In their recent ISB News Report article about transgenic sorghum, Visarada and Kishore (2007) provide incomplete information about the likelihood that transgenes would spread to wild relatives of the crop. Introgression of transgenes that enhance the fitness of weedy relatives could possibly make them more difficult to manage (e.g., Snow et al. 2005). Visarada and Kishore note that, "The most important issue related to biosafety concerns in sorghum is pollen-mediated gene flow to the wild species Sorghum halepense (Johnsongrass), a wild weedy relative ... " However, by citing only a single reference – Godwin (2005) – they give the impression that crop-to-wild gene flow will not occur because Godwin reported that "hybridization of S. halepense (2n=40) and cultivated sorghum, S. bicolor (2n=20), would produce unviable hybrids." Contrary to this citation, many other studies show that pollen- and seed-mediated gene flow to wild and weedy relatives of sorghum is expected to be extensive (e.g., De Wet and Harlan 1971; Holm et al. 1977; Arriola and Ellstrand 1996; Ejeta and Grenier 2005; and references therein).

Despite having different ploidy numbers, sorghum and Johnsongrass easily hybridize and produce fertile offspring in the USA (Arriola and Ellstrand, 1996, 1997). Johnsongrass is self-incompatible, and crop-wild hybrids were detected 100 m away from the crop, which was the greatest distance examined. Crop genes appear to accumulate and persist in Johnsongrass populations (Morrell et al. 2005). These authors found 32% frequencies of putative crop-specific alleles in Johnsongrass populations that were adjacent to sorghum fields in Texas and Nebraska, USA. Surprisingly, they also found low frequencies of crop-specific markers in Johnsongrass populations with no recent exposure to the crop. Therefore, they concluded that gene flow among Johnsongrass populations could provide a bridge for the wide dissemination of transgenes following initial hybridization with the crop.

Sorghum also is expected to hybridize with its conspecific wild relatives with 2n=20 chromosomes, which are widespread in Africa and elsewhere (De Wet and Harlan 1971, Schmidt and Bothma 2006, Ejeta and Grenier 2005). "Shattercane" is a common name that is often applied to weedy conspecifics of Sorghum bicolor, some of which may represent feral crop plants (e.g., Dahlberg 2000, Ejeta and Grenier 2005). Research on genetic transformation of sorghum should and will continue as it may render solutions to the more intractable problems of sorghum production and nutrition. However, it is imperative that parallel research is also underway to 1) further understand the nature and extent of gene flow between sorghum and its wild relatives, 2) assess the effects of gene flow from proposed transgenic lines with new traits, and 3) mitigate potential problems that may arise as a result of gene flow between and among sorghum and its wild and weedy relatives. In general, transgenes that are incorporated into sorghum crops are expected to disperse via seed transport, seed mixing, as well as hybridization with cultivated varieties and weedy relatives. It will, therefore, be important to consider all of these processes when new transgenic sorghums are deployed for large scale cultivation or are proposed even for limited environmental release.

ENVIRONMENTAL SAFETY OF TRANSGENIC SQUASH: A GEOSTATISTICAL ANALYSIS
Ferdinand E. Klas, Marc Fuchs, & Dennis Gonsalves

Viruses can substantially reduce fruit production and quality in summer squash (Cucurbita pepo L.), with yield losses that often range from 20 to 80%. A $2.6 million economic loss from squash virus was reported in the state of Georgia in 1997¹. Two of the most important viruses affecting squash production are zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus (WMV)². These two viruses are transmitted by several aphid species in a noncirculative and nonpersistent manner². The ideal strategy to control ZYMV and WMV is to use resistant cultivars; however as no summer squash cultivar with satisfactory resistance to these two viruses has yet been developed by conventional breeding^3,4, control is routinely achieved by cultural practices, including delayed transplanting relative to aphid flights, use of film mulch to repel aphids, and application of stylet oil in combination with insecticides to reduce aphid vector populations⁵. In the state of Georgia, it is estimated that ten applications per field per year of stylet oil and insecticides are made routinely to control aphids, and hence, limit virus incidence and transmission².

