SummaryThe aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript ⁄ protein ⁄ metabolite profiles than the different genotypes.

The lipoxygenase pathway is involved in the early steps of plant responses to herbivorous insects and phytopathogens. Induced defenses in the crucifer Brassica oleracea have been well documented. Here, we have cloned a LIPOXYGENASE (LOX) from B. oleracea (BoLOX). The sequence reveals that the BoLOX protein has a transit peptide for chloroplast targeting, which is characteristic for class 2 LOXs involved in jasmonic acid (JA) biosynthesis which takes place in the chloroplast. Phylogenetic analysis shows that BoLOX is closely related to B. napus BnLOX2fl and Arabidopsis thaliana AtLOX2, which mediates JA biosynthesis. BoLOX also shares functional characteristics with AtLOX2; BoLOX is inducible by wounding, JA treatment, and herbivores such as caterpillars (Pieris rapae, P. brassicae, and Mamestra brassicae), spider mites (Tetranychus urticae), locusts (Schistocerca gregaria), and a bacterial pathogen (Pseudomonas syringae pv. tomato). Of these, Pieris spp. caterpillars also induce AtLOX2 and JA biosynthesis in Arabidopsis. However, the aphid Myzus persicae did not induce BoLOX, which agrees with previous reports that this aphid induces neither AtLOX2 nor JA biosynthesis in Arabidopsis. Quantitative expression analysis of temporal, spatial, and density-dependent BoLOX transcript levels through real-time quantitative polymerase chain reaction demonstrated that BoLOX is maximally expressed after feeding by only two first-instar caterpillars for 24 h. Systemic expression was approximately 10-fold lower than local expression for herbivore-induced responses. The good correlation of BoLOX transcript levels with reports in the literature on induced defenses of B. oleracea is discussed.

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Background: To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-nonauthorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products.

The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments.