More Information on Current DuPont Products and Potential Impacts of Cross Pollination

1.1   Impacts of Gene Flow from Commercially Improved Maize to Local Cultivars and Landraces

Landraces of maize have been the focus of several studies in Mexico (Benz, 1988; Sanchez and Ordaz, 1987; Wellhausen et al., 1952). An early survey by Wellhausen and co-workers (1952) reported 25 races and three sub-races. Currently, more than 50 races can be differentiated (Sanchez et al., 2000), based on multivariate analysis of morphological, cytological, isozymes and DNA analysis. The largest differentiation on the number of landraces from the 1950s to present may be attributed to more sensitive techniques, increased efforts in collecting, or to the creation of new local varieties as farmers continue to select for specific environmental conditions and food uses.

Research on maize varieties in Mexican agricultural ecosystems have indicated that seed exchange among farmers in small communities and pollen flow are equally important to facilitate gene flow (Louette, 1997; Castillo and Goodman, 1997; Pressoir and Berthaud, 2004). Current evidence suggests that many small-scale subsistence-oriented farmers have taken improved varieties and planted them alongside their local varieties (Bellon and Brush, 1994).

Also important is a farmer's willingness to deliberately promote gene flow among improved open pollinated varieties and hybrids into their local landraces (Aguirre-Gomez et al., 2000) to transfer valuable genes into their local varieties. Hybridization and subsequent introgression between local and improved maize through empirical plant breeding practices is highly valued by farmers in Mexico and Central America (Almekinders et al., 1994; Bellon and Risopoulos, 2001). At present, it is widely accepted that gene exchange among improved open pollinated varieties, hybrids and land races has been common practice in Mexico and Central America (Castillo and Goodman, 1997; Louette, 1997). Yield advantages of improved landraces from the same region compared to old landraces suggests that introgression of genes through farmers intervention has been beneficial to Mexican landraces (Castillo and Goodman, 1997).

Concerned with maintaining genetic diversity in local cultivars and landraces of maize, Iltis (1974) proposed the creation of in situ conservation to complement the ex situ conservation of maize germplasm systematically practiced since the 1940's-50's. It is estimated that 80% of the teosinte genetic diversity in Mexico has been collected (Serratos et al., 1997) and preserved ex situ. In situ conservation refers to preserving entire ecosystems, within certain agricultural areas, by prohibiting changes in cultural practices or the introduction of foreign genetic material. This concept has been criticized widely on the grounds that it is not feasible for socio-economic reasons. In their studies of maize varieties and seed sources in the traditional farming community of Cuzalpa (Mexico), a reserve established in part for in situ conservation of maize landraces, Louette and Smale (1996) found that farmers enhance the genetic diversity of local cultivars with introductions of both improved cultivars and landraces from farmers in other areas. Genetic material from introduced varieties was incorporated into local cultivars to improve yields and, as a result, was a source of increased diversity and not genetic erosion. Traditional agricultural systems in this area are not closed and isolated with respect to the flow of new genetic material but are open and evolving. Therefore, in situ preservation and collection of landraces represents only a snapshot of the diversity present at that moment.

One needs to differentiate the intentional introduction of genes from "improved" maize varieties/hybrids into local landraces/varieties from the unintentional introduction of genes via pollen flow from "improved" varieties/hybrids. No matter what the route, however, the consequences are likely to be similar if the same agronomic forces dictated by the local farmers are consistently applied.

1.2   Impacts of Gene Flow from Biotech Maize Varieties to Local Cultivars and Landraces

In late 2001, a paper was published in the journal Nature that claimed to present evidence for introgression of biotech DNA into Mexican landraces (Quist and Chapela, 2001), despite the fact that Mexico had banned the cultivation of biotech varieties. While some considered the finding a threat to maize genetic diversity, the scientific evidence presented in the paper was widely criticized by scientists. Nature editors later retracted the paper.

