INFORMATION SYSTEMS FOR BIOTECHNOLOGY


April 2008
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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SEND IN THE CLONES
Phill Jones

On July 5, 1996, scientists at Scotland's Roslin Institute succeeded in cloning the first livestock mammal using a somatic cell obtained from an adult animal. To produce Dolly the sheep, researchers replaced the nucleus of an egg with a nucleus isolated from the udder of a six-year old sheep. Roslin Institute announced Dolly's birth in early 1997. The news incited visions of herds of cloned animals. The point might have been missed that researchers had tried over 270 times to produce one cloned sheep.

Dolly's arrival raised questions about food products made from cloned livestock. Would the food be safe for human consumption? In January, US and European agencies offered their views. The opinions are so similar, they're almost clones.

Agencies Make Themselves Heard about Clones
In 2001, US Food and Drug Administration officials decided that cloning may become a standard method for improving livestock. The FDA's Center for Veterinary Medicine (CVM) asked livestock producers to keep food products derived from animal clones or their offspring out of the human food supply until the FDA could evaluate food safety. For five years, CVM researchers analyzed hundreds of peer-reviewed publications and data from unpublished studies on the health of clones and their offspring, and on the composition of food produced from the animals.

In December 2006, the FDA released three documents on animal cloning: a draft risk assessment, a proposed risk management plan, and a draft guidance for industry. The draft risk assessment revealed that the FDA deemed meat and milk from clones of adult cattle, swine, and goats, as well as their offspring, to be as safe to eat as food from conventionally-bred animals. The FDA also considered the effects of cloning technology on animals. "Cloning poses no unique risks to animal health," said CVM director Stephen F. Sundlof in a press release, "when compared to other assisted reproductive technologies currently in use in US agriculture."

The FDA sought comments from the public on its three draft documents. The agency received over 30,000 remarks.

On January 15, 2008, the FDA issued final versions of the three draft documents. The agency reiterated its assessment that the cloning process poses no unique risks to animal health, compared to the risks of other reproduction methods; that the composition of food products derived from cattle, swine, and goat clones, or their progeny, does not differ from food products of conventionally-bred animals; and that food derived from cattle, swine, and goat clones, or their offspring, does not pose unique risks to consumers. Due to a lack of information, the FDA did not include assessments of other cloned livestock animals, such as cloned sheep.

Under current law, the FDA may require food labeling if the agency has safety concerns, or if the agency finds a material difference in the composition of food. In the FDA's view, neither condition applies to food derived from clones or their offspring. Accordingly, the FDA will not compel labels to alert consumers that a product contains ingredients from cloned livestock or progeny.

The FDA underscores that their conclusions for the three livestock species have varying degrees of uncertainty. One source of uncertainty arises from the present understanding of the epigenetic processes involved in early embryonic development. Clones are genetically identical, but they differ in the epigenetic state of their genomes. For example, the genomes of clones differ in DNA methylation. Another source of uncertainty arises from ongoing developments in cloning technology. New techniques may introduce hazards not found in current cloning methods. The FDA has stated that it will continue to monitor the developing state of knowledge about animal cloning and new cloning technology.

The agency also addresses the manufacture of animal feed from clones. In its Guidance for Industry, the FDA takes the position that clones of any species could be used in the production of feed for animals without additional restriction. "Animal Cloning: A Risk Assessment" and other related documents are available from the FDA website (http://www.fda.gov/cvm/cloning.htm).

Animal cloning techniques have been associated with an increased risk of unfavorable health outcomes in surrogate dams carrying late-term clone fetuses and in very young clones, particularly in cattle and sheep. To minimize the impact of animal health risks, the FDA has been collaborating with the International Embryo Transfer Society to prepare a manual on animal care standards for animals involved in the cloning process. The two organizations plan to release a copy of the manual on the Society's website early this year.

On the day that the FDA announced the release of its animal cloning documents, Bruce Knight, the USDA's Under Secretary for Marketing and Regulatory Programs, stated that his organization agrees with the FDA's opinions about food safety. "Now that FDA has evaluated the scientific data and public comments and issued its final risk assessment," he said, "USDA will join with technology providers, producers, processors, retailers and domestic and international customers to facilitate the marketing of meat and milk from clones. We'll be working closely with stakeholders to ensure a smooth and seamless transition into the marketplace for these products." In the meantime, the USDA encourages the industry to maintain the voluntary moratorium on sending milk and meat from animal clones into the food supply.

