Proteomic analysis of four Brazilian MON810 maize varieties and their four non-genetically-modified isogenic varieties

Profiling techniques have been suggested as a nontargeted approach to detect unintended effects in genetically modified (GM) plants. Seedlings from eight Brazilian maize varieties, four MON810 GM varieties and four non-GM isogenic varieties, were grown under controlled environmental conditions. Physiological parameters (aerial part weight, main leaf length, and chlorophyll and total protein contents) were compared, and some differences were observed. Nevertheless, these differences were not constant among all GM and non-GM counterparts. Leaf proteomic profiles were analyzed using two-dimensional gel electrophoresis (2DE) coupled to mass spectrometry, using six 2DE gels per variety. The comparison between MON810 and its counterpart was limited to qualitative differences of fully reproducible protein spot patterns. Twelve exclusive proteins were observed in two of four maize variety pairs; all of these leaf proteins were variety specific. In this study, MON810 leaf proteomes of four varieties were similar to non-GM counterpart leaf proteomes.     

Compositional variability in conventional and glyphosate-tolerant soybean (Glycine max L.) varieties grown in different regions in Brazil

The compositions of a diverse range of commercially available conventional and genetically modified (GM; glyphosate-tolerant) soybean varieties from maturity groups 8 and 5, respectively, grown in the northern and southern soybean regions of Brazil during the 2007-2008 and 2008-2009 growing seasons were compared. Compositional analyses included measurement of essential macro- and micronutrients, antinutrients, and selected secondary metabolites in harvested seed as well as measurement of proximates in both forage and harvested seed. Statistical comparisons utilized a mixed analysis of variance model to evaluate the relative contributions of growing season, soybean growing region, production site, phenotype (GM or conventional), and variety. The study highlighted extensive variability in the overall data set particularly for components such as fatty acids, vitamin E, and isoflavones. There were few differences between the GM and non-GM populations, and most of the variability in the data set could be attributed to regional and variety differences. Overall, the results were consistent with the expanding literature on the lack of any meaningful impact of transgene insertion on crop composition.     

南非冬闲期表施和混施入Bt层玉米落叶和Cry1Ab蛋白的分解研究

Unintended effects of genetic modification on chemical composition of Bt maize leaf litter may have impacts on its decomposition. In most agricultural systems in South Africa, maize litter is either left on the soil surface or incorporated into the soil during tillage. A litterbag experiment, using leaf litter of three maize hybrids （DKC80-12B, DKC80-10 and DKC6-125）, was carried out at the University of Fort Hare Research Farm, South Africa, to determine the effects of genetic modification on decomposition of maize leaf litter when left on the soil surface under field conditions between July and November, the normal fallow period, in 2008. Another litterbag experiment was conducted at the University of Fort Hare Research Farm and Zanyokwe Irrigation Scheme, South Africa, using leaf ~itter of two maize hybrids genetically modified with the erylAb gene （MONS10）, DKC75-15B and PAN6Q-3OSB, and their corresponding near isolines, CRN3505 and PAN6Q-121. The degradation of CrylAb protein in the litter, both surface-applied and soil-incorporated, was also investigated. Decomposition of Bt maize litter was similar to that of non-Bt maize litter both when applied on the surface and when incorporated into soil. Soil-incorporated litter, as well as its CrylAb protein, decomposed faster than that applied on the surface. The leaf litter C：N ratios of PAN6Q-308B and PAN6Q-121 were similar throughout the study, whereas those of DKC75-15B and CRN3505 declined by similar amounts during a 12-week period. These findings suggested that decomposition of leaf litter of Bt maize, with the MON810 event, was not affected by maize genetic modification, and that the CrylAb protein broke down together with plant leaf litter during the winter fallow regardless of whether the litter was applied on the soil surface or incorporated into soil.     

Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays)

Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.     