Transgenic squash ZW-20 expresses the coat protein (CP) gene of ZYMV and WMV and is highly resistant to single and mixed infection by these two viruses^6-10 (Fig. 1A). Virus-resistant squash line ZW-20 was the first disease-resistant transgenic crop to receive exemption status in the USA in 1994¹¹ and cultivars derived thereof, including crookneck, straightneck, and zucchini types, were commercially released starting in 1995^3,7.

The reaction of virus-resistant transgenic crops, including transgenic squash ZW-20, to virus infection is usually based on a comparative analysis of disease incidence in transgenic versus conventional cultivars. This approach relies on differences in the percentage, mean, and/or variance of infected plants between two genetically distinct cultivars^6-10. While it is powerful to evaluate the level of resistance, this approach does not provide information on the spatial relationship among test plants nor does it illustrate disease progress in space. Geostatistical analysis, in contrast, accounts for the position of a plant in space and quantifies the degree of spatial dependence with its neighbors^12,13. If transgenic plants are immune to virus infection, the spatial relationship among test plants would not be significant. However, if transgenic plants show resistance to systemic infection but have local infection in the form of chlorotic dots or blotches, it would be interesting to determine if they can serve as sources of inoculum for secondary virus spread by vectors.

Hypothetically, for any given pair of plants in a field, one can influence the infectious status of another by serving as virus source for aphid-mediated virus transmission. This degree of dependence on virus spread between plant pairs can be quantified and modeled by geostatistical analysis of data. The parameters measured are the infectious status of the test plants (healthy or infected) and the location (coordinates) of the two samples within a field. From the latter measurement the distance between paired samples could be determined. The intrinsic hypothesis is that the spatial influence (quantified as the semivariance) between two samples of a plant pair depends only on their mutual distance. As distance between sample pairs increases, the influence that one sample will have on another in terms of virus spread usually decreases. As a result, the value that quantifies the degree of relationship between paired samples increases. These quantification values (semivariance) over distance are plotted as semivariograms. The semivariograms are further used to estimate the range of spatial association by model fitting, and the significance of the model fitting is indicated by the determination coefficient (R²). If paired samples have no influence on each other, regardless of the distance, or their influence is over a very short distance, the semivariogram plot is flat or the values oscillate. Such a semivariogram is not well structured. Thus, if a plant that is systemically infected with an aphid transmissible virus serves as source of virus inoculum for secondary spread by vectors, its influence on the infectious status of another plant would decrease over distance and result in a well structured semivariogram. Likewise, if two plants are resistant to an aphid transmissible virus, there would be limited, if any, association between them in terms of secondary virus spread, regardless of distance. This would result in a poorly structured semivariogram.

Recently, we reported on a comparative analysis of the incidence of ZYMV and WMV in space and time in fields of transgenic ZW-20 and conventional squash¹⁴. Initial work on transgenic ZW-20 identified two lines, designated ZW-20H and ZW-20B⁷. Line ZW-20H was used to develop commercial transgenic squash cultivars resistant to ZYMV and WMV^3,6,7. Although both lines are resistant to systemic infection of ZYMV and WMV, they exhibit local infection with ZW-20H developing chlorotic dots and ZW-20B developing chlorotic dots and chlorotic blotches⁶. The presence of chlorotic blotches suggested that ZW-20B is less resistant than ZW-20H to infection with ZYMV and WMV. Thus, we were interested in using a geostatistical approach to determine if these plants might serve as source of virus inoculum for secondary aphid-mediated spread¹⁴.

