•Shape, orientation, and size of pollen source and recipient field
A continuous design seems to favor short distance pollen dispersal; a clear and high edge effect and a rapid decline of cross-fertilization over the next 50 meters is observed. Most field experiments in this design class used small transgenic plots and relatively wide nontransgenic border areas. It has been observed that large sink populations subsidize the species pool of small source populations via a mass effect. In general, the larger the recipient field compared to the donor field, the lower the probability of cross-fertilization. In the discontinuous design field trials, pollen pressure from both donor and recipient is presumably equal because of similar field sizes. As a consequence, the outcrossing rate in this design class declines slowly and steadily over the first tens of meters and levels off at around 0.1% over a hundred meters. Not only the size but also the alignment of donor and recipient fields is important for levels of outcrossing. With a donor field size of 2 ha, the rate of outcrossing might be much higher than from a 10 ha field, if the long side of a recipient field is facing the source field as compared to the short side. In other words: the deeper the recipient field, the lower the cross-fertilization level of the total harvest product.

•Isolation distance and border crops between pollen source and recipient field
Isolation distance is one means to ensure seed purity (e.g., 100 m for certified rapeseed). Some outcrossing studies investigated the effectiveness of isolation zones for reducing gene flow compared to the use of nontransgenic buffer areas. When crops are isolated by open ground or low growing crops, it appears that the first rows of the recipient field intercept a high proportion of foreign pollen due to the low convarietal pollen load of the field margin. When there is no gap between transgenic and nontransgenic fields, the plants located in the contact area act on one side as a pollen trap; on the other side, plants produce additional pollen that dilutes the transgenic airborne pollen, so that at within-field distances comparable to a certain isolation distance lower rates of cross-fertilization are observed.

•Genotype and zygosity
Different herbicide resistant plant varieties, in particular with resistance to glufosinate and glyphosate, have been used in outcrossing studies. Each transgenic variety can show different levels of outcrossing under the same experimental and environmental conditions, influenced by differences in flowering time, pollen quantity, and selfing rate. In addition, in the case of homozygous glyphosate and glufosinate resistant plant lines, all pollen carries the herbicide resistance gene. By contrast, in studies of cross-fertilization from glufosinate resistant hybrids, the amount of transgenic pollen was lower, resulting in only about 5/8 of the outcrossing frequencies of homozygous herbicide resistant lines. Moreover when measuring outcrossing via herbicide spray tests, one has to consider that gene dosage effects have been demonstrated in several cases by comparing hemizygous and homozygous transgenic plants with homozygotes usually having higher transgene expression levels. Therefore hemizygous seedlings with low expression levels of the herbicide resistance gene might not always be easily distinguishable from unmodified susceptible plants.

•Local environment and climatic conditions
The range of cross-fertilization at a given location is also determined by the narrow range of weather conditions and local topography around the field trial site, and the numbers of bees and other insects that are likely to increase the amount of pollen transfer.

Management strategies to reduce gene flow
Due to additional sources of adventitious GE presence—seed impurity, volunteers, adventitious seed transfer during harvest and transport—a maximal value of 0.5% for crop-to-crop cross-fertilization is relevant within the 0.9% threshold for the adventitious presence of GE crop products in nontransgenic food and feed set by EU labeling legislation. Technical measures for achieving co-existence have to ensure that thresholds will not be exceeded on a long-term basis. The first few oilseed rape rows intercept a high proportion of pollen when open ground or low growing barrier crops separate oilseed rape fields. The removal of the first 10 m of crop along the side of a nontransgenic field facing a GE crop might be more efficient for reducing the total level of cross-fertilization in a recipient sink population than to recommend separation distances. It appears that the use of predominantly self-pollinating, male sterile, or cleistogamous cultivars as a biological containment strategy will also reduce gene flow^2,3.

The adventitious presence of GE oilseed rape is not only affected by outcrossing via pollen-mediated gene flow, it is also affected by volunteer populations within fields via seed-mediated gene flow. The dynamics and persistence of volunteer oilseed rape is mainly influenced by field management practices (time of first tillage following oilseed rape, cultivar type, tillage depth, crop rotation)⁴. If volunteers flower, cross-pollination to other oilseed rape plants and fields can occur. Herbicide resistant volunteers arising from unintended gene flow can be easily managed with herbicides if the subsequent crop is a non-herbicide-resistant cereal⁵ but will require special weed control strategies in the case of crops with resistance to the same herbicide. In order to avoid the formation of multiple herbicide resistant plants, farmers should not grow cultivars with different herbicide resistances in adjacent fields.