Carbohydrate Concentration in Lentils (Lens culinaris Medikus.): Genotypic and Environmental Effects

Lentils contain a range of prebiotic carbohydrates; however, genotypic and environmental implications of these components have not been well understood. The objective of this study was to determine the genetic and environmental variation of carbohydrate concentrations in three commonly grown lentil cultivars in South Dakota, USA at two locations over two growing seasons. Significant genetic and environmental variation was found for most of the constituents evaluated. Raffinose-family oligosaccharides (RFO) ranged from 3161 mg 100g^?1 at Bison and 3670 mg 100g^?1 at Wall and from 3217 mg 100g^?1 to 3650 mg 100g^?1 between cultivars. Moreover, RFO content was strongly correlated to a simple plant stress index (SI) in which higher estimated plant stress (e.g., low precipitation) produced higher levels of RFO. These results demonstrate important differences in prebiotic concentrations, which can be used for future lentil breeding efforts when matching cultivars to diverse growing climates with highly variable growing season precipitation.     

Sampling and modeling for the quantification of adventitious genetically modified presence in maize

The coexistence of genetically modified (GM) and non-GM crops is an important economic and political issue in the European Union. We examined the GM content in non-GM maize crops in Spain in 2005. Both the standing crop and the harvest were tested, and the %GM DNA was quantified by real-time polymerase chain reaction. We compared the level of GM as a function of distance from known GM source fields in a 1.2 km2 landscape. The distribution of GM was compared to predictions from previous studies, and good agreement was found. Control and monitoring of adventitious GM presence in non-GM crops can only be achieved by fit-for-purpose sampling and testing schemes. We used a GM dispersal function to simulate non-GM crops in the studied zone and tested the accuracy of five different sampling schemes. Random sampling was found to be the most accurate and least susceptible to bias by GM spatial structure or gradients. Simulations showed that to achieve greater than 95% confidence in a GM labeling decision of a harvest (when treated as a single marketed lot), 34 samples would be needed when the harvest was outside 50% of the GM threshold value. The number of samples required increased rapidly as the harvest approached the GM threshold, implying that accurate labeling when the harvest is within +/-17% of the threshold may not be possible with high confidence.     

Practicable group testing method to evaluate weight/weight GMO content in maize grains

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.     

Comparison of sugar, acids, and volatile composition in raspberry bushy dwarf virus-resistant transgenic raspberries and the wild type 'meeker' (rubus idaeus L.)

Raspberry bushy dwarf virus (RBDV) causes a significant reduction in yield and quality in raspberry and raspberry-blackberry hybrid. Genetic modifications were made to 'Meeker' red raspberries to impart RBDV resistance. The RBDV-resistant transgenic and wild type 'Meeker' plants were grown in Oregon and Washington, and the fruits were harvested in the 2004 and 2005 growing seasons. Year-to-year and site-to-site variations were observed for the degrees Brix and titratable acidity, with Oregon raspberries having slightly higher degrees Brix and lower titratable acidity than Washington raspberries. Twenty-nine volatile compounds were quantified using stir bar sorptive extraction (SBSE) paired with gas chromatography-mass spectrometry (GC-MS). There were very few differences in volatile concentrations between the transgenic varieties and the wild type 'Meeker'. Much larger variations were observed between sites and harvest seasons. Raspberries grown in Oregon appeared to have higher concentrations of delta-octalactone, delta-decalactone, geraniol, and linalool. Chiral analysis of alpha-ionone, alpha-pinene, linalool, terpinen-4-ol, delta-octalactone, and delta-decalactone demonstrated a much higher percentage of one isomer over the other, particularly alpha-ionone, alpha-pinene, delta-octalactone, and delta-decalactone, with more than 90% of one isomer, while a racemic mixture was observed for linalool. The isomeric analysis revealed very little variation between varieties, locations, or years. The flavor compounds tested in this study did not show any difference between the transgenic lines and the wild type 'Meeker' raspberry.     

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.     

Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape

We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.     