On January 11, the European Food Safety Authority issued its draft document on a review of food safety concerns. "The currently available data indicate," says EFSA, "that food products from clones of cattle and pigs and their progeny are as safe as food products of livestock derived by conventional breeding." EFSA also concluded that "there is no expectation that clones or their progeny would pose any new or additional environmental risks compared with conventionally bred animals." After reviewing comments from the public, EFSA plans to finalize its opinion by May 2008. A copy of the draft opinion is available on the EFSA website (http://www.efsa.europa.eu).

The European Group on Ethics in Science and New Technologies released its opinion on ethical aspects of animal cloning on January 16 (http://ec.europa.eu/european_group_ethics/index_en.htm). It is not a resounding endorsement of the technology. "Considering the current level of suffering and health problems of surrogate dams and animal clones," the group said, "the EGE has doubts as to whether cloning animals for food supply is ethically justified." The organization decided that further research is required to determine whether this applies to the progeny of clones. "At present, the EGE does not see convincing arguments to justify the production of food from clones and their offspring." EFSA's scientific conclusions and the report on ethical aspects of animal cloning will help to inform decisions about animal cloning by the European Community and the European Parliament.

Fears of a Clone
The FDA's long-awaited final opinion on food safety did not please everyone. "The FDA has acted recklessly and I am profoundly disappointed in their rush to approve cloned foods," declared Senator Barbara A. Mikulski (D-Md.) in a January 15, 2008, press release. "[T]he FDA has rushed into a decision that could have dangerous consequences. The long term effects of these products are still unknown and could be harmful to consumers." Last year, Senator Mikulski sponsored a bill to amend the Federal Food, Drug, and Cosmetic Act and the Federal Meat Inspection Act to require that food that contains products from a cloned animal or its progeny be labeled accordingly.

The flipside of labeling the presence of cloned animal ingredients is to label food as free from such ingredients. Since the FDA issued its draft documents in 2006, consumers have been pushing for clone-free labels on food products derived from conventional livestock. The FDA has said that, should a manufacturer want to voluntarily label a product as "clone-free," the request will be reviewed to ensure compliance with requirements that labeling be truthful and not misleading.

For now, stud farms and breeders constitute the target market for animal cloning technology. A clone, which can represent an investment of $20,000, is too valuable to milk or to slaughter for meat, especially when the milk and meat are not worth more than that from a conventional animal. It may be years before food products from clone progeny flock onto grocery store shelves.

Selected Sources

Cohen, R (2008) Animal Cloners Eager to Whet Public's Appetite. The Star Ledger. January 8, 2008. Available at: http://www.nj.com.

Statement by Bruce Knight, Under Secretary for Marketing and Regulatory Programs on FDA Risk Assessment on Animal Clones. USDA Press Release. January 15, 2008. Available at: http://www.usda.gov.

Tessler, J (2008) Bull Market for Clones: Studs, Not Stock. The Associated Press. January 16, 2008.

Weiss, R (2008) Food From Clones Safe, E.U. Draft Says. The Washington Post, A06 (January 12, 2008).

Weiss, R (2008) FDA Says Clones Are Safe For Food. The Washington Post, A01 (January 15, 2008).

Weiss, R (2008) Labels Weighed for Food From Clones. The Washington Post, A09 (January 20, 2008).

Phill Jones
Biotech-Writer.com
PhillJones@nasw.org



THE IPKb VECTOR SET: MODULAR BINARY PLASMIDS FOR CEREAL TRANSFORMATION
Jochen Kumlehn

The systematic collection of plant genetic resources, along with the acquisition of plentiful genomic sequence and gene expression data and the elaboration of methods allowing for both transient and stable transgene expression, have encouraged a series of novel experimental approaches to understand the genetical and physiological bases of plant phenotype. Genetic transformation, a critical technology in this process, remains, however, cumbersome and time-consuming in the cereals, which supply the bulk of the global requirement for food and feed. The vector systems that work well as transformation agents for many dicotyledonous species are, unfortunately, of only limited utility in the monocotyledons, largely because commonly used promoter sequences and/or selectable markers are ineffective in a monocotyledonous host. In addition, Agrobacterium-mediated transformation, which operates very efficiently in many dicotyledonous hosts, remains a demanding technology in the monocotyledons.

In a recent paper, Himmelbach and co-workers described a novel set of modular binary vectors (the IPKb series), specifically tailored for cereal transformation and targeted to either over-expression or RNA-interference (RNAi)-mediated gene knock-down1. In both types, the insertion of effector sequences is facilitated by the exploitation of GATEWAY destination cassettes, which permit the efficient, site-specific and reliable exchange of DNA fragments between plasmids (Fig. 1). Any DNA sequence can be readily transferred from an easily cloned entry vector to the binary destination vector via an LR reaction, a procedure that avoids the need for the digestion and ligation-based cloning of the typically rather large binary vectors. This is particularly advantageous in the context of RNAi vectors, in which two inverted DNA repeats need to be connected by a spacer or intron sequence. The IPKb RNAi vectors pIPKb006 through pIPKb010 contain an inverted repeat of GATEWAY destination cassettes, separated by the wheat RGA2 intron2. The IPKb vector set also includes derivatives both of the over-expression and RNAi types in which various promoters, which are fully functional in monocotyledonous species, have been inserted to drive ubiquitous or epidermis-specific transgene expression (Table 1). Any other established or de novo isolated promoter sequence can be readily inserted upstream of the GATEWAY destination cassette of either the over-expression vector pIPKb001, or the RNAi vector pIPKb006, thereby increasing the versatility of the IPKb vector set.


Figure 1. Schematic representation of the IPKb over-expression and RNAi-mediated gene knock-down binary plasmid types. A gene sequence of interest (GOI) can be exchanged between an appropriate entry vector and an IPKb destination vector using the GATEWAY recombination system, as indicated by the attL and attR recombination sites (L1, L2, R1 and R2) and the dashed arrows. Derivatives are available for both plasmid types, involving any of the four promoters shown, or with a multiple cloning site (MCS), allowing the integration of further promoters. CmR = chloramphenicol resistance gene for selection of bacteria; ccdB = toxin gene for negative selection of bacteria; RGA2Int = intron of wheat RGA2 gene; RB and LB = right and left T-DNA borders; ColE1 = E. coli origin of replication; pVS1 = Agrobacterium origin of replication; SpecR = spectinomycin resistance gene for selection of bacteria; T = transcription termination sequence; HptR = hygromycin resistance gene for plant selection; ZmUbi1P = maize ubiquitin 1 promoter; OsAct1P = rice actin 1 promoter; d35SP = doubled enhanced CaMV 35S promoter; GstA1P = wheat glutathione-S-transferase 1 promoter; SfiIA and SfiIB = SfiI restriction sites.

Since specific selection markers may be preferred for some target hosts, or may be necessary for certain gene stacking strategies, the IPKb vectors have been constructed to allow for the ready introduction of further marker gene expression cassettes. This can be accomplished via the exchange of the selectable marker-containing SfiI fragment with that of compatible vectors such as 5U, 7U, and 9U provided by the DNA Cloning Service, Hamburg, Germany (http://www.dna-cloning-service.com). For example, this approach enables access to the dihydrofolate reductase gene (dhfr, resistance to metothrexate), the phosphinothricin-N-acetyl transferase gene (pat, resistance to phosphinothricin) and the neomycin phosphotransferase II gene (nptII, resistance to kanamycin) in which the selectable marker genes are under control of the strong ubiquitous maize ubiquitin 1 (ZmUbi1) promoter (Fig. 2). Other combinations of promoters and selectable marker genes are also feasible. The swapping of plasmid fragments is achieved in a single recombination step, using the rare cutter SfiI, which has a 13nt recognition site including five variable nucleotides in central position. Directed ligation of plasmid fragments is accomplished between appropriate pairs of single-strand overhangs derived from either SfiIA or SfiIB. As a further option, any of the available binary vectors can be engineered to lack a plant selectable marker expression cassette, which is unnecessary where the efficiency of transformation is known to be high, and which allows for the production of instantly marker-free transgenic plants. To achieve this, the SfiI fragment containing the plant selection marker is simply replaced by the compatible marker-free fragment of the B-BA binary vector (DNA Cloning Service, Hamburg, Germany).

