Toward metrological traceability for DNA fragment ratios in GM quantification. 3. Suitability of DNA calibrants studied with a MON 810 corn model

The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio.     

Toward metrological traceability for DNA fragment ratios in GM quantification. 2. Systematic study of parameters influencing the quantitative determination of MON 810 corn by real-time PCR

This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.     

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.     

Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods

In this study, the event-specific primers for insecticide-resistant maize, MON810, and herbicide-tolerance maize, NK603, have been designed. Simplex PCR and multiplex PCR detection method have been developed. The detection limit of the multiplex PCR is 0.5% for MON810 and NK603 in 50 ng of the template for one reaction. Quantitative methods based on real-time quantitative PCR were developed for MON810 and NK603. Plasmid pMulM2 as reference molecules for the detection of MON810 and NK603 was constructed. Quantification range was from 0.5 to 100% in 100 ng of the DNA template for one reaction. The precision of real-time Q-PCR detection methods, expressed as coefficient of variation for MON810 and NK603 varied from 1.97 to 8.01% and from 3.45 to 10.94%, respectively. The range agreed with European interlaboratories test results (25%). According to the results, the methods for quantitative detection of genetically modified maize were acceptable.     

Development of the visual loop-mediated isothermal amplification assays for seven genetically modified maize events and their application in practical samples analysis

As more and more genetically modified (GM) crops are approved for commercialization and planting, the development of quick and on-spot methods for GM crops and their derivates is required. Herein, we established the polymerase chain reaction and agarose gel electrophoresis-free system for the identification of seven GM maize events (DAS-59122-7, T25, BT176, TC1507, MON810, BT11, and MON863) employing a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay was performed using a set of four specific primers at 60-65 °C in less than 40 min, and the results were observed by direct visual observation. In these developed assays, the specificity targeted at each GM maize event based on the event-specific sequence was well confirmed, and the limits of detection were as low as four copies of maize haploid genomic DNA with an exception of 40 copies for MON810 assay. Furthermore, these developed assays were successfully used to test six practical samples with different GM maize events and contents (ranged from 0.0 to 2.0%). All of the results indicated that the established event-specific visual LAMP assays are more convenient, rapid, and low-cost for GM maize routine analysis.     

A microarray platform for parallel detection of five transgenic events in foods: a combined polymerase chain reaction-ligation detection reaction-universal array method

We recently developed a multiplex polymerase chain reaction (PCR) system for the simultaneous detection of four transgenic maize (MON810, Bt176, Bt11, and GA21), one transgenic soybean (Roundup Ready), and two control genes (lectin and zein). Because PCR can lead to ambiguous interpretations due to low specificity, we have developed the ligation detection reaction (LDR) combined with a universal array as a molecular tool to confirm results of PCR analysis. Here, we describe the PCR-LDR-universal array procedure and demonstrate its specificity in revealing the presence of transgenic DNA in experimental samples, raw materials, and commercial foodstuffs.     

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.     

Detection methods for biotech cotton MON 15985 and MON 88913 by PCR

Plants derived through agricultural biotechnology, or genetically modified organisms (GMOs), may affect human health and ecological environment. A living GMO is also called a living modified organism (LMO). Biotech cotton is a GMO in food or feed and also an LMO in the environment. Recently, two varieties of biotech cotton, MON 15985 and MON 88913, were developed by Monsanto Co. The detection method is an essential element for the GMO labeling system or LMO management of biotech plants. In this paper, two primer pairs and probes were designed for specific amplification of 116 and 120 bp PCR products from MON 15985 and MON 88913, respectively, with no amplification from any other biotech cotton. Limits of detection of the qualitative method were all 0.05% for MON 15985 and MON 88913. The quantitative method was developed using a TaqMan real-time PCR. A synthetic plasmid, as a reference molecule, was constructed from a taxon-specific DNA sequence of cotton and two construct-specific DNA sequences of MON 15985 and MON 88913. The quantitative method was validated using six samples that contained levels of biotech cotton mixed with conventional cotton ranging from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-20%. Limits of quantitation of the quantitative method were all 0.1%. Consequently, it is reported that the proposed detection methods were applicable for qualitative and quantitative analyses for biotech cotton MON 15985 and MON 88913.     

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.     

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.     

Detection and quantification of transgenes in grains by multiplex and real-time PCR

Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.     

Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.     

Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.     

构建番茄红素β/ε环化酶基因RNAi植物表达载体及其表达分析

根据GenBank中Lyc-β基因 (X86452) 及Lyc-ε基因 (Y14387) 设计引物,通过高保真PCR从番茄cDNA中克隆到两段长度为302 和330bp的Lyc-β基因片段和两段长度为302 和288bp的Lyc-ε基因片段。从质粒pCAMBIA-2301 (AF234316) 克隆出gusA基因长度为168 和235bp的2个内含子片段。根据RNAi原理将正、反向序列与内含子连接,之后插入启动子与终止子之间构建成4组植物表达载体。利用农杆菌介导的叶盘法转化烟草植株,通过PCR鉴定共获得107株转基因植株。利用荧光定量PCR分析干扰效果,结果显示经4组干扰载体干扰后,转基因植株中目的基因mRNA含量分别为对照的3.4%、16.4%、15.2%和21.8%。进一步用HPLC对应分析转化株的番茄红素含量,结果表明转基因植株中番茄红素平均增长量分别为3.4、2.1、3.4和2.0μg/g。同时测定转基因植株中的β-胡萝卜素与叶黄素含量,发现它们根据干扰的基因不同呈现对应的变化。     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize

In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.     