Feral populations are widespread at relatively low densities in regions cultivating oilseed rape. Pollen flow from sporadic occurrences of feral oilseed rape to neighboring rapeseed fields can be considered a rare event due to the high amount of competing field pollen. Therefore feral plants as a source for further transgene flow may only be a realistic scenario if large feral populations are present near an oilseed rape field. Nevertheless, volunteer and feral population dynamics should also be taken into account when assessing sources for adventitious GE presence and feasibility of coexistence.

Alexandra Hüsken and Antje Dietz-Pfeilstetter
Federal Biological Research Centre for Agriculture and Forestry
(from January 1 2008: Julius-Kühn-Institute)
Messeweg 11-12
D-38104 Braunschweig, Germany
a.huesken@bba.de; a.dietz@bba.de

In this model, grain and stover, plus the enzymes needed to process them into ethanol, are all produced from the same acreage. This design allows for synergies in transportation and coordination, as well as for efficient utilization of natural resources. The system is self-contained, having all the necessary components to produce ethanol; there is no additional input required to provide the raw materials for stover ethanol over that which is needed to grow corn for grain ethanol. The benefits in this example go beyond the savings obtained when planting a field that otherwise would remain fallow and include: 1) more efficient utilization of raw materials without additional inputs, reducing the environmental impact of growing separate crops for grain ethanol, lignocellulosic ethanol, and enzyme; 2) savings from the elimination of the highly capital intensive fermentation equipment traditionally used for making the vast quantities of enzyme required for biomass conversion into ethanol; 3) increased revenue for growers; 4) reduced transportation cost because the grain, stover, and enzymes required for ethanol production are supplied in one central location; and 5) lowered unit cost of the enzymes since all unit operations are similar to existing practices for growing commodity crops, except mixing the enzyme fraction with the stover. These economic benefits are realized in tandem with the additional benefit of being able to make the clear distinction that, in practice, the industrial product (enzyme) is clearly separated from those practices, locations, and fields used for food production.

Conclusions
Transgenic plants used to produce biopharmaceuticals and bioindustrial products have great potential, but current production practices limit their cost effectiveness in some cases and raise concerns that the use of food organisms as hosts for nonfood products increases the potential for inadvertent exposure. This concept, however, is in direct conflict with other current production practices that use yeast and eggs (food sources) to produce products such as industrial enzymes, vaccines, and pharmaceuticals. The food - host argument diverts attention from the real issue, which is to ensure that transgenic nonfood products remain outside the food system, rather than which host is used for production.

The proposed model discussed above for production of nonfood products in transgenic plants may potentially alleviate associated cost constraints and public concerns. The practice of using a dedicated area for nonfood production would draw a clear distinction between plants used to produce food and nonfood applications. This should help put plant-based production on par with other non-plant production systems used for transgenic products.

John A Howard
Applied Biotechnology Institute, Building 36
Cal Poly State University
San Luis Obispo, CA 93407
jhoward@appliedbiotech.org

Ds INSERTION LINES VALUABLE FOR RICE BREEDING
Shu-Ye Jiang and Srinivasan Ramachandran

On the other hand, sterile rice lines were frequently observed in our Ds insertion lines. Several such lines were investigated further. Male sterile^3,4 lines provide resources from which the cytoplasmic male sterile (CMS) or photoperiod (temperature)-sensitive male sterile rice lines are derived. These two kinds of male sterile rice lines form the basis for three-line or two-line hybrid rice combinations for commercial release. One such line—a photoperiod sensitive male sterile rice plant—was further characterized. It exhibited male sterility under short day length conditions, and the sterility was recovered under long day length conditions. Further investigation showed that photoperiod sensitive localization of a myosin protein controlled the fertility transformation³.