The IPKb series was based on the generic binary vector 6U (DNA Cloning Service Hamburg, Germany), in which the hygromycin phosphotransferase (hpt) coding sequence fused to the ZmUbi1 promoter acts as a very effective selectable marker gene cassette. A further important feature of 6U and its derivatives is that they harbor the pVS1 origin of replication, which ensures good plasmid persistency in agrobacteria even under non-selective conditions3, and thereby maintains the transformation competency of the Agrobacterium population during the entire co-cultivation period. In conjunction with various promoter-reporter and promoter-effector constructs, 6U produces a stable and high transformation efficiency in both barley4,5 and wheat (Hensel and Kumlehn, unpublished).

The functionality of pIPKb002 to pIPKb005 was tested by introducing the gus gene into the GATEWAY destination site, followed by Agrobacterium-mediated transformation of barley and the subsequent expression analysis of stable transgenic plants. The transformation efficiency of the IPKb plasmids was on a par with that of conventional 6U-based binary vectors without a GATEWAY cassette5. The resultant T1 seedlings expressed GUS, with the strongest expression present in the leaves of lines transformed with pIPKb002_GUS (driven by the ZmUbi1 promoter), followed by pIPKb003_GUS (OsAct1 promoter), pIPKb005_GUS (TaGstA1 promoter), and pIPKb004_GUS (d35S promoter). For the transgenic pIPKb005_GUS lines, fluorescence spectroscopy revealed that GUS activity in isolated abaxial epidermis was, on average, ten times stronger than in remaining leaf tissue. This result not only provides an example of tissue-specific transgene expression achieved through the use of an IPKb vector, but also opens the way to their use as a tool for studying and manipulating the interaction between barley and many of its pathogens.

The binary destination vectors pIPKb007 through pIPKb010 drive the expression of RNAi sequences, with transcriptional regulation provided by the same promoters used for the IPKb over-expression vectors. The functionality of the RNAi-vectors was verified via the biolistic delivery into barley leaf tissue of vector derivatives targeted against Mlo, a negative regulator of resistance against the causal pathogen of barley powdery mildew6. All of the plasmids tested (pIPKb007_Mlo to pIPKb010_Mlo) produced a phenocopy of the loss-of-function mlo resistance, indicating that the presence in planta of the Mlo-RNAi constructs acted to reduce the transcription of MLO, and hence increased the level of resistance to powdery mildew.

In addition to the generation of numerous stable transgenic barley and wheat plants using various derivatives of the IPKb destination vectors, pIPKb002_GUS and pIPKb004_GUS proved also effective for the stable transformation of tobacco, where fluorescence spectroscopy was able to demonstrate the ubiquitous expression of GUS. Thus the IPKb vector set provides an appropriate vehicle to compare transgene expression in mono- and dicotyledonous species using an identical binary vector.

The IPKb vector set provides a framework for the development of derivatives with further promoters, plant selection markers, sequences suited for homologous recombination-mediated marker deletion strategies, affinity or screenable tags that can be N- or C-translationally attached to the coding sequence, and for the development of systems permitting T-DNA insertion mutagenesis in cereals. The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) will supply pIPKb001 through pIPKb010 gratis on request for non-commercial research use.

References

1. Himmelbach A, Zierold U, Hensel G, Riechen J, Douchkov D, Schweizer P and Kumlehn J (2007) A set of modular binary vectors for the transformation of cereals. Plant Physiology 145, 1192-1200

2. Douchkov D, Nowara D, Zierold U, Schweizer P (2005) A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Molecular Plant Microbe Interactions 18, 755-761

3. Itoh Y, Watson JM, Haas D, Leisinger T (1984) Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid 11, 206-220

4. Kumlehn J, Serazetdinova L, Hensel G, Becker D and Loerz H (2006) Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnology Journal 4, 251-261

5. Hensel G, Valkov V, Middlefell-Williams J and Kumlehn J (2008) Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. Journal of Plant Physiology 165, 71-82

6. Bueschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, Van Daelen R, Van der Lee T, Diergaarde P, Groenendijk J, Toepsch S, Vos P, Salamini F, Schulze-Lefert P (1997) The barley Mlo gene: A novel control element of plant pathogen resistance. Cell 88, 695-705

Jochen Kumlehn
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)
Plant Reproductive Biology
Gatersleben, Germany
kumlehn@ipk-gatersleben.de



