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CROP ECOLOGY, MANAGEMENT & QUALITY

Optimizing Pollen Confinement in Maize Grown for Regulated Products

^a Univ. of Missouri-Delta Center, 147 State Hwy. T, P.O. Box 160, Portageville, MO 63873
^b 1870 Oxborough Ct., Chesterfield, MO 63017
^c Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167

^* Corresponding author (stevensw{at}missouri.edu)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 
Genetically modified maize (Zea mays L.) produced for regulated products such as pharmaceutical or industrial proteins will require methods to confine transgenic pollen. In one production system, nontransgenic maize would be used to pollinate detasseled transgenic inbred plants. Resulting hybrid kernels would be used for protein extraction or seed increase. The effect of different female inbred detasseling efficiency levels on gene flow was tested at three locations in southeastern Missouri in 2000 and 2001. Pollen sources were yellow inbred isolines representing transgenic females planted in alternating rows with white inbred maize representing nontransgenic males. During detasseling, female plants were intentionally missed at rates of 0, 730, 1460, and 7300 tassels ha^–1. Each detasseling treatment was matched with a maize isoline and traceable marker. White hybrid trap plots were planted on three dates at 200 and 300 m from pollen sources. Dates that maximized silking synchronization with yellow isoline tasseling were selected for sampling. Gene flow was detected by counting yellow kernels in white maize plots. When no tassels were removed from an isoline, the highest recorded gene flow was 0.03% at the 200 m and 0.02% at the 300 m isolation distances. At greater detasseling levels, gene flow decreased. Gene flow was 0.0013% or less when 730 tassels ha^–1 remained. When complete detasseling was intended, one positive kernel with a tracer gene was detected at 200 m, and none was detected at 300 m. For effective control of regulated transgenes in pollen by detasseling, complete and timely tassel removal will be necessary.

Abbreviations: Bt, Bacillus thuringiensis • IT, imidazolinone tolerant • PCR, polymerase chain reaction • PMIC, plant-made industrial compound • PMP, plant-made pharmaceutical • TPS, tris-potassium acetate-sodium chloride

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 
PLANTS ENGINEERED for use as green factories have the potential to become important sources of plant-made industrial compounds (PMICs) and plant-made pharmaceutical (PMP) proteins. Products such as biodegradable plastic, monoclonal antibodies, blood plasma proteins, peptide hormones, and human hepatitis B vaccine have been produced in transgenic plants (Nawrath et al., 1994; Richter et al., 2000; Saruul et al., 2002; Ohlrogge and Chrispeels, 2003).

Pharmaceutical proteins are currently being produced from modified mammalian cells in stainless-steel containers called bioreactors. Unfortunately, the high cost and relatively low capacity of bioreactors for producing pharmaceutical proteins limits the availability of some drugs to patients. Furthermore, limited manufacturing capacity could delay 20 to 50 pharmaceutical products in the pipeline. To help solve these capacity problems, scientists have used biotechnology techniques to develop plants capable of producing the desired proteins. By increasing or decreasing the numbers of hectares planted to PMP and PMIC crops, manufacturers can meet the demand for these proteins.

Methods are needed to ensure that grain or plant tissue from PMP and PMIC crops do not enter our food and feed supplies. Food industry groups representing grocers and grain processors want assurance that biotechnology companies can accomplish the needed confinement standards (Applebaum, 2002; Green, 2002). Also, pharmaceutical companies and Food and Drug Administration regulations require a pure therapeutic product (Cline, 2003).

Currently, some interested parties are considering the development of PMPs or PMICs in maize. An important part of a PMP and PMIC maize confinement strategy is preventing movement of viable pollen by wind from regulated maize fields into nonregulated maize fields. In this paper, the term gene flow will denote pollen-mediated fertilization of one group of maize plants by another group of genetically different maize plants.

Approaches to pollen confinement of regulated maize include spatial isolation, temporal isolation, and controlled pollination. Relative to other grass species, maize pollen grains are large in size (86–122 µm diam.) (Lewis et al., 1983). The large pollen grain size is a positive trait for confinement because most maize pollen usually does not travel far before settling to the ground. Raynor et al. (1972) found that wind transported maize pollen a shorter distance than pollen grains from species such as timothy (Phleum pratense L.) and ragweed (Ambrosia spp.). The amount of maize pollen captured at 60 m (0.037 miles) from a pollen source was 5% of that captured at 1 m.