Influence of elevated CO2 and GM barley on a soil mesofauna community in a mesocosm test system

We hypothesized that the combined effect of rising levels of atmospheric carbon dioxide (CO₂) and increasing use of genetically modified (GM) crops in agriculture may affect soil food-webs. So we designed a study for the assessment of the effects of elevated CO₂ (eCO₂) concentrations and GM barley on a soil-mesofauna community employing a 2nd tier mesocosm test system. The GM barley, Hordeum vulgare cv. Golden Promise, had a modified content of amino acids and it was compared with three non-GM barley cultivated varieties including the isogenic line. Our mesocosm experiment was conducted in a greenhouse at ambient (aCO₂) and eCO₂ (+80 ppm) levels and included a multispecies assemblage of Collembola, Acari and Enchytraeidae with either a GM or conventional spring barley varieties. To detect food-web changes we added dried maize leaves naturally enriched in δ¹³C and δ¹⁵N relative to the soil substrate. Soil, plants and animals were collected after five and eleven weeks. We found that the eCO₂ concentration did not affect the plant biomass, but the predatory mite and two collembolan species showed significantly lower abundances at eCO₂. The densities of three collembolan species (Folsomia fimetaria, Proisotoma minuta and juveniles of Mesaphorura macrochaeta) was significantly lower in the GM treatment compared to some of the non-GM varieties. F. fimetaria was less abundant in presence of GM barley compared to the cultivated barley variety “Netto” at both CO₂ levels, while the density of P. minuta was significantly reduced with the GM barley compared to variety “Netto” at aCO₂ and the isogenic variety at eCO₂. Maize litter acted as a food source for the community, as it was revealed by δ¹³C values in microarthropods. Microarthropod δ¹³C decreased over time, which indicates a diet change of the species towards carbon derived from barley, due to maize litter decomposition. The industrially produced CO₂ gas also had a role as an isotopic marker, as the different δ¹³C values were reflected in the barley and in the collembolan species. GM barley did not affect δ¹³C and δ¹⁵N values of soil animals indicating that the overall trophic structure of the mesofauna community was not changed compared to the non-GM cultivated varieties. The mesocosm methodology integrating stable isotope analysis demonstrates the potential of the multi-species mesocosm as a tool to detect and track changes in the soil trophic interactions in response to environmental pressures, climate and novel agricultural crops.     

Responses by earthworms to reduced tillage in herbicide tolerant maize and Bt maize cropping systems

Genetically modified (GM) crops may affect earthworms either directly through the plant, its root exudates and litter, or indirectly through the agricultural management changes that are associated with GM plant production. In order to investigate such possible effects, we established two field studies of Bacillus thuringiensis (Bt) maize and a glufosinate ammonium tolerant maize and included a reduced tillage (RT) treatment and a conventional tillage (CT) treatment as examples of a likely concomitant change in the agricultural practise. At a French study site at Varois, (Bourgogne), a field grown with the Bt-toxin producing transgenic maize line MON810 was studied for 1 year. At a Danish study site, Foulum (Jutland), 1 year of Bt maize was followed by 2 years of herbicide tolerant (HT) maize. At the French study site, the most prominent effects observed were due to the tillage method where RT significantly reduced the earthworm populations to levels about half of CT. At the Danish study site effects of CT complied with known reduction of anecic earthworms due to this technique and likewise effects of RT were observed for endogeic earthworms. Earthworm populations were diminished with the HT crop, probably due to exposure to the herbicide Basta^® during the two consecutive autumn seasons. This study confirms the importance of including the tillage techniques and pesticide usage when evaluating the environmental effects of new agricultural technologies.     

Characterisation of agro-morphological traits of corchorus accessions

The genus Corchorus includes nutrient-rich indigenous leafy vegetables often grown or harvested in the wild in Africa. It has potential to contribute to food security, but there is need to improve it as a crop through plant breeding. However, not much is known about the genetic variability among Corchorus species. The aim of this study was thus to characterise the available germplasm of Corchorus accessions and investigate heritability of their agro-morphological traits. Eleven Corchorus accessions (4 South African and 7 from the World Vegetable Center) were evaluated in a field study at Roodeplaat, South Africa, for their morpho-agronomic traits using a randomised complete block design with three replications across two seasons (2015/2016 and 2016/2017). Data were subjected to ANOVA, principal component analysis and cluster analysis. Broad sense heritability, genetic advance, genetic parameters and correlations were determined among the accessions and traits. The Corchorus accessions showed significant (P < .05) differences in all the quantitative traits evaluated. The data showed significant variability among the studied Corchorus accessions in their agro-morphological traits for exploitation in future breeding programmes that in turn can contribute to the improvement of this crop.     