Despite the controversy over the paper's technical merits, its conclusion that DNA from plants derived through biotechnology was detected in Mexican maize was confirmed in early 2004 by the Mexican Biosecurity Commission Cibiogem. Follow up studies commissioned by Cibiogem revealed that the DNA was detectable in seven percent of 4000 samples from 188 localities in the states of Oaxaca and Puebla. Preliminary analysis suggests that some indigenous farmers planted imported grain that contained biotech corn, together with local cultivars. The result was not surprising as it is generally thought that introgression of transgenes from commercial hybrids into landraces is possible if the plants are grown in proximity to one another. The most important issue is, what impact introgression of transgenes has on biodiversity. In the position of the Mexico-based International Maize and Wheat Improvement Center (CIMMYT) on the issue of transgenes in Mexican landraces, Director General Iwanaga said: "When transgenes are present in Mexican maize landraces grown by farmers, does this mean that an important resource is lost forever? As scientists, we would answer 'no,' because the landraces may have changed, as they do all the time, but they have not disappeared. On the contrary, with the addition of a transgene, they could actually be considered more diverse. This additional diversity may not be desirable, however. It is precisely this issue that the Mexican government must resolve.

1.3   Impacts of Gene Flow between Biotech Maize Varieties and Wild Relatives

Most teosintes are sexually compatible with maize (Doebley, 1990) and are able to form hybrids. As described earlier in the document, experimental data suggest that cross-pollination events are more likely to happen when pollen flows from teosinte to maize rather than vice versa. If the maize plants were derived through biotechnology, the resulting hybrids would carry transgenes. Doebley (1990) identified several conditions that would facilitate introgression of transgenes in teosinte. First, introgression of transgenes into teosinte populations is possibly only when biotech maize is grown in an area where the wild relative is native (Mexico/Central America.)

Second, genes from maize cannot interfere with traits, such as seed dispersal, that allow teosinte to reproduce. Humans have bred maize plants to retain seeds together on single structure. The lack of seed dispersal differs from wild maize (teosinte), in which each seed is designed to separate from the others. This is meant to maximize reproductive success by ensuring most seeds have enough space, light, nutrients, water, and other resources to develop. Whenever cultivated maize is crossed with teosinte, the seeds do not disperse from the hybrid. Wilkes (1972) states that although hybrids of maize and teosinte have been found among all known teosinte populations in Mexico and Guatemala, the gene flow has not altered the strong selection pressure for a hard enclosed teosinte fruitcase. This significantly reduces the possibility that the hybrids will produce progeny that will survive to reproduction. Therefore, maintenance of genetic material from cultivated maize in the teosinte population is difficult, whether the cultivated maize is a biotech or non-biotech variety.

Third, as described above, the transgene must confer an adaptive advantage to the hybrid that increases its fitness beyond those teosinte plants lacking the transgene. In the case of Bt, for example, wild teosinte populations would not benefit by having a Bt gene unless lepidopteran pests have a significant impact on the size of the teosinte populations in that area. Given the lack of adaptive advantage conveyed by the Bt transgene in conjunction with the adaptive disadvantage of significantly decreased seed dispersal, the Bt trait would be lost in just a few generations. Phenotypic traits provided by the transgene must result in a plant's enhanced ability to survive in order for the transgene to be actively selected.

Ellstrand et al. (1999) reviewed the literature and presented evidence for natural hybridization and introgression for a number of important crops. For maize, they cited the following research papers.

• Work by Wilkes (1977) describing plants morphologically intermediate between maize and teosintes that were observed in and near Mexican maize fields when teosinte is abundant.
• Doebley's (1990) allozyme studies that demonstrated very low levels of introgression from maize into the seven teosinte species or subspecies they studied.
• Kato's work (1997) indicating that cytogenetic analyses "…offer no evidence of …maize-teosinte introgression in either direction…"

According to Ellstrand et al. (1999), no one has conducted genetic analyses of wild intermediates using currently available molecular techniques to determine whether the intermediates are true hybrids, introgressants, or crop mimics. Determining this would help address Ellstrand's (1997) concern of the possibility of extinction by hybridization. To him, the problem of extinction by hybridization depends upon whether hybridization occurs at a substantial rate, and whether the wild relative is locally rare compared to the nearby crop. The "nearby crop" could be a landrace, a commercially improved variety developed by conventional breeding, or a biotech variety. The potential impact on the wild relative will be the same.