Field experiments were conducted over two consecutive years with several plots of either transgenic or conventional squash¹⁴. The first year experiments compared ZW-20B and nontransgenic plants, while the second year experiments included ZW-20H, ZW-20B and nontransgenic plants. Each plot was surrounded by a single row of nontransgenic plants, which were mechanically inoculated with ZYMV or WMV prior to transplanting and used as primary virus source for aphid-vectored infections. This layout simulated field settings where viruses are transmitted from external sources into solid blocks of transgenic squash, thereby approaching conditions of natural virus infection in commercial fields of transgenic squash. No insecticide was used so as not to impede efficient virus spread by indigenous aphid populations. Virus incidence was monitored on test plants by visual observation of symptoms every 3-4 days and by double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) using immunoglobulins specific to ZYMV and WMV¹⁴. Data on symptom development over time, symptom type, and DAS-ELISA scores were mapped for transgenic and nontransgenic plants at three time intervals in both growing seasons. Geostatistical analysis was conducted from maps of ELISA-positive plants and experimental semivariograms were established for both ZYMV and WMV at each sampling date to characterize the spatial dependence of test plants that reacted positively in DAS-ELISA, except those in border rows that served as virus source¹⁴.

Test plants used in our study were transgenic squash ZW-20H and ZW-20B, and conventional squash cultivar Pavo, which has the same genetic background as the two transgenic hybrids. Our study indicated that, across all experiments, transgenic ZW-20H and ZW-20B plants showed no systemic symptoms upon mixed infection by ZYMV and WMV (Fig. 1B), but 40% of transgenic ZW-20H developed localized chlorotic dots (Fig. 1D) and 60% of transgenic ZW-20B developed localized chlorotic dots and /or blotches (Fig. 1E) that were confined mainly to old leaves¹⁴. Both viruses were detected by DAS-ELISA in symptomatic but not in asymptomatic leaf tissues. Some transgenic plants reacted positively for ZYMV and WMV but, remarkably, the rate of mixed infection was low (4 – 12%)¹⁴. In contrast, conventional squash exhibited severe systemic symptoms, including mosaic, vein yellowing, chlorosis, leaf deformation, and stunted growth (Fig. 1C). Target viruses were readily detected in most, if not all, control plants (95-100%) and the rate of mixed infection was high (57 – 86%)¹⁴.

The temporal patterns of symptomatic transgenic plants were strikingly different from those of symptomatic conventional plants. Transgenic plants that reacted positively for ZYMV and WMV in DAS-ELISA tests were scarce and did not increase in number at the same magnitude as controls. In contrast, the patterns of symptomatic ELISA-positive conventional plants were increasingly dense over time¹⁴. Similarly, the spatial spread patterns of ZYMV and WMV in transgenic squash were scarce while those in conventional squash were uniformly dense (Fig. 2).

While symptom readings and virus detection by DAS-ELISA clearly showed that ZW-20H and ZW-20B are resistant to systemic infection by ZYMV and WMV, the potential of transgenic plants with chlorotic dots or blotches to serve as effective inoculum source for secondary aphid-mediated dissemination of the two viruses needed to be determined. Geostatistical analysis of virus spread suggested that transgenic plants did not serve as virus source for secondary spread of WMV, although ZW-20B could serve as a virus source for spread of ZYMV.

Experimental semivariograms describing the spatial structure of WMV (Fig. 3B, F, and J) in fields of transgenic squash were completely flat or oscillating, thus exhibiting a poor fit of the theoretical model¹⁴. These results suggested a lack of spatial dependence among most transgenic plants and, therefore, no plant-to-plant virus spread of WMV. However, the semivariogram for the spread of ZYMV suggested that ZW-20B (which developed blotches) could serve to some extent as inoculum source in 1994, but not in 1995 (Fig. 3I). Also, a well structured linear semivariogram described the spatial structure of ZYMV in ZW-20H (Fig. 3E). As this semivariogram reveals a long range of spatial dependence, influx of ZYMV likely occurred from beyond the limits of the experimental field site and was not achieved by plant to plant transmission within the plot. In contrast, well-structured semivariograms characterized the spread of ZYMV (Fig. 3 C and G) and WMV (Fig. 3D, H and L) in fields of conventional squash, indicating virus spread has a strong spatial dependence¹⁴. However, there was one exception with a semivariogram showing oscillating values for ZYMV spread in 1995 (Fig. 3K). A high incidence of ZYMV with 91% infected plants likely accounts for reduced spatial variability and therefore yielded a random semivariogram, as reported previously¹⁵.