GENE FLOW AMONG TRANSGENIC PLANTS AND THEIR WILD RELATIVES: IMPLICATIONS FOR RISK ASSESSMENT
Allison Snow

Research on gene flow from transgenic plants marches on! For example, the North Central Weed Science Society hosted the second biannual symposium on gene flow on December 12 13, 2007, in St. Louis, Missouri. Abstracts of the 38 oral presentations and posters are available at http://www.ncwss.org/. The meeting brought together academic, industry, government, and other interested scientists to discuss recent research on 1) within-species gene flow, 2) crop-wild hybridization and gene introgression, 3) consequences of gene flow, 4) approaches to managing gene flow, and 5) modeling gene flow. The organizing committee included Michael Horak (Monsanto Company; Committee Chair), David Gealy (USDA), Hector Quemada (Crop Technology Inc.), Neal Stewart (University of Tennessee), Mark Westgate (Iowa State University), and Allison Snow (Ohio State University). More than fifty people from at least six countries participated.

The arrival of transgenic crops in the 1990's triggered an explosion of research on the extent and consequences of gene flow from crop species to their wild, weedy, or feral relatives. More recently, many gene flow studies have focused on problems that can arise from the unwanted, adventitious presence of transgenes in non-GE seed and food supplies. This later phase of gene flow research was well represented in presentations about crop-to-crop transgene dispersal in corn, alfalfa, canola, and wheat. Underscoring the need for managing pollen- and seed-mediated dispersal of transgenes, David Gealy summarized a new report from CAST (Council on Agricultural Science and Technology) on "Implications of Gene Flow in the Scale-Up and Commercial Use of Biotechnology-Derived Crops" (http://www.cast-science.org/).

Meanwhile, gene flow to wild, weedy, or feral relatives is still a very active area for research. Investigators discussed new findings about the extent of hybridization in wild/weedy relatives of corn, rice, wheat, sugar beet, canola, squash, sunflower, sorghum, radish, and cowpea. Most of these crops have weedy relatives that could become more challenging to manage if they acquire new types of herbicide resistance, whether transgenic or not. This issue was raised in several presentations, and three invited speakers described gene flow from GE canola in Canada, where herbicide resistance traits have spread to wild Brassica rapa, volunteer canola, and feral populations that establish from spilled seeds. Michael Owen's group also presented studies of the potential for spontaneously evolved herbicide resistance to spread via hybridization among closely related weed species in the Asteraceae and Amaranthaceae families.

A poster by Remy Pasquet et al. examined possible risks of Bt genes that could enter wild cowpea populations in West Africa, where Bt cowpea is being developed. Otherwise, few presentations included crop-wild systems in which it was possible to examine the consequences of gene flow, as opposed to its mere occurrence (which is often referred to as "exposure" to a given "hazard"). Two speakers discussed current regulations and the challenges of evaluating the "hazard" component of risk assessment, while others indicated that any transgene could be considered a commercial hazard if it spreads adventitiously to non-GE seeds or food.

Given the regulatory, commercial, and environmental incentives for confining transgenes, research on bioconfinement methods such as sterility, chloroplast transformation, and site-specific recombination to remove transgenes is also receiving attention. Christiane Koziolek presented ongoing research by the EU project known as Transcontainer (http://www.transcontainer.org), and Neal Stewart's group described plans for related studies in tobacco and canola.

Several important research areas were not represented at the meeting. These included studies of 1) gene flow from new types of GE plants that are being developed for biofuels, forage, landscaping, and forestry applications; 2) fitness effects of transgenic drought resistance, cold tolerance, or better nutrient use efficiency in wild/weedy relatives; 3) whether increased fitness due to transgenes could result in weedier or more invasive plant populations; and 4) whether transgene introgression could threaten the genetic diversity of wild relatives, above and beyond the effects of ongoing gene flow from conventional crops. To address these and other types of questions, investigators noted that weed scientists need to have greater access to transgenic materials and more opportunities for funding from federal agencies.

In summary, this symposium offered a great opportunity for researchers to share recent results and for other interested parties to gauge the status of gene flow research in the USA and elsewhere.

Dr. Allison A. Snow
Department of EEO Biology
Ohio State University, Columbus, OH
Snow.1@osu.edu



GENE FLOW IN SUGAR BEET PRODUCTION FIELDS
Henri Darmency & Marc Richard-Molard

Concern has grown in Europe over the agricultural and environmental impacts of genetically engineered (GE) crops, especially about gene flow to conventional varieties and wild relatives. Contrary to most crops that are grown for their seed or fruit, sugar beet (Beta vulgaris L.) is grown for its root. Therefore, pollen-mediated gene flow in root production areas is not a concern in the debate on GE and non-GE crop co-existence, including table beet and chard in private gardens. However, pollen flow could be responsible for the admixture of GE and non-GE materials in the seed lots provided to farmers; but it can be easily prevented by breeding and multiplying GE and non-GE seeds in different regions. In contrast, pollen flow to wild relatives could certainly generate agronomic trouble much more easily and quickly than with any other crop.

