There is much discussion of the probability of transgene flow from transgenic crop varieties to landraces and wild relatives in centers of origin or diversity, and its genetic, ecological, and social consequences. Without costly research on the variables determining gene flow, research on transgene frequencies in landrace (or wild relative) populations can be valuable for understanding transgene flow and its effects. Minimal research requirements include (1) understanding how farmer practices and seed systems affect landrace populations, (2) sampling to optimize Ne/n (effective /census population size), (3) minimizing variance at all levels sampled, and (4) using Ne to calculate binomial probabilities for transgene frequencies. A key case is maize in Mexico. Two peer-reviewed papers, based on landrace samples from the Sierra Juárez region of Oaxaca, Mexico, reached seemingly conflicting conclusions: transgenes are present (Quist and Chapela, 2001, Nature414: 541–543; 2002, Nature416: 602) or “detectable transgenes” are absent (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343 and 18242). We analyzed these papers using information on Oaxacan maize seed systems and estimates of Ne. We conclude that if Quist and Chapela’s results showing presence are accepted, Ortiz-García et al.’s conclusions of no evidence of transgenes at detectable levels or for their introgression into maize landraces in the Sierra de Juárez of Oaxaca are not scientifically justified. This is because their samples are not representative, and their statistical analysis is inconclusive due to using n instead of Ne. Using estimates of Ne based on Ortiz-García et al.’s n, we estimate that transgenes could be present in maize landraces in the Sierra Juárez region at frequencies of ~1–4%, and are more likely to be present in the 90% of Oaxacan landrace area that is not mountainous. Thus, we have no scientific evidence of maize transgene presence or absence in recent years in Mexico, Oaxaca State, or the Sierra Juárez region.

Cleveland et al. (2005, Environ. Biosafety Res.4: 197–208) offer useful suggestions for monitoring transgenes in landraces of maize, but we disagree with their statement that the scientific conclusions of our paper (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343) are not justified. First, contrary to their perception, our survey was not designed to evaluate transgenes in the whole State of Oaxaca, but rather to monitor a specific portion of the District of Ixtlán de Juárez where the presence of transgenes had been reported previously by Quist and Chapela (2001, Nature414: 541–543). Second, our paper described two methods for estimating frequencies of undetected transgenic seeds, while Cleveland et al. recommend a third approach that explicitly estimates effective population size. They argue that the effective population size of our seed samples is smaller than we assumed, leading to false claims about our detection accuracy. However, we employed a robust statistical approach to compensate for possible bias by using numbers of maternal plants, in addition to numbers of seeds, to provide a conservative estimate of the minimum number of independent samples. When we re-analyzed our 2004 data using effective population sizes, our conclusion that transgenic seeds were “absent or extremely rare” did not change, nor did the general range of possible frequencies of undetected transgenic seeds. Unlike Cleveland et al., we advocate using combined probability tests to analyze data across localities. Third, our critics argue that we accepted the null hypothesis that transgenes were absent. Actually, we assumed that transgenes were present in local landraces, and we used parameter estimation methods to calculate the probability of failing to detect transgenic individuals at a range of frequencies. In agreement with Cleveland et al., we reiterate that there is a clear need for additional surveys with rigorous sampling methods to provide estimates of transgene frequencies over broad geographic areas in Mexico.

An investigation was carried out to monitor the escape and spread of oilseed rape (Brassica napus) transgenic plants and the introgression of transgenes to its closely related feral species in Japan. We screened a total of about 7500 feral B. napus, 300 B. rapa, and 5800 B. juncea seedlings from maternal plants in 143 locations at several ports, roadsides, and riverbanks. The presence of glufosinate-resistance or glyphosate-resistance transgenes in these seedlings was confirmed by means of herbicide treatments and also immunochemical and DNA analyses. B. napus plants with herbicide-resistant transgenic seeds were found at five of six major ports and along two of four sampled roadsides in the Kanto District. Transgenic oilseed rape plants have not been commercially cultivated in Japan, suggesting that the transgenes would probably have come from imported transgenic seeds that were spilled during transportation to oilseed processing facilities. No transgenes were detected in seeds collected from B. napus plants growing along riverbanks in the Kanto District or in seeds from closely related species (B. rapa and B. juncea). To our knowledge, this is the first published example of feral, transgenic populations occurring in a nation where the transgenic crop has not been cultivated commercially.

Horizontal gene transfer (HGT) is an important mechanism by which bacteria recombine and acquire novel genes and functions. Risk scenarios where novel plant transgenes transfer horizontally into bacteria have been addressed in numerical theoretical assessments and experimental studies. A key outcome of these studies has been that the frequencies of such inter-domain transfer are very low, if occurring at all, suggesting that such transfers would not occur at a level that is biologically significant. The relationship between transfer frequencies and the subsequent selection or genetic drift of transgene carrying bacteria often remains unresolved in these studies and assessments. Here we present a stochastic model to better understand the initial establishment and population dynamics of rare bacterial transformants carrying horizontally acquired (trans)genes. The following key parameters are considered: initial transformant numbers, strength of selection, bacterial population size and bacterial generations (time). We find that the initial number of transformants is important for the subsequent persistence of transformants only in the range of 1 to ~50 independent HGT events. Our simulations show that transformant populations under a wide range of HGT rates and selection coefficients undergo stochastic developments where they persist at low frequencies for up to several years (at frequencies that are below detection using available field sampling methodology), after which they eventually may go to fixation. Stochastic variability may thus play a crucial but disregarded role in the design of field monitoring strategies e.g. in biosafety assessments. We also estimate the time required for transformants to reach 0.0002% prevalence in a bacterial population, a threshold that allows experimental detection of transgene carrying bacteria through sampling of the larger bacterial populations.

Fine roots of forest trees form together with certain soil fungi symbiotic structures (ectomycorrhizas), where fungal hyphae are in intimate contact with plant cells. Due to root cell degeneration, plant DNA is released and could be taken up by the fungus. The possibility that horizontal gene transfer might result in a risk for the environment should be evaluated before a massive release of genetically engineered trees into nature occurs, even though only a few convincing examples of horizontal gene transfer are known. Transgenic poplars containing a construct of the Streptomyces hygroscopicus bar gene under the control of the Cochliobolus heterostrophus GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter were generated by Agrobacterium-mediated transformation. The functionality of this construct in the ectomycorrhizal model fungus Amanita muscaria was previously verified by protoplast-based fungal transformation. 35 000 ectomycorrhizas, formed between transgenic poplars and non-transgenic A. muscaria hyphae, were isolated and transferred to selective agar plates. Putative herbicide-resistant fungal colonies were obtained after the first round of selection. However, none of these colonies survived a transfer onto fresh selection medium, nor did they contain the bar gene, indicating that no horizontal gene transfer from poplar to A. muscaria occurred during symbiosis under axenic conditions. However, since ectomycorrhizas are associated under natural conditions with viruses, bacteria and other fungi, these additional associations should be evaluated in future.

The intrinsic toxicity of lepidopteran-active Bt proteins necessitates assessment of non-target risks associated with environmental release of transgenic crops expressing these proteins. Principles of ecological risk assessment provide a means for assessing non-target risks when information regarding exposure to the toxin and species-specific effects are lacking. This is shown for the case of Bt Cry1F maize release in Japan, where off-field pollen dissemination and effect on butterfly species is of concern. The specific ecological entity of concern for the assessment of the non-target impact of Cry1F maize pollen was Yamato-shijimi (pale grass blue butterfly), Pseudozizeeria maha (Kollar), a commonly occurring, susceptible species. Yamato-shijimi is widely adapted in Japan where it occurs in both rural and metropolitan settings, corresponding to the distribution and habitat of katabami (Oxalis corniculata (L.)), the larval host plant. The northern extent of Yamato-shijimi habitat lies to the south of major maize production regions in Japan, but exposure may occur elsewhere where maize and Yamato-shijimi co-occur. Screening level assessment of potential adverse effects to Yamato-shijimi in the field environment considered the probability for spatial-temporal co-occurrence of the life stages of concern (1st and 2nd instars) and the stressor (Cry1F protein expressed in maize pollen) at environmentally relevant concentrations. In the event of exposure to maize pollen, early instars of Yamato-shijimi feed exclusively on the underside of katabami leaves, which further limits the portion of the butterfly population that would be exposed. Projected levels of exposure to Cry1F pollen are below the toxicity level of concern and, thus, indicate negligible risk. Most sensitive species characterization (intergenera sensitivity) similarly shows negligible risk to other Japanese butterfly species of concern when distributed beyond the maize field or field margin.