Barley carbohydrate composition varies with genetic and abiotic factors

Abstract

Changes in chemical quality arising from genetic composition of different barley varieties as well as non-genetic factors were investigated by comparing samples from different seasons and locations. The samples (15), consisting of different varieties originating from Norway and Canada, were analysed for polysaccharide composition as well as total protein content. The results revealed differences in chemical parameters between the hulled and hull-less varieties, especially in carbohydrate composition. Also, variations within the types were found, which indicated that factors other than the presence or lack of hull may also influence the carbohydrate composition. As expected, varieties grown at the same location in both seasons had a lesser variation in their grain composition between the growing periods than varieties grown at different locations. Changing the growth location from Canada to Norway also gave an increase in starch and insoluble fibre, but decreased the amount of β-glucan, protein and soluble fibre. Promising varieties for the food industry are samples with an atypical starch characteristic without hull, due to higher levels of the proposed health-beneficial components. Also, the commercial Norwegian variety, Olve, had an advantageous grain composition and is a promising variety for food uses.     

Effects of genotype and environment on the contents of betaine, choline, and trigonelline in cereal grains

This study examined the environmental and genetic variation in methyl donor contents and compositions of 200 cereal genotypes. Glycine betaine, choline, and trigonelline contents were determined by (1)H NMR, and significant differences were observed between cereal types (G) and across harvesting years and growing locations (E). Glycine betaine was the most abundant methyl donor in all of the 200 lines grown on a single site, and concentrations ranged from 0.43 ± 0.09 mg/g dm in oats to 2.57 ± 0.25 mg/g dm in diploid Einkorn varieties. In bread wheat genotypes there was a 3-fold difference in glycine betaine content. Choline contents, in the same lines, were substantially lower, and mean concentrations ranged from 0.17 mg/g dm in oats to 0.27 mg/g dm in durum wheat. Trigonelline was by far the least abundant of the methyl donors studied. Despite this, however, there were large differences between cereal types. Twenty-six wheat genotypes were grown in additional years at four European locations. The average glycine betaine content was highest in grains grown in Hungary and lowest in those grown in the United Kingdom. Across the six environments, there was a 3.8-fold difference in glycine betaine content. Glycine betaine levels, although moderately heritable (0.36), were found to be the most susceptible to the environmental conditions. Free choline concentrations were less variable across genotypes, but heritability of this component was the lowest of all methyl donor components (0.25) and showed a high G × E interaction. Trigonelline showed the most variation due to genotype. Heritability of this metabolite was the highest (0.59), but given that it is at a very low concentration in wheat, it is probably not attractive to plant breeders.     

Effect of Wheat Genotype and Environment on Relationships Between Dough Extensibility and Breadmaking Quality

Dough extensibility affects processing ease, gas retention, and loaf volume of finished products. The Kieffer dough extensibility test was developed to assess extensibility of small dough samples and is therefore adapted for use in breeding programs. Information is lacking on relationships between wheat growing environments and dough properties measured by the Kieffer dough extensibility test. This study documents the variability of dough extensibility (Ext), maximum resistance to extension (Rmax), and area under the extensibility curve (Area) in relation to breadmaking quality, and the effect of wheat growing environments. Mixograph, Kieffer dough extensibility, and bake tests were performed on flour milled from 19 hard red spring wheat (Triticum aestivum L.) genotypes grown during three growing seasons (2007‐2009) at six South Dakota locations. Although both genotype and environment had significant effects on Kieffer dough extensibility variables, environment represented the largest source of variation. Among genotype means, Area was most correlated (r = 0.63) with loaf volume, suggesting that by selecting lines with increased Area, loaf volume should improve. Rmax was positively correlated (r = 0.58) with loaf volume among genotype means but negatively correlated (r = –0.80) among environmental means. Ext was positively correlated (r = 0.90) with loaf volume among environmental means. Weather variables were correlated with Rmax, Ext and loaf volume and therefore could help predict end‐use quality.     