The USDA regulates PMP and PMIC production and reviews their guidelines for growing regulated maize each year. In 2003, regulations for growing open-pollinating, regulated maize required at least 1.6 km (1 mile) spatial isolation from other maize fields (USDA-APHIS, 2003). The required isolation distance was reduced to 802 m (2640 feet) if regulated maize was also temporally isolated, that is, it was not to be planted <28 d before or 28 d after any other maize growing within 802 m. Temporal isolation prevents synchronization of pollen production (nicking) in fields with regulated plants to silking of plants in neighboring fields. A variation of temporal isolation is to use cultivars for PMPs or PMICs from maturity groups that pollinate much earlier or later than cultivars in the region.

In one candidate maize production system for regulated products, practices similar to conventional hybrid seed production would be used. Rows of inbred maize with regulated genes would be planted and managed to prevent pollen from being released into the environment. The systems would incorporate confinement by controlled pollination using male sterility, bagging, or detasseling. Rows of male nonregulated maize would be planted in alternating rows to pollinate the female maize plants containing the regulated gene. Maize plants in the female rows would be harvested for regulated proteins. The authors are not aware of any detasseling efficiency studies reported in the literature from experiments designed specifically to address confinement issues with maize production systems for making regulated products.

Detasseling could be used as a confinement method in either single-cross or double-cross hybrid seed production systems. In a double-cross, both parents are hybrids lines. In a single-cross, hybrid seeds are produced from inbred lines. Although a double-cross hybrid maize seed production with hybrid parents could be used for making regulated products, a single-cross hybrid system was evaluated in our study. The resulting hybrid kernels from inbreds would be used for protein extraction or seed increase for making double-crosses.

Several gene flow studies have been conducted with detasseled maize in seed production systems. Hutchcroft (1959) observed that pollen could be released from female plants if tassels are incompletely removed or if detasseling is delayed. Garcia et al. (1998) conducted a study to determine if small-scale seed production of transgenic maize could be conducted without significant risk of gene introduction into the wild relatives and land races of maize in Mexico. They concluded that there was no danger of pollen dissemination and gene escape into wild relatives of maize if transgenic plants are used as females and detasseled before flowering.

Research was begun in 2001 in southeastern Missouri to assess the effectiveness of using detasseling and spatial isolation to prevent pollen-mediated gene flow in PMP and PMIC inbred maize production systems. Three test locations isolated from other maize fields were used to evaluate confinement practices in a candidate protein production system. In the model system that we evaluated, the hybrid kernels produced on the female inbred plants would contain the regulated product. The premise of our study was to evaluate production of maize kernels with regulated proteins from inbred parents. By using a maize isoline series, four different detasseling efficiencies of females were simultaneously assessed to determine the likelihood of gene flow from PMP and PMIC maize lines in a pollen confinement strategy across multiple environments.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 
Experiment Overview
In 2001 and 2002, an experiment was established in three production fields. In the center of each location, an inbred maize pollen source block was planted (Fig. 1) . This area represented a regulated hybrid seed production field. In the pollen source blocks, detasseling treatments were applied to four yellow inbred isolines which had traceable genes (Fig. 2) . Female isolines represented plants that would contain regulated genes. Around each side of the pollen block, male-sterile maize (taller than the inbreds) was planted in rows to reduce pollen blowing from the pollen source block. At 200 and 300 m from the pollen source area, white hybrid trap plots were planted on three dates. Trap plots were arranged in a Latin square arrangement (Fig. 3) . At harvest, ears in the white hybrid trap plots were inspected for yellow kernels. Yellow seeds on white corn maize were indicators of gene flow from the yellow maize. White endosperm vs. yellow endosperm difference is due to a single gene (y1), with yellow being dominant to white (Weber, 1994). The trap plot planting dates with the best nick with yellow female isolines were selected for sampling. Yellow seeds from these trap plots were collected and planted in pots in a greenhouse. Tissue from the plants was analyzed for tracer genes from the inbred isolines. Percentage gene flow detected from each detasseling treatment was calculated.

View larger version (156K): [in this window] [in a new window]   Fig. 1. Aerial view of pollen confinement research field at Hayti, MO, in 2001.