Screen cage experiments were conducted to further assess the role of test plants as source of virus inoculum under more controlled field conditions. Accordingly, a few test plants in the center of each field plot were covered late in the second growing season with screenhouses¹⁴. Covered plants were heavily sprayed twice with insecticides to eliminate indigenous aphids. Then, healthy nontransgenic squash were transplanted in close spatial proximity to established test plants. Next, aviruliferus Myzus persicae were deposited on symptomatic leaves of test plants (50 per plant) at 2 – 3 days intervals over 18 days. None of the virus recipient squash plants (0 of 24) became symptomatic in the screenhouses covering transgenic ZW-20H plants, while 100% (67 of 67) of recipient squash plants became symptomatic in screenhouses covering conventional squash¹⁴. These results indicated that Myzus persicae efficiently transmitted viruses from symptomatic conventional squash plants but not from transgenic ZW-20H plants. Notwithstanding, 14 of 38 (37%) of recipient squash became symptomatic in a screenhouse covering transgenic ZW-20B plants. These observations confirmed the semivariogram predictions.

Altogether, our data suggest that transgenic ZW-20H plants do not serve as sources of virus inoculum for secondary aphid-mediated spread of ZYMV and WMV, while transgenic ZW-20B can serve as limited source of ZYMV inoculum for secondary aphid-mediated spread. In contrast, nontransgenic plants infected with ZYMV and WMV readily served as inoculum source for aphid-mediated spread of these two viruses.

Summer squash cultivars derived from transgenic line ZW-20H, including the crookneck hybrid tested in our study¹⁴, were commercially released more than a decade ago^3,6,7. A good adoption rate by growers mirrors their successful commercialization. In 2005, virus-resistant transgenic squash, including cultivars derived from line ZW-20H, accounted for 12% of the total acreage in the USA, with the highest adoption rate in New Jersey (25%), Florida (22%), South Carolina, Tennessee, and Georgia (20%)¹⁶. In addition, initial yields in the absence of viruses were restored with the cultivation of virus-resistant transgenic squash¹⁶.

The release of virus-resistant transgenic squash has raised concern about their impact on the environment^17-21. Accordingly, potential risks related to heteroencapsidation and recombination, among others, have been expressed^17-21. Heteroencapsidation refers to the encapsidation of the genome of a challenge virus by the CP subunits expressed in a transgenic plant expressing a viral CP gene. Resulting virions may have new properties. For example, an otherwise vector nontransmissible virus could become transmissible through heteroencapsidation. Also, a virus could infect an otherwise nonhost plant. Recombination refers to the exchange of genetic material between transcripts of a viral transgene and the genome of a challenge virus during replication. Resulting recombinant viruses with chimeric genomic molecules may possess altered biological properties compared to their parental lineages, such as changes in vector specificity, expanded host range, and increased pathogenicity. Consequently, new virus epidemics could result from recombination and heteroencapsidation.

Our study confirmed in commercial-type, small-scale field settings the high level of resistance of transgenic squash ZW-20 plants to ZYMV and WMV infection previously described^6-10,14. In addition, geostatistical analysis provided new insights into the impact of commercial virus-resistant transgenic squash on the dynamics of virus spread. Semivariograms expressing random spatial structures and field cage experiments inferred that transgenic squash ZW-20H supported no plant-to-plant transmission of ZYMV and WMV¹⁴. Actually, it limited the incidence of ZYMV and WMV by restricting the amount of infected tissue, lessening the rate of mixed infection, and reducing the availability of virus inoculum for acquisition and subsequent spread by aphid vectors^6,7,14. These features substantially reduce opportunities for challenge viruses to interact with viral transgene-derived products, i.e., protein and transcripts. Therefore, it is reasonable to predict that transgenic squash line ZW-20H and any cultivar derived thereof that display a similarly high level of resistance against ZYMV and WMV as the crookneck hybrid tested in our study¹⁴ are unlikely to efficiently assist the transmission of aphid nontransmissible strains of ZYMV and WMV through heteroencapsidation, and transmission of recombinant strains of ZYMV and WMV.