Relationships Between Simple Grain Quality Parameters for the Estimation of Sorghum and Maize Hardness in Commercial Hybrid Cultivars

Grain hardness affects sorghum and maize processing properties especially for dry milling. A variety of simple grain quality parameters were assessed on 17 sorghum and 35 white maize hybrid cultivars grown in six and four locations, respectively, in South Africa. The purpose was to determine tests that can be used to distinguish hardness in commercial sorghum and maize. The grains were characterized by test weight (TW), thousand kernel weight (TKW), decortication with the tangential abrasive dehulling device (TADD), and kernel size. Maize was also characterized for susceptibility to breakage, stress cracking, and near‐infrared transmittance (NIT) milling index. Principal component analysis showed that, in nontannin and tannin sorghums, TADD hardness and TW were closely correlated (P < 0.001). In maize, TADD hardness was closely correlated (P < 0.001) with NIT milling index and TW. Hence, TADD hardness and NIT milling index or TADD hardness and TW would be suitable for maize hardness evaluation. A combination of TADD hardness, TW, TKW, and kernel size >3.35 mm can be used together to select sorghum grain for hardness. It thus appears that TADD hardness is an excellent method of estimating both sorghum and maize hardness that can be applied for routine batch analysis and cultivar evaluation.     

Genotype and Environmental Influences on Pasting Properties of Rice Flour

The pasting behavior of flour from several Australian rice (Oryza sativa L.) cultivars, differing in amylose content and grown in three different locations and three seasons, were determined using the Rapid Visco Analyser. Genotype, growth season, and growth location all affected the pasting behavior of rice flour. The amylose content of the same cultivar was significantly higher in the coolest growing season, resulting in RVA traces with lower peak viscosity and higher setback than samples with lower amylose content. When the same cultivar of rice was grown in different locations in the same season, there were no significant differences in the total starch, protein, lipid, and amylose content of the flour, but there were significant differences in the pasting behavior. This indicates that environmental as well as genetic factors influence the pasting behavior of rice flour. Flour from parboiled and quick‐cooking rice did not paste and had low viscosities compared with unprocessed rice. Results from this study showed that the pasting behavior of rice flour was related to genotype and was influenced by environmental factors that brought about subtle changes in the grains that were not picked up by chemical analyses.     

Variation of Free Asparagine Concentration and Association with Quality Parameters for Hard Red Spring Wheat Grown in North Dakota

Free asparagine in wheat is known to be a precursor for the formation of acrylamide, which is unacceptable to consumers owing to its potential risks to human health. This research was performed to determine variation of free asparagine concentration (FAC) in hard red spring (HRS) wheat grown in North Dakota. Quality traits and FAC were analyzed for 75 HRS wheat genotypes grown at three locations. The ANOVA indicated that growing location had a strong effect on FAC. The main effect of genotype and interaction of genotype × location were also highly significant (P < 0.001). The genotype × location interaction was also explored graphically using a biplot of principal components calculated from the genotype and genotype × environment interaction model. The biplot analysis revealed that the pattern of interaction of genotype × location might be a noncrossover type. Certain HRS genotypes were identified to have consistently low FAC across growing locations. The FAC showed low genotypic correlations with quality traits, indicating low level of linkage between FAC and quality traits for HRS wheat genotypes.     

Monitoring the weather at five winter wheat experimental field sites

Weather monitoring equipment was set up at 5 winter wheat experimental field sites ranging from Lethbridge, Alberta, Canada, to Lubbock, Texas, U.S.A. Uniform weather-measurement techniques were employed at all locations for the following weather variables: wind speed and direction; solar radiation; air temperature and relative humidity; soil temperature and precipitation. Weather data for the Nebraska and Kansas locations were automatically retrieved via daily computer telecommunication. Data from the remaining locations were retrieved at the end of each growing season. Data from 2 growing seasons show wide differences in the environmental conditions among the study locations. Near-real-time data collection required less quality control because the daily eavesdropping on the station resulted in less sensor down time.