There is always a small proportion of sugar beet plants that flower, in spite of being a root crop, because of either their sensitivity to vernalization or the presence of a dominant bolting gene. Since wild sea beet, weed beet, and sugar beet are the same botanical species, and since they are allogamous, they can easily produce hybrids and are completely interfertile1. The progeny of crosses among these beet types is perfectly adapted to field conditions, has no hybrid fitness cost, and therefore can display the favorable traits encoded by the transgenes. In particular, herbicide resistance, which is desired by farmers to significantly reduce the number of herbicide sprays and working hours, could result in the spread of herbicide-resistant weed beets. The weed beet is already a serious problem to sugar beet growers since no herbicide available differentiates between weed beet and sugar beet. Severe infestations can reduce to nothing the sugar beet yield and lead the farmer to stop growing this crop. Therefore, breeding transgenic herbicide-resistant varieties can solve this particular problem, provided that gene flow is contained for a long time.

The question of pollen-mediated gene flow to weed beets in sugar beet root production areas was addressed in a six-year farm-scale study. Since GE sugar beet is not yet grown on commercial fields, this study is the only documented background of field growth for that crop. It was a joint action of governmental research institutes (INRA), professional associations (ITB, for beet; CETIOM, for oilseed rape) and industry (Hilleshog and KWS, for providing the GE beets). The program started in 1995 in two locations: Châlons, in Champagne, and Dijon, in Burgundy, in the north-eastern part of France. Besides the gene flow study, the trial confirmed that the number of herbicide sprays was reduced from 4.1 to 2.5 thanks to the post-emergence, non-selective herbicides used for the GE varieties, without any difference in weed control or yield than with conventional herbicide programs2.

Field monitoring
In each location there were four 1-ha adjacent fields grown in rotation with GE sugar beet, GE oilseed rape, conventional wheat and fallow. The sugar beet field was divided in two parts, each sown with a heterozygous herbicide-resistant line: a Roundup Ready glyphosate-resistant line from Hilleshog, and a Liberty Link glufosinate-resistant line from KWS. Each variety was sprayed with its respective herbicide, except for the central lane of the field that was sprayed with various herbicides used on conventional sugar beets in the region. Weed beets were transplanted into the central lane of the field when no local weed beet emerged in the sugar beet field. The monitoring took place between 1996 and 2001. More detail is available in the full publication3.

The number of sugar beet bolters varied widely according to the transgenic line and year, from 0 to 121 per ha, thus providing a good opportunity to study the consequences of pollen flow under a wide range of realistic conditions and amounts of pollen escape. Indeed, the rate of sugar beet bolters of commercial varieties in France during the same period ranged from 0.001 to 0.1%4. These flowering plants included vernalized herbicide-resistant sugar beets and susceptible annual hybrids coming from pollination of the seed mother plants by susceptible wild beets surrounding the nursery. Susceptible hybrids and spontaneous weed beets could grow and flower in the central lane of the field, which was treated with selective herbicides. All the bolting plants were mapped, and their seeds were carefully collected and then tested for herbicide resistance in the greenhouse.

Production of herbicide-resistant seeds by sugar beet bolters
On average, 58.2% of seeds produced by resistant sugar beet bolters were resistant. The deviation from the 75% expected from mating among heterozygotes denoted both the contribution of pollen coming from susceptible beets and probably the better viability and fertilizing ability of the pollen of weed beets. This category of plant accounted for 84.8% of the total resistant seed production over the years studied, but, as shown in Figure 1, their importance decreased during the second round of the rotation.


Resistant seedlings also appeared in the progeny of susceptible sugar beet bolters at a mean percentage of 1.7%, and they accounted for 1.2% of the total resistant seed production over the six years under study.