The key issue here is that both outbreeding depression (resulting from detrimental gene flow that reduces the fitness of a local rare species) and swamping (which occurs when a local rare species loses its genetic integrity) are frequency-dependent phenomena and show positive feedback. Ellstrand and Elam (1993) showed that with each succeeding generation of hybridization and backcrossing, genetically pure individuals of the locally rare species became increasingly rare until extinction occurred. An unpublished example from Ellstrand showed that if 900 individuals of a locally common species mate randomly with 100 individuals of the locally rare one, extinction by outbreeding depression and/or swamping could occur in two generations. His example also showed that if the rare species mated with its own kind even five times more frequently than the rate expected under random mating, the time to extinction is expected to double relative to random mating. Ellstrand goes on to point out that in contrast to the issue of enhanced weediness, the problem of extinction by hybridization does not depend upon relative fitness, but only on the patterns of mating.

According to Serratos et al. (1997), the principal threats to teosinte populations are changes in land use through increased grazing and urbanization. They view biotech maize as a marginal threat compared with the effects of urban growth. The impact of biotech maize, or any improved maize, can be controlled through known practices of isolation. Finally, some have questioned whether transgenes from maize would enhance teosinte weediness. According to Ellstrand et al. (1999), to date, gene flow from non-biotech varieties to teosinte has not increased its weediness.

1.4   Impact of Gene Flow via Pollination on Neighboring, Non-biotech Maize

The seed industry follows internationally determined and accepted standards for genetic purity. For example, these standards call for the use of specific distances between fields to manage gene flow. These distances are necessary to meet the standards for purity.

The seed industry is applying the lessons learned from working with these standards, as well as our own experiences, to understand and manage gene flow issues related to crops derived through biotechnology. In addition to using isolation distances before marketing these crops, we surround the field with a number of border rows of non-biotech plants that serve as pollen-catchers. This further reduces the chances for cross-pollination. In spite of the many measures (discussed below) used during seed production to minimize the presence of biotech genetic material in non-biotech seed batches and vice versa, it is recognized by many that trace amounts of biotech genetic material or seeds may be found in some fields containing non-biotech varieties

Because maize is a cross-pollinated crop with wind-dispersed pollen, cross-pollination can occur unless steps are taken to avoid it. If non-biotech maize plants in one field have receptive silks at the time of biotech pollen shed in a neighboring field, some cross-pollination of the non-biotech hybrid with the biotech hybrid will probably occur. The amount of this cross-pollination depends upon many factors, including the distance of the fields from each other, presence of non-biotech crop borders, intensity and duration of pollen shed of the non-biotech maize (pollen competition), and the predominant wind speed and direction.

U.S. state seed certifying agencies are responsible for official standards for certified seed (Table 1). The standards, which vary somewhat by state, generally set minimum distances for isolation. These distances are modified by additional border rows, size of the field and production block, adequate natural barriers (in some states), and differential flowering dates (in some states).

 Table 1. General isolation distances set by U.S. state seed certifying agencies for single-cross
 seed production. Requirements designed to target genetic purity of 98-99 percent.

When no border or one-border rows are used, minimum distances from 410-660 feet are typically required between the female parent of the hybrid being produced and any other corn of the same seed color, maturity or endosperm type. If the corn that might serve as a potential adventitious pollen source has different kernel color or endosperm type, isolation distance of 660 feet is required. Recognizing the inevitability of adventitious presence of different genetic material, the Organization for Economic Co-operation and Development (OECD) suggested purity levels for parent seed at 99.0-99.5%. For hybrid maize seed, the U.S. Federal Seed Act (USDA) requires 95 percent purity in order to label it as a single hybrid. These widely accepted standards for certification of varietal purity could also be useful when trying to establish thresholds for purity of non-biotech maize.

It is important to realize, however, the situation for seed production, which is what seed companies do, and grain production, which is what producers do, are different. These same isolation standards that apply to seed production fields may result in much higher purity in hybrid grain production fields. A hybrid grain field produces a pollen load that is many times, perhaps 10 to 100 times, greater than that of a single-cross seed production field. In addition, the timing of pollen shed in a hybrid field is usually highly synchronized with silk emergence. The large pollen load and synchronous timing of pollen and silks serve to greatly limit pollen mixing from outside sources.

