Genetically Modified Lines Research Articles

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.

Field trial with genetically modified wheat contradicts glasshouse findings

The introduction of transgenes into plants can cause unintended phenotypic effects which may pose risks to the environment. We assessed pathogen resistance and fitness related traits of genetically modified (GM) wheat with enhanced resistance against the fungus powdery mildew (Pm3b gene). Four offspring pairs, each consisting of a GM line and its corresponding non-GM control, with differing transgene expression levels were compared in glasshouse and field experiments.

Transgene × Environment Interactions in Genetically Modified Wheat

BackgroundThe introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants.Methods and FindingsWe studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results.The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control.ConclusionsOur results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.

Effects of genetically modified amylopectin-accumulating potato plants on the abundance of beneficial and pathogenic microorganisms in the rhizosphere

In this study, the potential effects of a genetically modified (GM) amylopectin-accumulating potato line (Solanum tuberosum L.) on plant beneficial bacteria and fungi as well as on phytopathogens in the rhizosphere were investigated in a greenhouse experiment and a field trial. For comparison, the non-transgenic parental cultivar of the GM line and a second non-transgenic cultivar were included in the study. Rhizospheres were sampled during young leaf development (EC30) and at florescence (EC60). The microbial community composition was analysed by real-time PCR to quantify the abundances of Pseudomonas spp., Clavibacter michiganensis, Trichoderma spp. and Phytophthora infestans. Additionally, total bacterial and fungal abundances were measured. None of the examined gene abundance patterns were affected by the genetic modification when wild type and GM line were compared. However, significant differences were observed between the two natural potato cultivars, especially during the early leaf development of the plants. Furthermore, gene abundance patterns were also influenced by the plant developmental stage. Interestingly, the impact of the cultivar and the plant vegetation stage on the microbial community structure was more pronounced in field than in greenhouse. Overall, field-grown plants showed a higher abundance of microorganisms in the rhizosphere than plants grown under greenhouse conditions.

Attitudes towards the use of genetically modified animals in research

Here we provide the first experimental evidence that public concerns about the use of animals in research are accentuated when genetically modified (GM) animals are used. Using an online survey, we probed participant views on two uses of pigs as research animals (to reduce agricultural pollution or to improve organ transplant success in humans) with and without GM. We surveyed 327 animal technicians, researchers, advocates, university students and others. In both scenarios and across demographics, support dropped off when the research required the use of GM pigs or GM corn. For example, 66% of participants supported using pigs to reduce phosphorus pollution, but this declined to 49% when the pigs were fed GM corn and to 20% when the research required the creation of a new GM line of pigs. Those involved in animal research were more consistently supportive compared to those who were not or those who were vegetarians.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Rhizosphere Communities of Genetically Modified Zeaxanthin-Accumulating Potato Plants and Their Parent Cultivar Differ Less than Those of Different Potato Cultivars

The effects of genetically modified (GM), zeaxanthin-accumulating potato plants on microbial communities in the rhizosphere were compared to the effects of different potato cultivars. Two GM lines and their parental cultivar, as well as four other potato cultivars, were grown in randomized field plots at two sites and in different years. Rhizosphere samples were taken at three developmental stages during plant growth and analyzed using denaturing gradient gel electrophoresis (DGGE) fingerprints of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria, Bacillus, Streptomycetaceae, Pseudomonas, gacA, Fungi, and Ascomycetes. In the bacterial DGGE gels analyzed, significant differences between the parental cultivar and the two GM lines were detected mainly for Actinobacteria but also for Betaproteobacteria and Streptomycetaceae, yet these differences occurred only at one site and in one year. Significant differences occurred more frequently for Fungi, especially Ascomycetes, than for bacteria. When all seven plant genotypes were compared, DGGE analysis revealed that different cultivars had a greater effect on both bacterial and fungal communities than genetic modification. The effects of genetic modification were detected mostly at the senescence developmental stage of the plants. The site was the overriding factor affecting microbial community structure compared to the plant genotype. In general, the fingerprints of the two GM lines were more similar to that of the parental cultivar, and the differences observed did not exceed natural cultivar-dependent variability.