Production of herbicide-resistant seeds by weed beet
On average, 6.2% of the seeds produced by weed beets were resistant, which accounted for 14% of the total resistant seed production over the six years in the two locations. A more detailed analysis has been published3. Some of these resistant seeds were not produced within the sugar beet field, but rather in the fallow field, and their proportion decreased as the distance between the fields increased. Fallow field production represented 0.2% of the total resistant seed production. The largest distance at which a cross was recorded between the GE sugar beet bolters and a weed beet was 112 m. This showed that a foreign pollen grain entering a pollen cloud at low frequency over a weed beet population that grows in a distant fallow field has an effective fertilizing ability. Pollen flow monitoring using male sterile plants within and around the farm scale experiments showed fertilization at 277 m and up to 1172 m3,5. The distribution curve of the number of fertilized seeds in terms of distance from the pollen source had generally a negative power shape3,5,6. For instance, the number of resistant seeds recorded in different groups of plants in 1999 in Châlons, up to 120 m away from the resistant bolters, followed the equation N = 14.4 d-0 ,75, R2 = 0.89 (Fig. 2).



Thus, in spite of the low density of GE bolters (8 in one ha in 1999), pollen flow can reach adjacent as well as distant weed beet populations and transfer the herbicide-resistance gene. Within the sugar beet field, there was an average of 3.5% resistant seeds in the seed produced by the susceptible weed beet, accounting for 7.4% of the total resistant seed production over the six years and the two locations. The weed plants that produced resistant offspring were not located at shorter distances from the resistant bolters or at farther distances from susceptible plants than the other weed beets. They flowered simultaneously with other weed plants, but they produced more flowers and 2.4 times more viable seeds than plants that did not produce resistant offspring. Higher production of flowers and higher seed sets could partly explain the ability of those plants to catch more numerous pollen grains and mature more embryos, thus simply having a higher probability of producing resistant offspring. However, if the number of viable seeds per plant somewhat depended on a genetic factor, the consequence would be the propagation of herbicide resistance together with the most reproductive individuals, which would be a very unfavorable conjunction of factors with respect to weed control.

Finally, resistant seeds could also originate from resistant weed beets. In 2000 and 2001, a few resistant weed beets emerged, either spontaneously from the soil seed bank containing seeds left to shed in the same field in 1999, or sowed in the field in order to simulate the creation of a soil seed bank containing herbicide-resistant seeds, as would have occurred if the seeds had not totally been harvested in former years. All these resistant weed beets were heterozygotes and produced, on average, 74% resistant seedlings. This category of plants accounted for 6.4% of the total resistant seed production, but unlike resistant seeds produced by sugar beet bolters, it was concentrated in the last two years, during the second crop rotation (Fig. 1).

Management
At the end of the first round of crop rotation, the cumulated number of seeds released on the farm-scale trial (4 ha x 2 locations) was 222,000, of which 22.3% were herbicide-resistant, representing 0.6 resistant seed per m2. However, there was a large variability among fields. Most resistant seeds were produced by resistant sugar beet bolters (see Fig. 1). This result strengthens the urgent need to eradicate all transgenic bolters. On one hand, eradication can be achieved through production of high quality certified seeds of varieties that are not sensitive to vernalization and free of annual hybrids. On the other hand, destruction of bolters should also be pointed out as a compulsory task among farmers' good agronomic practices. Clearly, some of the bad results reported above belong to a worst-case scenario, because most farmers would have reduced the risk of seed release and pollen flow by destroying bolters when they were too numerous.

However, if bolting still occurs, even at very low frequency, and is not destroyed by farmers, or if transgenic volunteer roots grow and flower in crops subjected to the same herbicide or in fallow fields, the transgenes will unavoidably be transmitted to weed beets within a short period of time. Pollen flow from resistant sugar beets to susceptible ones and to weed beets outside the field accounted for 0.2% of resistant seeds in the farm-scale study. Subsequent multiplication of resistant weed beets in the second round of crop rotation accounted for 13.6% of resistant seeds. These seeds would be the source of further multiplication of herbicide resistant weed beets. Therefore, farmers must prevent the constitution of a soil seed bank containing herbicide-resistant seeds. Useful practices, besides destruction of bolters, could include management of fallow fields to control weed beets and change of crop rotation. The effect of various farming systems on gene escape from GE crops to volunteers and weed beets could be anticipated by simulation models fed with basic data on weed beet biology, such as those collected in the farm scale study7,8.

