollen grains affects the ability of pollen to germinate in stigma (Barnabas, 1984). Under normal conditions, the gradient in floret development and silk length along the ear at silking determines interval between early and late appearing silk, which results in pollination asynchrony between them (Cárcova et al., 2003). Therefore, some degree of asynchrony should be expected in an experiment involving different hybrids and varying environments. In this study, the impact of the asynchrony on the level of cross-fertilization should have been the same to or smaller for out-cross between the two populations than within a population as percent cross-fertilization was influenced by the synchronization of pollination of donor pollen with the receptive silking across two populations (Table 1) rather than synchronization within a hybrid. We assume that as a cross-pollinated crop, foreign pollen grains are favored for maize receptive silks (out-cross over 50%; Table 2) when pollen grains from both yellow and white kernel maize are available.
The approach of using yellow kernel maize as a marker of cross-fertilization in white maize (Fig. 5)
has been proved to be a useful tool. The experimental results clearly showed that majority of the maize pollen grains had settled close to the source itself, and an exponential decrease in pollen dispersal was observed as the distance from the pollen source of the yellow-kernel Bt maize increased. The risk of cross-fertilization of white maize (or other non-GM maize) by pollen from neighboring yellow Bt maize was very low beyond the 37th row (28 m) from the source. However, trace amount of the yellow maize pollen dissemination may have occurred beyond the 48th row (37 m) of the white kernel maize plants; this study was not able to confirm this because of the limited field size. Nonetheless, even if some pollen had reached that distance, the likelihood of significant cross-fertilization would be very low because of the short viability of the pollen grains once shed. Our results are also in agreement with the findings of Bateman (1947), Raynor et al. (1972), Garcia et al. (1998), Luna et al. (2001), Jemison and Vayda (2001), and Halsey et al. (2002) that a sharp decline in pollen dispersal occurs as the distance from the source increases. From a practical point of view, and considering differences in planting dates from neighboring maize fields, and/or different maturity of two hybrids involved, our data suggest that it is possible to produce non-GM maize by removing the outside rows of plants (about 30 m) adjacent to the GM maize field if the acceptance level is set at 1% cross-fertilization. Although the chances of cross-fertilization of white maize by pollen from neighboring yellow maize at the distance of 28 m was minimal, this study did not examine the situation in which the Bt and non-Bt maize were separated by a non-maize space. In the study reported here, the white maize rows from 1 to 37 might have also served as a physical barrier for the pollen of yellow maize in addition to compete for viable pollen from the yellow maize. A situation with a non-maize barrier pollen flow could be an area of further study on this topic.
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ACKNOWLEDGMENTS |
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Received for publication April 7, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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