Welfare Phenotyping of Genetically-Modified Mice

Technologies that enable the targeted manipulation of the genome have created new opportunities to study the role and interplay of specific genes in both the regulation and function of physiological and behavioural processes and in the development of pathological conditions. Despite the potential benefits, there are ethical issues in relation to the application of these technologies, some of which relate to the impact on the welfare of the animals involved. Matters of concern include the methods involved in the derivation and production of genetically-modified (GM) animals and resulting phenotypes, where animal welfare is compromised. In the case of the latter, this may be the predicted consequence of the genetic modification, but the occurrence of unforeseen animal welfare complications is a major challenge in the management of GM animals. There has been a rapid escalation in the development of new GM lines, most of them involving mice. Databases of available lines have been developed by national and international consortia, and researchers have developed standard protocols to describe the phenotype of a new line; increasingly, such data are entered into these databases. The inclusion of animal welfare assessments with these data would provide a powerful and sophisticated tool to promote refinement. The scope, level and frequency of monitoring would facilitate the identification of unpredicted effects and the management of humane endpoints, and would identify opportunities to manage the animals so as to ameliorate negative impacts. Furthermore, by highlighting the subtleties of gene-environment interactions, such data have wider implications in achieving the goals of refinement.

Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in ‘Elstar’ apple and the effect of grafting

SummaryMany people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple.Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus × domestica Borkh., ‘Elstar’) with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type ‘Elstar’, two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages.

Real-Time PCR Array as a Universal Platform for the Detection of Genetically Modified Crops and Its Application in Identifying Unapproved Genetically Modified Crops in Japan

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

Microbial and nematode communities associated with potatoes genetically modified to express the antimicrobial peptide magainin and unmodified potato cultivars

The antimicrobial peptide magainin II has activity against a range of micro-organisms. Tubers harvested from potatoes genetically modified (GM) to express a synthetic magainin gene show improved resistance to the bacterial pathogen Erwinia carotovora. The microbial and nematode communities associated with three magainin-expressing potato lines, their near-isogenic, unmodified parental cultivar (Iwa) and an unrelated cultivar (Karaka) were investigated on field-grown plants. Heterotrophic plate counts were used to enumerate aerobic culturable bacterial and fungal populations, while cultivation-independent analysis of bacterial communities was based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from community DNA from phyllosphere, rhizosphere and geocaulosphere (tuber surface) samples. Small but statistically significant differences in the population sizes of culturable bacteria, fungi and yeast were detected among some GM magainin-expressing lines and the unmodified control. However, these differences were typically smaller than the differences between the unmodified parental line control (Iwa) and the unrelated cultivar control (Karaka). Similarly, the difference in the proportion of the nematode population belonging to the fungal feeding trophic group between Iwa and Karaka was greater than that amongst Iwa and its near-isogenic GM lines, and was significantly so for the genus Aphelenchus. The nematode channel ratio (NCR) indicated a more fungal-dominated decomposition channel in soil beneath Karaka compared to Iwa at harvest. In general, eubacterial phylloplane communities were similar for all lines, while the rhizosphere communities associated with two of the three GM lines differed from communities associated with their unmodified parental line control. When roots were senescent, there was no significant difference among potato lines in rhizosphere eubacterial communities or individual trophic groups of the nematode community. Greater diversity was found in geocaulosphere; α-proteobacteria and actinomycete communities of two of the three GM lines differed significantly from their unmodified parental line control and the unrelated cultivar control, while the communities associated with the third GM line were more similar to those of the two control lines. This highlights the importance of testing several GM lines when assessing non-target effects. Results suggest that there is little likelihood of any major sustained non-target effect of genetic modification using a magainin II transgene on plant-associated and soil microflora and function.

Transgene flow to hybrid rice and its male-sterile lines

Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640 m2 in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037-0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1-2 m; (3) The maximum distance of transgene flow was 30-40 m to rice cultivars and 40-150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used.

Pyrolysis-field ionization mass spectrometry of rhizodeposits – a new approach to identify potential effects of genetically modified plants on soil organisms

The objectives of the present study were (1) to investigate the qualitative composition of rhizodeposits leached from soils cropped with non-transgenic and genetically modified (GM) potatoes, and disclose if there were GM-specific modifications in potato rhizodeposition, and (2) to compare these results with conventional bulk parameters of microbial activity in soil. We have raised potatoes from a non-transgenic line (Solanum tuberosum L. cv. Désirée) and three GM lines, which expressed a gene for the resistance to kanamycin (DLH 9000) and a gene for T4 lysozyme (DL10 and DL12). A sandy soil placed in 340 cm3-"CombiSart" containers was used, from which the rhizodeposit was leached after a six-week growth period. The freeze-dried leachates were analyzed by pyrolysis-field ionization mass spectrometry (Py-FIMS). The Py-FI mass spectra gave detailed molecular-chemical information about the composition of leachates, indicating that the potato growth generally altered the composition of the soil solution. Moreover, a principal component analysis of the mass spectra showed differences between the leachates from the non-transgenic parent line and the GM potatoes as well as among the latter group. However, these differences in molecular composition could not be assigned to the release of T4-lysozyme into soil. Dehydrogenase activity and substrate-induced soil respiration as more common bulk parameters of soil microbial activity failed to disclose any significant effects of the various potatoes grown. The limitations of the described rhizodeposit leaching and analysis for risk assessment of GM potato cropping under field conditions are discussed critically. However, it could be concluded that the Py-FI mass spectrometric "fingerprint" can be developed as a fast, comprehensive, highly sensitive and reproducible analytical approach to discern any effects GM-crops may exert on soil ecological parameters.