Considerable attention has been paid to potential environmental risks associated with the release of virus-resistant transgenic crops over the past 15 years²². Significant progress has been made and remarkable advances provide new insights into the significance of the major safety issues. In particular, field environmental safety assessment studies have provided evidence of limited, if any, environmental risk beyond background events²². So far, there is no compelling evidence to indicate that transgenic plants expressing viral genes increase the frequency of heterologous encapsidation or recombination. Similarly, there is little evidence to infer that transgenic plants expressing viral genes alter the properties of existing virus field populations or create new viruses that otherwise could not emerge in conventional plants subjected to multiple virus infection. Our findings indicate that safety issues over virus-resistant transgenic squash and virus-resistant transgenic crops, in general, are substantially less significant than initially predicted^17-21, particularly from the standpoint of heteroencapsidation and recombination.

With nitrogen fertilizer prices hovering at about $500/ton, one of the most eagerly awaited next generation biotech crop traits is greater nitrogen use efficiency, which would help farmers save on input costs and have environmental benefits as well. Several companies are working on nitrogen efficient crops, including Arcadia Biosciences, which recently announced that development of Nitrogen Use Efficient (NUE) canola is showing early success.

In addition to eight successful field trials completed over five growing seasons, Davis, Calif.-based Arcadia established a collaboration with Monsanto Company in 2005 to develop NUE canola, and early field trials indicate notable progress. Field trials have demonstrated that NUE canola can maintain normal yield while using 50% less nitrogen fertilizer, or increase yields by 15% or more under conventional fertilizer use rates.

Conventional crops can only absorb about one-half of the nitrogen that is applied in the form of fertilizer. The other one-half may enter the atmosphere, ground water, and surface waters. Because it enables farmers to increase the amount of crop yield per unit of nitrogen fertilizer used, NUE technology provides the opportunity to increase profitability and help improve the environment.

Most of the nation’s canola is grown in North Dakota. Canola oil is recognized as a healthy cooking oil and is rapidly becoming established as a feedstock for biodiesel production. More than half of all canola grown in the U.S. is genetically modified, according to industry estimates.

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Book Description This book brings together experts from a variety of perspectives on bioengineered food, which holds the promise of radically reducing hunger in the third world but which is mired in political controversy.

From the Publisher More than one million of the world’s poorest children die each year from a lack of Vitamin A. Another 100 million children suffer from Vitamin A deficiency, which increases the risk of blindness, infections, and diseases such as measles and malaria. Yet a revolutionary solution to this malignant crisis—a vitamin-enhanced rice—remains unutilized, the victim of anti-science advocacy groups.

The sad fate of Golden Rice, the genetically modified version of the world’s most popular staple, is one of many revelations in Let Them Eat Precaution: How Politics Is Undermining the Genetic Revolution in Agriculture (AEI Press, January 2006). Bioengineering has created new kinds of soybeans, wheat, and cotton that generate natural insecticides (making them more resistant to pests and drought and increasing yields); nutrition-added fruits, vegetables, and grains; and futuristic "farmaceuticals"— life-saving medicines made by melding agricultural methods with advanced biotechnology. Countless scientific studies have found that biotech farming can dramatically reduce reliance on costly and environmentally harmful chemicals, and the products that result are safe and healthy.

Editor Jon Entine, along with ten experts from the United States and Great Britain, explain why cultural politics and trade disputes, not science, pose the biggest hurdles in developing these products. Instead of meeting the desperate needs of the world’s poor with new medicines and vitamin-fortified crops, anti-biotech campaigners offer liberal doses of the "precautionary principle"—the controversial notion that innovation should be shelved unless all risks can be avoided. Well-funded environmental groups such as Greenpeace and Friends of the Earth; organic advocates; religious groups such as Christian Aid; and "socially responsible" investors exploit anxiety about science, caricaturing genetic technology as inherently unpredictable and a "genetic Godzilla" that could usher in an age of "Frankenfoods."

Let Them Eat Precaution includes:

"Beyond Precaution" by Jon Entine, scholar in residence at Miami University of Ohio, and adjunct fellow at the American Enterprise Institute.

