Overexpression of epsps transgene in weedy rice: insufficient evidence to support speculations about biosafety.

The recent paper published in New Phytologist, Wang et al. (2014; this issue pp. 679–683), purports in its title that ‘A novel 5enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide’ and in the paper claims that weedy rice expressing the transgene is more competitively fit than the wild type in the absence of glyphosate treatment. While this may be so, there is a lack of evidence in the paper that the transgene confers glyphosate resistance, or that the transgenic weedy rice is more competitively fit than its wild type. As this paper has generated extensive media coverage facilitated by the authors, we feel it imperative to analyze the paper in depth to ascertain whether the claimsmade in title, summary, and text have beenmet, whether criteria necessary for publication have been achieved, as well as whether the media reportage was justified in the form released by the authors. In many respects, the findings are not as novel as the authors imply. This is not the first instance when genes from a crop have increased the rate of growth and/or fecundity of a related weed with which the crop can cross-pollinate. Many such cases have been discussed before (Ellstrand, 2003). Whenever yield-enhancing traits are introduced into crops they can quickly introgress in related infesting weeds. For example, weedy Aegilops spp. growing in or near wheat fields typically introgress wheat genetic material (Weissmann et al., 2005), have larger flag leaves, ears, and grain than the same Aegilops spp. in wild habitats (Rehman et al., 2006; Arrigo et al., 2011). Indeed after disease resistance was crossed into a US wheat from a nonindigenous Aegilops spp., it quickly introgressed from wheat into indigenous Aegilops cylindrica, a major weed in parts of the United States (Perez-Jones et al., 2006). Most crop-related weeds, and especially weedy rice, have co-domesticated with the respective crops. There is considerable evidence thatmost weedy rice strains derive fromde-domestication of cultivated rice and are not derived directly from the wild progenitors (Gross et al., 2010; Lawton-Rauh & Burgos, 2010; Thurber et al., 2013, and references cited therein). Thus,weedy rice fecundity has increased through the ages because cultivated rice yield has increased. None of this very relevant information was intimated or cited, and the authors made it appear as if it is novel that a trait introduced in a crop enhanced and raised fecundity when it introgressed to a related weed. A basic tenet of published science is that other scientists should be able to reproduce the experimental findings, based on the Materials and Methods section of peer-reviewed papers. This was not the case here. Particularly crucial, no sequence data were provided or deposited at EMBL/GenBank for the transgene construct used.The authors say they used a modified rice epsps, but there was no information provided on the sequence modifications, or its protein products Km or Ki for glyphosate, yet they claim that the modified gene confers resistance to glyphosate. No data are provided that demonstrate resistance to glyphosate or its extent, nor is a reference given to any peer-reviewed study where such data are presented, despite implying that such a critical and influential connection is supported by this study. They do show a doubled level of the EPSPS protein. In other cases where there was a doubled level of EPSPS expression (Boerboom et al., 1990; Baerson et al., 2002) there was only a c. seven-fold increase in the I50 resistance level to glyphosate, which is usually not enough for commercial use in the field. They say that the gene was described in a published paper (Xu et al., 2002) (correct citation given here and not in the manuscript in question). This paper (Xu et al., 2002) only presents data on cloning the rice epsps gene and presents nothing about its modification. Is this the gene the authors used to produce the transgenic rice in question, whichwas then further crossed toweedy rice? They cite no paper on the original transgenic cultivated rice containing this gene nor on the variety they used. Additionally, there is a surprising paucity of molecular data about the transgenic rice plants, for example, there is an absence of Southern blot data, resequencing hybrids to confirm identity, gene insert number, number of transgenic events recovered, the variation of gene expression and phenotypes among the transgenic events, or even enough gene expression analysis of the glasshouse-grown plants to be certain of increased transgene expression. However, we can assume here that transgenic plants had higher expression. In some places in the paper, the authors state that the overexpressed epsps gene in the transgenics is the ‘native rice epsps gene’ (e.g. the Summary), and in other places they confusingly refer to it as a modified gene. In the Materials and Methods section, they state that the transgene is different from the native gene in size (700 and 1000 bp), but there is no sequence information on how it is different, or even if it includes the entire coding sequence. Since the entirety of the findings of Wang et al. rests on the material composition of the transgenic plants, we do not understand how the findings could be validated or replicated by others in rice or another species of plant and thus how this paper can be seen as a valid contribution deserving publication. No data are given on the fecundity of the transgenic rice vs the parent variety. Did all cultivated rice transformants give such an increase or just one – that is, is this a positional effect that has nothing to do with the gene inserted? There is no sequencing and mapping of flanking regions that would supply insights into this