References

1. Boudry P, Mörchen M, Saumitou-Laprade P, Vernet Ph and Van Dijk H (1993) The origin and evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar beets. Theor Appl Genet 87, 471-478

2. Gestat de Garambé T and Richard-Molard M (1999) Produire des betteraves OGM tolérantes à un herbicide non sélectif: conséquences sur les systèmes de culture. Rev Ind Aliment Agric, juillet/Aout.

3. Darmency H, Vigouroux Y, Gestat de Garambé T, Richard-Molard M and Muchembled C (2007) Transgene escape in sugar beet production fields: data from six years farm scale monitoring. Environ Biosafety Res 6, 197-206

4. Perarnaud V, Souverain F, Prats S, Dequiedt B, Fauchere J and Richard-Molard M (2001) Influence du climat sur le phénomène de montée à graine de la betterave: synthèse http ://www.itbfr.org (accessed January, 2008)

5. Darmency H, Klein E, Gestat de Garambé T, Gouyon PH, Richard-Molard M and Muchembled C (2008) Pollen dispersal in sugar beet production fields, submitted

6. Bateman AJ (1947) Contamination of seed crops II. Wind pollination Heredity 1, 235-246

7. Tricault Y, Sester M, Darmency H, Angevin F, and Colbach N (2007) La gestion des betteraves adventices résistantes à un herbicide: une approche par simulation. In AFPP, 20th Conf. COLUMA, Dijon, December, 213-222

8. Sester M, Tricault Y, Darmency H, and Colbach N (2008) GENESYS-BEET: a model of the effects of cropping systems on gene flow between sugar beet and weed beet. Field Crops Res in press

Marc Richard-Molard
ITB, 45 rue de Naples 75008 Paris

Henri Darmency
Unité Mixte de Recherche sur la Biologie et la Gestion des Adventices
INRA, BP 86510, 21065 Dijon, France

Darmency@dijon.inra.fr



More meetings can be found at http://www.isb.vt.edu

19th NEW PHYTOLOGIST SYMPOSIUM
Physiological Sculpture of Plants:
New visions and capabilities for crop development
1720 September 2008
Timberline Lodge, Mount Hood, Oregon, USA

In recent years there has been a great expansion of knowledge of genes that influence the regulatory pathways that control organismal properties of adaptive and economic importance. The goal of this meeting is to discuss this rapidly moving body of knowledge with an eye to future translation, i.e., how the knowledge might be used to create major advances in breeding, biotechnology, and genetic engineering. By bringing together a number of very diverse basic science and breeding science perspectives into a small, informal meeting format we will consider how to improve efficiency, or extend the limits, for phenotype- or marker-based breeding, not to duplicate what breeding can already do well.

Organizers: Steven Strauss (Oregon State Univ., USA), Richard Amasino (Univ. of Wisconsin, USA), Richard Flavell (Ceres Inc., CA, USA), Harry Klee (Univ. of Florida), Holly Slater (New Phytologist, UK)

Further details including registration instructions are available online at: www.newphytologist.org/physiological



10th ISBGMO
16 November - 21 November, 2008
Museum of New Zealand Te Papa, Wellington, New Zealand

In celebration of the 10th ISBGMO, the International Society for Biosafety Research (ISBR) is highlighting past achievements in biosafety research on GMOs and charting future directions. Established as a biennial event since 1990 to showcase environmental biosafety research, ISBGMO brings together scientific researchers, policy makers, regulators, non-governmental organizations (NGOs), and industry representatives to foster productive dialogue and multidisciplinary approaches while embracing diverse perspectives from all parts of the globe.

The 10th ISBGMO includes eight plenary sessions, four evening workshops, and posters. Each plenary session will offer presentations from 2 4 keynote speakers as well as contributed talks selected from submitted abstracts (due April 30th, 2008). In addition, a special joint ISBR/OECD session will examine risk assessment practices and explore the challenges to formulating sound regulatory decision-making.

Plenary Sessions

Biosafety: Experience and results
•Introgression, naturalization, and invasion
•Abiotic and biotic stress tolerance
•GM animals

Workshops

Designing field experiments for environmental risk assessment
•Regulators' forum
•Novel approaches to environmental risk assessment
•Risk communication
•Impacts on soil ecosystems
•ISBR/OECD Session: Risk assessment - state of the art
•Biocontainment methods
•Post market environmental monitoring

For more information: http://www.isbgmo.info/




ISB News Report
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