"Global Views on Agricultural Biotechnology" by Thomas Jefferson Hoban, director of the Center for Biotechnology in a Global Society and professor in the departments of sociology, anthropology, and food science at North Carolina State University. Mr. Hoban is also a member of the Advisory Committee on Agricultural Biotechnology at the U.S. Department of Agriculture (USDA).

"Agricultural Biotechnology Caught in a War of Giants" by C.S. Prakash, professor of plant biotechnology at Tuskegee University and president of AgBio World Foundation; and by Gregory Conko, senior fellow and director of food safety policy at the Competitive Enterprise Institute.

"Trade War or Culture War? The GM Debate in Britain and the European Union" by Tony Gilland, science and society director at the British Institute of Ideas.

"Hunger, Famine, and the Promise of Biotechnology" by Andrew S. Natsios, administrator of the U.S. Agency for International Development (USAID).

"Let Them Eat Precaution: Why GM Crops Are Being Over-Regulated in the Developing World" by Robert L. Paarlberg, professor of political science at Wellesley College; associate of the Center for International Affairs at Harvard University; and consultant for the International Food Policy Research Institute, USAID, USDA, and U.S. State Department.

"Can Public Support for the Use of Biotechnology in Food Be Salvaged?" by Carol Tucker Foreman, director of the Food Policy Institute at the Consumer Federation of America and former assistant secretary for food and consumer services at the USDA.

"Deconstructing the Agricultural Biotechnology Protest Industry" by Jay Byrne, president of v-Fluence Interactive Public Relations (dealing with issues management, including biotechnology).

"Functional Foods’ and Biopharmaceuticals: The Next Generation of the GM Revolution" by Martina Newell-McGloughlin, director of the Systemwide Biotechnology Research and Education Program at the University of California-Davis; co-director of the NIH Training Program in Biomolecular Technology; member of the Genomics Panel on Technology of the WTO; and member of the Technology Discussion Panel on Sustainable Agriculture at the UN.

"Challenging the Misinformation Campaign of Antibiotechnology Environmentalists" by Patrick Moore, founding member of Greenpeace and former director of Greenpeace International. Mr. Moore now heads the environmental group Greenspirit in Vancouver, Canada.

Praise for Let Them Eat Precaution
"Let Them Eat Precaution does a superb job of educating the reading public on the basic issues of genetically modified foods. The distinguished authors provide a devastating point-by-point refutation of the anti-GMO activists’ false claims, providing a reasoned, scientifically grounded perspective on this critical issue. As the Marie Antoinette title implies, though the affluent may be leading the charge against GMO foods, it is the poor who are most likely to suffer the effects of activists that falsely claim to speak for the world’s poor."  
– Thomas DeGregori, professor of economics, University of Houston, and author of Origins of the Organic Agriculture Debate.

"A well-funded global antibiotech activist campaign, abetted by European Union regulators more interested in political pandering than good science, threatens to starve millions of the world’s poorest people by denying them access to environmentally safer and higher yielding biotech crops. The distinguished experts assembled in Let Them Eat Precaution make it abundantly clear that humanity’s health and well-being depend on innovation, not a technological freeze in the name of the "precautionary principle," which demands perfect safety from all new technologies. The contributors carefully document not only the policy challenges facing agricultural biotechnology but the real benefits—from a massive reduction in pesticide use to a slew of new pharmaceuticals and vitamin-enriched foods—that may never come to fruition if antiscience advocacy groups prevail in this battle of ideas."  
– Ronald Bailey, author of Liberation Biology: The Scientific and Moral Case for the Biotech Revolution and science correspondent for Reason magazine

"This fine volume fills a very useful role in the ongoing debate over the use of biotechnology in foods and pharmaceuticals. Let Them Eat Precaution covers every aspect of the issue, catalogs what is known about GM crops, and helps us understand the ideological bias is for opposition to the use of this life-saving technology. The antibiotechnology campaigns are denying food to starving millions– a high price to pay for ideology."
– Peter Raven, director of the Missouri Botanical Garden, St. Louis, Mo.