View larger version (122K): [in this window] [in a new window]   Fig. 2. Pollen source blocks were planted to model a possible pharmaceutical or industrial maize production system with four rows of a nondetasseled male white kernel inbred alternated with four rows of varying detasseled females which were treatment-specific yellow isoline inbreds.

View larger version (131K): [in this window] [in a new window]   Fig. 3. A 3-by-3 latin square design was used in white hybrid trap blocks with individual plots having planting dates relative to the planting date of the yellow maize pollen source block to assure pollen synchronization and equal pollen competition for each planting date.

A 4-ha maize pollen source block was planted in the center of each field location. Pollen source blocks were 200 by 200 m and surrounded by a 9-m fallow strip and 12-m wide strip of male-sterile hybrid maize. Within the pollen source block, a hybrid seed production system was established. Four inbred maize isolines were planted in female rows. Four rows of male white inbred corn were planted in a repeating eight-row pattern with the four yellow corn inbred females. The yellow maize rows represented PMP or PMIC detasseled female rows and the white maize rows represented nontransgenic male pollinator rows in a protein production system. White maize trap plots were planted 200 and 300 m from the outside row of the detasseled inbreds in the pollen sources. To assure that silks in the white trap plots would be receptive at the time the pollen block was releasing pollen (nicking), the plots were planted on three dates spaced 90 to 120 Growing Degree Units relative to the pollen source planting date (approximately –1 wk, same day, and +1 wk). Each planting date was replicated three times at each location in a Latin square arrangement; so that there were nine plots in each block (Fig. 3). Twenty-eight irrigated trap blocks in each field were used. Additional trap blocks were planted outside the center-pivot watering zone. However, because drought stress affected maize reproductive development, these trap blocks were not sampled at harvest. The location of each trap plot sampling point was recorded with a differential global positioning system.

The maize row direction at each test location was oriented with the field slope to allow for water drainage. Rows at Hayti and Clarkton, MO, were oriented east to west; and rows at Wardell, MO, were oriented north to south. At Clarkton and Hayti, most of trap plots were north and south of the pollen source block. At Wardell, most of the trap plots were east and west of the pollen block. At each location, two partial strips of white maize were also planted along the center of the field originating on each end of the pollen source block and extending out to the ends of the field.

Fields were irrigated as needed to prevent water stress. Herbicides and insecticides were applied at recommended label rates to control weeds and insects. Fertilizer was applied according to soil test recommendations. Weather stations (Campbell Scientific, Inc., Logan, UT) were established at each test site to measure wind speed and direction. Wind data were recorded in 15-min intervals in the morning hours during the peak 9- to 10-d pollination period.

Genetic Materials
A yellow maize inbred 7054 (proprietary) and three inbred trait conversions of 7054 were planted in the 4-ha pollen source blocks located in the center of each location. The yellow maize inbred conversions contained a scorable nontransgenic imidazolinone-tolerant (IT) gene, or the MON 810 transgene (Bacillus thuringiensis [Bt]), or the NK 603 transgene (glyphosate resistance), which were used for tracing gene flow (Fig. 2). Each yellow female inbred isoline had been backcrossed five times. Converted inbred lines had similar flowering dynamics, plant height, tassel morphology, and pollen production. The average plant population for the yellow maize inbreds was 58361 plants ha^–1 at all sites. Proprietary white inbred maize (9DZD2W) was planted in the male rows in the pollen blocks. Proprietary (LH198SDms x LH185) male-sterile hybrid maize was in the borders around the pollen blocks.

White hybrid maize (cv. DK665W) was planted in trap plots. Each bag of white maize (inbred or hybrid) was inspected and yellow kernels removed before planting the pollen block or trap plots.