Open field trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality

BackgroundParthenocarpic tomato lines transgenic for the DefH9-RI-iaaM gene have been cultivated under open field conditions to address some aspects of the equivalence of genetically modified (GM) fruit in comparison to controls (non-GM).ResultsUnder open field cultivation conditions, two tomato lines (UC 82) transgenic for the DefH9-RI-iaaM gene produced parthenocarpic fruits. DefH9-RI-iaaM fruits were either seedless or contained very few seeds. GM fruit quality, with the exception of a higher β-carotene level, did not show any difference, neither technological (colour, firmness, dry matter, °Brix, pH) nor chemical (titratable acidity, organic acids, lycopene, tomatine, total polyphenols and antioxidant capacity – TEAC), when compared to that of fruits from control line. Highly significant differences in quality traits exist between the tomato F1 commercial hybrid Allflesh and the three UC 82 genotypes tested, regardless of whether or not they are GM. Total yield per plant did not differ between GM and parental line UC 82. Fruit number was increased in GM lines, and GM fruit weight was decreased.ConclusionThe use in the diet of fruits from a new line or variety introduces much greater changes than the consumption of GM fruits in comparison to its genetic background. Parthenocarpic fruits, produced under open field conditions, contained 10-fold less seeds than control fruits. Thus parthenocarpy caused by DefH9-RI-iaaM gene represents also a tool for mitigating GM seeds dispersal in the environment.

Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines.

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or "omics" methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.

Comparison of rapid liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of glycoalkaloids in transgenic field-grown potatoes

Two rapid methods for highly selective detection and quantification of the two major glycoalkaloids in potatoes, α-chaconine and α-solanine, were compared for robustness in high-throughput operations for over 1000 analytical runs using potato tuber samples from field trials. Glycoalkaloids were analyzed using liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. An electrospray interface was used in the detection of glycoalkaloids in positive ion mode. Classical reversed phase (RP) and hydrophilic interaction (HILIC) columns were investigated for chromatographic separation, ruggedness, recovery, precision, and accuracy. During the validation procedure both methods proved to be precise and accurate enough in relation to the high degree of endogenous biological variability found for field-grown potato tubers. However, the RP method was found to be more precise, more accurate, and, more importantly, more rugged than the HILIC method for maintaining the analytes’ peak shape symmetry in high-throughput operation. When applied to the comparison of six classically bred potato cultivars to six genetically modified (GM) lines engineered to synthesize health beneficial inulins, the glycoalkaloid content in potato peels of all GM lines was found within the range of the six cultivars. We suggest complementing current unbiased metabolomic strategies by validating quantitative analytical methods for important target analytes such as the toxic glycoalkaloids in potato plants.

Quality of genetically modified (GM) and conventional varieties of canola (spring oilseed rape) grown in western Canada, 1996–2001

The success of GM herbicide tolerant canola is demonstrated by its acceptance by the farm community in Canada. There have been continuing comments, however, including some from major customers, suggesting that GM canola has lower quality than conventional canola. Data drawn from both the Western Canada Canola/Rapeseed Co-operative Test data from 1998–2001 and from the Canadian Grain Commission's harvest surveys of canola from 1996–2001 were used to compare the quality of GM and conventional canola registered and grown. Weed seed contamination of harvest survey samples decreased significantly as the herbicide tolerant lines increased in production. While variety registration data suggested GM and conventional lines had no differences in oil content, data from harvest surveys suggested that GM lines tended to have slightly higher oil contents. Protein and oil contents remain inversely related with no differences in the inverse relationship due to GM. While registration requires that all lines have less than 12 micromoles per gram of glucosinolates, data from harvest surveys show GM lines to have significantly less glucosinolates than conventional lines, possibly due to decreased contamination with cruciferous weeds. A comparison of glucosinolate contents between non-GM herbicide tolerant canola and conventional non-herbicide tolerant canola showed similar differences. There were no significant differences in chlorophyll content, erucic acid levels or saturated fatty acids but harvest survey data showed GM lines were slightly more unsaturated than conventional lines. It would seem safe to conclude that differences in quality between GM and conventional canola are due to the functioning of the GM trait – herbicide tolerance – that allows the GM canola to perform to its potential in the field.

Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis.

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.