Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants.

Multiple variants of the Cauliflower mosaic virus 35S promoter (P35S) are used to drive the expression of transgenes in genetically modified plants, for both research purposes and commercial applications. The genetic organization of the densely packed genome of this virus results in sequence overlap between P35S and viral gene VI, encoding the multifunctional P6 protein. The present paper investigates whether introduction of P35S variants by genetic transformation is likely to result in the expression of functional domains of the P6 protein and in potential impacts in transgenic plants. A bioinformatic analysis was performed to assess the safety for human and animal health of putative translation products of gene VI overlapping P35S. No relevant similarity was identified between the putative peptides and known allergens and toxins, using different databases. From a literature study it became clear that long variants of the P35S do contain an open reading frame, when expressed, might result in unintended phenotypic changes. A flowchart is proposed to evaluate possible unintended effects in plant transformants, based on the DNA sequence actually introduced and on the plant phenotype, taking into account the known effects of ectopically expressed P6 domains in model plants

Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects

The stability of Fab antibody fragment expression during plant development was studied using two homozygous Arabidopsis thaliana lines that contain single copies of the transgenes. These lines exhibited expression characteristics that are typical for homology-based post-transcriptional gene silencing. Their developmental silencing profiles differed markedly, presumably due to the influence of the genomic context on the T-DNAs. In both lines, a clear gene dosage effect could be observed: in contrast to the homozygous lines, derived hemizygous plants accumulated high levels of Fab fragments throughout development. Interestingly, silencing also occurred in double-hemizygous plants, which resulted from a cross between the two homozygous lines and had two copies of each T-DNA at non-allelic positions in their genome. In all cases, down-regulation of the Fab levels was strictly correlated with methylation of cytosine residues in the transcribed regions of the transgenes. Remarkably, this methylation was also found in regions in which the transgenes were non-homologous regions. Finally, the time point of down-regulation depended on the culture conditions and differed for leaves and roots of the same transgenic plant

Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa

Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for non-model plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes should be thoroughly investigated for each experimental condition under investigation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users.status: publishe

Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens

The recognition of the T-DNA left border (LB) repeat is affected by its surrounding sequences. Here, the LB regions were further characterized by molecular analysis of transgenic plants, obtained after Agrobacterium tumefaciens-mediated transformation with T-DNA vectors that had been modified in this LB region. At least the 24-bp LB repeat by itself was insufficient to terminate the T-strand synthesis. Addition of the natural inner and/or outer border regions to at least the LB repeat, even when present at a distance, enhanced the correct recognition of the LB repeat, reducing the number of plants containing vector backbone sequences. In tandem occurrence of both the octopine and nopaline LB regions with their repeats terminated the T-strand synthesis most efficiently at the LB, yielding a reproducibly high number of plants containing only the T-DNA. Furthermore, T-strand synthesis did not terminate efficiently at the right border (RB) repeat, which might indicate that signals in the outer RB region inhibit the termination of T-strand synthesis at the RB repeat

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens-mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6-30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20-36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4-6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24-31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells

Repercussions of politicized regulation exemplified by compulsory new TC1507-maize 90-day rat feeding study

Politicized and prescriptive regulation of genetically modified (GM) crops has unintended adverse effects, including misdirected resources and reduced benefits.  In the case of animal testing, this suboptimal use of resources includes the needless sacrifice of animals.  Whole-food animal feeding studies are generally of negligible value in the risk assessment of GM crops, a position that was affirmed by the European Food Safety Authority (EFSA).  Contrary to EFSA’s 2011 position, in 2013, the European Commission directed that 90-day rat studies be conducted for new GM events.  As no EFSA guidance was available for hypothesis-free 90-day rat feeding studies, EFSA interpreted this as a mandate to develop a prescriptive study design.  Recently, EFSA has retroactively required 90-day rat studies be completed under new study guidelines for previously approved component single events as part of the approval of breeding stacks.  Having been unable to secure a derogation (exemption) from EFSA, a new compulsory 90-day rat study was conducted with TC1507 maize to support a breeding-stack submission.  As previously shown, TC1507 maize does not adversely affect rats.  This politically driven additional animal testing is risk disproportionate, at odds with European and international standards for animal welfare, and provides no scientific value to the safety assessment for GM breeding stacks

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding.Peer reviewe