Earthworms of different functional groups affect the fate of the Bt-toxin Cry1Ab from transgenic maize in soil

The fate of the insecticidal Cry1Ab protein from crop residues (leaves and roots) of the transgenic maize variety MON810 was studied in the presence and absence of two earthworm species (Lumbricus terrestris, Aporrectodea caliginosa; separate incubations) in soil microcosms. The recombinant Cry1Ab protein was quantified using a highly sensitive ELISA. Control microcosms received corresponding non-transgenic plant material. All earthworms survived in the microcosms over a period of 5 weeks, irrespective of whether they received MON810 or non-transgenic plant material. Weight loss was observed for both earthworm species, independent of the plant material or transgenic modification. A strong decline of immunoreactive Cry1Ab in plant residues (mean initial concentration approx. 5000 ng g⁻¹) of MON810 was observed in all treatments, but in microcosms with earthworms this decline was significantly higher with less than 10% of the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab concentrations in casts were only 0.1% of those found in remaining plant material of the respective microcosms. No immunoreactive Cry1Ab proteins were found in earthworm tissues (threshold of detection: 0.58 ng g⁻¹ fresh weight). No further decline was found for Cry1Ab concentrations in casts of A. caliginosa during a subsequent period of 3 months of incubation in bulk soil (<0.1 ng g⁻¹) after removal of the earthworms from the microcosms, while in casts of L. terrestris the concentration decreased from 0.4 to below 0.1 ng g⁻¹. In conclusion, this study demonstrates that earthworms enhance the decline of immunoreactive Cry1Ab proteins from maize residues.     

Use of real-time polymerase chain reaction (PCR) and transformation assay to monitor the persistence and bioavailability of transgenic genes released from genetically modified papaya expressing nptII and PRSV genes in the soil

Soil samples were collected from an isolated field from December 2003 to April 2004 where transgenic papaya were planted, and the persistences of transgenic genes of 796 bp (located between 35S promoter and coat protein, 35S-P/PRSV-CP), 398 bp (located between plasmid pBI121 and NOS terminator, pBI121/NOS-T), and 200 bp (located between NOS promoter and nptII gene, NOS-P/nptII) were studied. At the end of planting, the residues of 398 bp in the soil was 0.06 microg g(-1) of soil, whereas the residues of 769 and 200 bp were less than 30 pg g(-1) of soil (detection limit). Kinetics studies on the persistence of these three fragments in sterile distilled water and nonsterile soil microcosms showed that two mechanisms might be involved: an initial fast exponential degradation pattern in the first week and then followed by a slow-release pattern throughout the experiment. Persistence of transgenic DNA in sterile water was longer than in nonsterile soil microcosms, indicating that enzymatic degradation and soil adsorption played important roles on the persistence of DNA in the environment. The reason for the fragment of 398 bp persisted longer than fragments of 769 and 200 bp is not clear, but the guanine plus cytosin (G plus C) content in the DNA fragment might be involved in the stability of DNA in the environment. Biological availability of soil DNA to bacteria conducted by the transformation assay indicated that gene transformation from soil DNA extracts to two Acinetobacter spp. did not occur.     

Simultaneous confirmatory analysis of different transgenic maize (zea mays) lines using multiplex polymerase chain reaction-restriction analysis and capillary gel electrophoresis with laser induced fluorescence detection

A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.     

Evaluation of compositional equivalence for multitrait biotechnology crops

Compositional analysis is an important tool in the evaluation of the safety and nutritional status of biotechnology-derived crops. As part of the comparative assessment of a biotechnology-derived crop, its composition is evaluated by quantitative measurement of the levels of key nutrients, antinutrients, and secondary metabolites and compared to that of conventional crops. To evaluate the effect of combining multiple biotech traits through conventional breeding, the forage and grain compositions of the double combinations MON 810 × NK603, MON 863 × MON 810, and MON 863 × NK603 and the triple combination MON 863 × NK603 × MON 810 were compared to their respective near-isogenic, conventional control hybrids. Overall, a total of 241 statistical comparisons between the multitrait biotechnology crop and its corresponding conventional controls were conducted. Of these comparisons 192 (79.7%) were not statistically significantly different (p > 0.05), and all 49 of the differences were within the 99% tolerance interval for commercial hybrids grown in the same field or related field trials. These data on combined trait biotechnology-derived products demonstrated that the forage and grain were compositionally equivalent to their conventional comparators, indicating the absence of any influence of combining insect protection and herbicide tolerance traits by conventional breeding on compositional variation.