Growers should also keep in mind that most biotech hybrids, including those containing the Bt trait, have heterozygous (or hemizygous) dominant gene systems. Only half of the pollen produced in a Bt commercial field contains the Bt gene because only one parent contains the transgene. This reduces the chance of co-mingling due to pollen drift from the biotech field to one half of what would otherwise be expected if all of the pollen possessed the gene.

The use of border rows is another technique widely used to minimize the probability of biotech pollen drift into a non-biotech field. Table 2 provides the minimum number of border rows that can be substituted for distance in the isolation of a non-biotech field from a biotech field for grain production.

Finally, in addition to distance and/or border row isolation, segregation of grain at harvest also can improve purity. If non-biotech and biotech varieties are grown in plots next to each other, harvesting a number of rows (12-16) from the side of the non-biotech plot that is nearest the biotech field, and then separating this grain from the rest of the non-biotech grain can help the grower achieve a much higher purity level in the grain harvested from the remainder of the non-biotech field.

It is obvious that there is an advantage to larger production fields and to greater isolation distances. It also should be clear, however, that if the grower has a market or use for the grain produced in the border rows, removing 12-16 outside rows should be adequate to insure high purity grain. Pollen mixing has always been a normal occurrence in corn production. The accepted practice has been that each grower is responsible for any isolation that may be required for various types of crops.

 Table 2. Minimum number of border rows recommended for grain production in Nebraska, Iowa,
 Indiana, and Wisconsin to maintain 1% or less non-biotech purity in a hybrid field at various
 distances (in feet) from a biotech field. (Information prepared by Dr. Joe Burris, Emeritus
 Professor Iowa State University under commission from the American Seed Trade Association.)

2.1   Potential Ecological and Evolutionary Impacts on Wild Relatives of Gene Flow from Biotech Varieties

Controlled hybridization of G. max to wild Glycine species has been extremely difficult, so the impact of gene flow from cultivated soy to wild soy populations or from commercially improved, non-biotech or biotech varieties to soy landraces should be essentially non-existent.

Because of the low frequency of hybridization between cultivated and wild soy and the relatively small area of overlap between wild and cultivated soy, the amount of transgene introgression into wild soy populations is expected to be minimal. When biotech soybeans are grown in areas containing wild soy populations, fairly simple techniques could be used to further minimize the already limited opportunity for gene transfer. A minimal increase in either isolation distances or border row numbers will reduce the possibility of cross-pollination, resulting gene flow and possible introgression to virtually zero.

As discussed in the introduction to the paper and also in the section on maize, many factors determine whether gene flow from cultivated crops to wild populations will result in an ecological or evolutionary change. In soybeans, the rate of gene flow is greatly minimized, first and foremost, by the reproductive biology of the soybean plant. In addition, ecological impact depends on the nature of the recipient plant and the adaptive value of the newly acquired trait. To date, the only transgene in soybean that affects yield (i.e., potential reproductive success) is resistance to the herbicide glyphosate. Because of the very low frequency of hybridization between wild and cultivated soy, in order for the glyphosate resistance transgene to become established in the wild soy population and have an ecological impact, there must be intense selective pressure placed on the wild soy population that would favor plants that had acquired the gene for glyphosate resistance. In other words, the wild soy population must be frequently treated with the herbicide glyphosate. Having a well-planned herbicide application program in place before growing biotech crops in centers of origin would be an expected practice.

2.2   Impact of Gene Flow via Pollination on Neighboring, Non-biotech Soybeans

Cross-pollination of soybeans is generally infrequent (<1%) although some have documented higher rates under optimal conditions (Ahrent and Caviness, 1994; Ray et al., 2003). Most likely, cross-pollination will not occur if biotech and non-biotech fields are 10 m apart. The OECD purity standard of 99 99.5% should be easily met at this distance, unless farmers inadvertently mix seed during planting, harvesting or in shipping and handling. The Association of Official Seed Certifying Agencies (AOSCA), which sets seed purity standards for virtually all crops, does not have specific guidelines for isolation distances for certified soybean production. Adventitious presence of genetic material is minimized during certified seed production by following their only guideline for isolation: "Fields of soybeans shall be separated from any other variety or uncertified seed of the same variety by a distance adequate to prevent mechanical mixture" (AOSCA, 1996). Adventitious presence can result from growers or subsequent handlers accidentally mixing seed by not completely cleaning harvest machinery, storage bins or seed handling systems.