Developing varieties with abiotic stress tolerance
Ds insertion lines were subjected to various abiotic stresses, including drought, high salinity, and cold conditions, to obtain valuable stress-responsive lines. To screen for drought-responsive lines, approximately 16,000 three week-old seedlings (WT and Ds lines) were treated with 3% or 10% PEG (polyethylene glycol, 6000) to obtain hyper-sensitive or tolerant lines, respectively. In total, we selected 84 of the best lines, including 61 sensitive and 23 tolerant lines. Among 307 of the moderate lines, 194 lines were sensitive and 113 lines were tolerant². More than 100 candidate lines were subjected to drought stress naturally under field and greenhouse conditions. The results validated the use of PEG screens to mimic drought conditions.

To screen salinity-responsive lines, 7,000 two-week-old seedlings were subjected to 50 mM NaCl to obtain hyper-sensitive lines, and to 200 mM NaCl to obtain tolerant lines. This screening produced 54 candidates, including 40 sensitive and 14 tolerant lines². One resistant line is shown in Fig. 2A.

Screening for cold-responsive lines was performed under natural winter conditions in southern China, with temperatures ranging between 10 – 20 °C. Out of 13,000 Ds insertion lines subjected to cold screens in two winter seasons, 470 cold-sensitive or -tolerant lines were obtained². By summarizing results from drought, high salinity, and cold screenings, we found that some Ds lines exhibited resistance/sensitivity to two or three stress conditions (Fig. 2B), which may be the best candidates for developing multiple-resistant rice varieties.

Figure 2. Screening for stress-responsive Ds insertion lines. (LEFT) An example of a salinity-resistant Ds line. After germination in MS media, both wild type and Ds lines were transferred for two weeks into 200 mM NaCl-containing MS media: Left, wild type; right, Ds line. (RIGHT) A summary of screening Ds lines under drought, high salinity, and cold growth conditions. We obtained 391 drought, 54 high salinity, and 470 cold-responsive Ds lines. Among them, two lines showed responsiveness to three stresses; one line was responsive to both drought and high salinity; two lines were responsive to both high salinity and cold; and 15 lines were responsive to both drought and cold conditions.

Improved breeding efficiency using molecular marker-assisted selection
Traditional breeding programs generally require seven to nine generations of conventional backcrosses to transfer an elite agronomic trait into a desirable parent. By contrast, molecular marker-assisted selection can capture desirable characters in new rice varieties within shorter periods of time. Ds insertion lines can be used as a valuable germplasm resource for rice breeding. More than 97% of Ds lines contain only a single copy of the Ds insertion; thus, the Ds transposon can be used as a molecular marker to capture desirable agronomic characters caused by the corresponding Ds insertion into a gene.

Ds insertion lines used to develop non-transgenic or marker-free rice
In our collection the Ds element is immobile. However, the Ds transposon can be released or remobilized into other regions in the presence of a transposase source by crossing with an Ac transposase-containing transgenic plant⁵. Subsequent to the remobilization of the Ds element, a footprint will usually be left behind. If a mutant has a Ds insertion into a coding region of a gene, we can develop new varieties without transgenic sequences. The footprint may cause a frame-shift mutation, making the encoded protein non-functional. Thus, the mutant phenotype can be retained by the footprint without additional foreign DNA sequences². We have successfully used this method to generate two footprint-containing plants. These plants, with no Ds elements, retained the mutant phenotype.

If the Ds element is not transposed into the coding region of a gene, new varieties can be developed by either over- or under-expressing this gene using a marker free method. Endogenous rice promoters (such as actin) can be utilized for this purpose. We have introduced two kinds of constructs to develop such rice varieties. As a result, only minimal T-DNA or maize transposon borders around 200 base pairs were retained in the final genetically engineered rice plants². These sequences do not encode a protein, so the product should be safe for commercial release.

In summary, we have generated a Ds insertion mutant population. We subjected these lines to phenotypic and abiotic stress screens. Some interesting lines have been obtained with higher yield, male sterility, or resistance/sensitivity to various abiotic stresses. Our results suggest that rice could be improved not only by introducing foreign genes but also by knocking out its endogenous genes. These results might provide a new method for rice breeders to further improve rice varieties.

Shu-Ye Jiang and Srinivasan Ramachandran*
Rice Functional Genomics Group, Temasek Life Sciences Laboratory
1 Research Link, National University of Singapore, Singapore 117604
*sri@tll.org.sg

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