No abstract

Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight.

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 materialThe online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users.

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