Detasseling Treatments
Yellow maize female rows were detasseled by hand to one of four levels of efficiency. Detasseling efficiencies in the yellow rows were intentionally designed to miss tassels at rates of 0, 730, 1460, and 7300 tassels ha^–1 (Fig. 2). The yellow maize inbreds were paired with detasseling treatments as follows: 7054 (nontransgenic), missed 7300 female tassels ha^–1; 7054 MON 810 (Bt gene), missed 1460 tassels ha^–1; 7054 IT (imidazolinone-tolerant gene), missed 730 tassels ha^–1; and 7054 NK 603 (glyphosate-resistant gene), missed no tassels. On a percentage basis, the 730 tassels ha^–1 treatment was equivalent to 1.25% of total plant population in the source block having 7054 IT tassels. The 1460 tassels ha^–1 treatment consisted of 2.5% of the total population having 7054 MON 810 tassels; and the 7300 tassels ha^–1 treatment consisted of 12.5% of total population having 7054 tassels. Numbers of tassels per area and percentages were based on total land area and populations in the source blocks including both yellow maize females and white maize male pollinator rows. Female rows were checked every two to three days during maize pollination. If more tassels were found in a female row than designated by the treatment rate, tassels were removed to the correct amount. Three tassel morphological traits (main stem diameter, main stem length, and total branch length) were measured for inbred 7054 to calculate a tassel area index. The index was then used in a regression equation reported by (Fonseca et al., 2003) to estimate the average pollen production per plant.

Gene Flow Confirmation
When the white maize matured, 70 ears per trap plot from 27 plots (three planting dates, nine replications) were harvested by hand and visually inspected for yellow kernels. The average number of yellow kernels from each planting date was calculated in the field and the planting date with the highest numerical gene flow (best nick) was then sampled (70 ears per plot) in all 28 trap blocks to evaluate the effect of distance, direction, and detasseling treatments on gene flow. Ears with any yellow kernels were tagged for identification and saved in bags. A representative white maize ear from each block was collected, shelled, and total kernels counted. This provided an estimate of the number of kernels inspected from each trap plot.

Yellow kernels found on the white maize ears were planted in greenhouse pots and allowed to grow. Tissue from each plant was assayed separately at a laboratory using a polymerase chain reaction (PCR) assay for MON 810 (Bt) and NK 603 (glyphosate resistant) and the nontransgenic IT gene. For samples collected in 2001, MON 810 and NK 603 genes were tested by PCR amplification and gel electrophoresis (Magin et al., 2000). The imidazolinone-tolerant gene was tested by end-point Taqman for 2001 samples. For 2002 samples, all three genes were tested by end-point Taqman. Since no other yellow maize was grown in the vicinity of the experiments, samples that were identified as negative for the three scoreable genes were assumed to be a result of gene flow from pollen released by 7054 yellow maize females (7300 female tassels ha^–1 treatment).

The end-point Taqman procedure involved the use of the PCR process to amplify a fluorescent molecule when a specific sequence was present. A computer and optical sensor measured the color of each sample to give a positive (fluorescent) or negative (nonfluorescent) reading. Approximately 3 cm² of corn leaf blade tissue was collected from each greenhouse maize plant and placed into a 1.2-mL tube retained in sampling box containing 96 individual tubes. A precipitation-based DNA extraction of tissue was aided by use of both the MultiDrop (LabSystems, Sunnyvale, CA) and RapidPlate (model Rapid Plate 96/384, Zymark, Hopkinton, MA) liquid handling devices. This equipment was used to speed up the addition of extraction reagents such as tris-potassium acetate-sodium chloride (TPS), potassium acetate, isopropanol, ethanol, and tris-ethylenediaminetetraacetic acid, and also for the transfer of supernatant (made up of ground leaf material, TPS, potassium acetate) from the original sample box to a receiver plate containing isopropanol used for the precipitation of DNA. After DNA was extracted, 2 µL from each sample was added to 3 µL of a prepared PCR mixture composed of water, Taq polymerase (2 x Universal Mastermix w/Ung, Applied Biosystems, Foster City, CA), target specific primers (forward and reverse), and FAM (6-carboxyflourescein) probe (Applied Biosystems), an internal control primer (forward and reverse) and VIC (chemical name withheld, proprietary info for Applied Biosystems) probe set (Applied Biosystems). The PCR reaction was performed in a 96-well rigid plastic plate (catalog #AB-1000, Marsh Biomedical, Rochester, NY), to mirror the setup of the original sample box. Endpoint-taqman assays included specific, fluorescent, dye-labeled probes for MON 810 (Bt), NK 603 (glyphosate resistant) and the nontransgenic IT alleles. Each probe annealed specifically to complementary sequences, and the Taq only cleaved probes that hybridize to the allele. Following PCR setup, the 96-well plates were then transferred to a thermocycler (model PTC-225, MJ Research, Waltham, MA) to amplify the DNA. Samples were then placed into a flourometer (Model Safire, Tecan, Maennedorf, Switzerland), which measured the amount of fluorescence generated during the Taqman reaction. Internally designed software (Monsanto, proprietary information) was used to deconvolute this generated raw data, and translate data into an allelogram. Clusters are scored according to their placement along the VIC/FAM axes, and also in relation to known positive/negative DNA controls added to each sample plate.

Data Analysis
The statistical analysis of gene flow data was performed using Proc Mixed from the Statistical Analysis System (SAS Institute, 1997). This procedure provided Type III F values but did not provide mean square values for each element within the analysis or the error terms. Mean separation was evaluated through a series of pair-wise contrasts among all treatments (Saxton, 1998). Probability levels > 0.05 were categorized as nonsignificant. Since there were many zero values in the gene flow data, square-root (X + 0.05)^1/2 transformation was applied before making pair-wise contrasts (Gomez and Gomez, 1984). The letter groupings from transformed means were then applied to original means.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 
The average white maize ear for all plots contained 554 kernels per ear. Tassel main stem and branch morphological measurements for inbred 7054 isolines showed an average tassel area index of 732 (Fonseca et al., 2003). The predicted pollen production per tassel using a regression equation reported by Fonseca et al. (2003) was 1.7 million pollen grains per tassel. The average height of the 7054 inbred isolines was 168 cm. The mean height of the pollen block border rows of LH198SDms x LH185 male-sterile maize plants was 230 cm.

Weather Conditions
Air humidity, wind, and temperatures at the test locations were favorable for producing gene flow between yellow inbreds and white trap plot hybrids. Relative humidity averaged across locations during the pollination periods in our study was 78% in 2001 and 83% in 2002. The average daily high temperature during pollination was 32.4°C in 2001 and 32.0°C in 2002. Luna V. et al. (2001) found that pollen remained viable for only 1 to 2 h after dehiscence, depending on atmospheric water potential. Johnson and Herrero (1991) reported that pollen viability declined greatly when temperatures were above 38°C. Schoper et al. (1987) reported that pollen tolerance to heat was influenced by maize genotype.

Wind measurements showed dominant wind directions from the south, southwest, north, and northwest during pollination (Table 1). At Clarkton and Hayti, most of the trap plots were located on the north and south sides of the fields because of row direction and drainage. These were in favorable positions for detecting north and south downwind pollen-mediated gene flow from yellow inbreds in the pollen source blocks. Most of the trap plots at Wardell were positioned on the east and west sides of the field. These plots were in a less favorable position for gene flow because of the dominant north and south winds.

No detectable gene flow was found at most of the locations from the zero intentionally missed detasseling treatment. One yellow kernel tested positive by PCR assay for the glyphosate resistance gene, and thus matched the complete detasseling treatment. The kernel was found on an ear isolated 200 m from the pollen source at Clarkton in 2001 (Table 5). The most likely source of error for the kernel, other than incomplete detasseling, is seed lot contamination in yellow maize inbreds or white maize trap plots. At each site, we observed and rouged out several taller off-type plants from the yellow inbred rows. In the trap plots, a yellow kernel could have been overlooked when the white maize seed lot was cleaned, and the resulting plant would have gone unnoticed in planting dates or nonirrigated plots that were not evaluated. Less likely sources of error are that the positive kernel resulted from stray pollen from outside the trial, a greenhouse mistake in planting or sampling glyphosate-resistant control plants, or a false positive due to laboratory error. We collected tissue from all the maize fields located several kilometers in each direction from the test sites. All samples tested negative for the genes used to trace gene flow in the experiment. Duplicate PCR from greenhouse samples also showed that the kernel was positive for the glyphosate resistence gene. If, in fact, the kernel resulted from an error in detasseling, it demonstrates the importance of multiple layers within a containment system, since no glyphosate-resistant kernels were noted at 300 m in this trial.

	SUMMARY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 
For effective control of regulated transgenes in pollen by detasseling, complete and timely tassel removal will be necessary. Any confinement strategy for maize pollen should be thoroughly tested across multiple environments and use trap plots that have multiple planting and hence silking dates. In our study, trap plots having the best nick with the pollen source were specifically selected for sampling. Multiple layers of confinement, including spatial isolation and detasseling, are recommended. The addition of male sterility to detasseling of transgenic lines would be expected to provide a higher level of security against unwanted gene flow in the layered confinement system.

Received for publication October 2, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY REFERENCES

 

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