Effects of gene flow of GM maize into non-GM maize and wild relatives

Evaluating the consequences of gene flow in general, regardless of whether or not transgenes are involved, is a challenge because it is difficult to predict the ecological and evolutionary effects of genes that are expressed in distinct ecological contexts and genetic backgrounds. Maize (Zea mays) is a wind-pollinated outcrossing plant with no known biological barriers to transgene flow from genetically modified (GM) to non-GM plants, i.e., hybrid varieties, open pollinated varieties (OPVs) and landraces. This research is complicated when the wild relative of maize (“teosinte”) and GM maize plants are also sympatric populations. However, only few studies exist that have investigated if and how transgenes are expressed in the various genetic backgrounds, following gene flow, and how this may affect the quantitative production of the transgene product, here Bt proteins, and their bioactivity against insect pests. In addition, it is possible that biotic stress factors such as herbivory further modulate these complex relationships. The outcomes of this research are of importance for the prediction of pest resistance evolution, smallholder seed diversity and preservation and risk assessments of GM organisms. The aim of this thesis was to investigate the entire triple-relationship of transgene expression, the resulting protein production (Cry1Ab toxin) and its bioactivity in pest insects, following transgene flow from GM hybrid maize into non-GM maize, i.e. near-isogenic non-GM maize hybrids (ISO), open pollinated maize varieties (OPVs) and wild/weedy relatives to maize. The thesis is divided into three chapters, which had the following specific aims: 1) to investigate the triple-relationship following transgene flow from GM hybrid maize into non-GM, near-isogenic maize hybrid (ISO) and OPVs under non-stress conditions. In addition, the Mendelian inheritance segregation pattern of the cry1Ab transgene expression was examined; 2) to examine how herbivore feeding damage by two maize insect pests, Helicoverpa armigera and Spodoptera littoralis, may affect this triple-relationship; and 3) to test if hybridization between the GM maize and wild/weedy teosinte plants from Spain is possible, and to study the above described triple-relationship in Cry1Ab positive crosses between GM maize and Spanish wild/weedy teosinte. Basically, in chapter 1 and 2, the plant materials and setup of the experiments were identical and conducted in the same climate chamber at ETH Zürich. The difference is that in chapter 1 the experiments were carried out under non-stress growing conditions and in chapter 2 the tested crosses were exposed to biotic stress exerted through feeding damage exerted by two different herbivorous pests of maize, Helicoverpa armigera and Spodoptera littoralis. Two groups of F1, F2 crosses and backcrosses with GM, ISO and OPV maize varieties from Brazil and South Africa were used. In both chapters, we measured the cry1Ab transgene expression as quantification of mRNA, the Cry1Ab protein concentration using quantitative ELISA tests and the pest survival in feeding bioassays. Bioassays were carried out with larvae of two lepidopteran maize pest species, Helicoverpa armigera and Spodoptera littoralis. Additionally, the segregation pattern of cry1Ab transgene was examined (in chapter 1 only). In chapter 3, we generated in climate chamber, under controlled conditions, crosses between GM maize and wild/weedy teosinte plants in both ways. This novel wild/weed teosinte plants were recently found in Spanish maize fields. Here, we also measured the cry1Ab transgene expression, Cry1Ab protein concentration (ELISA test) and pest survival (bioassays). Bioassays were carried out with larvae of the lepidopteran maize target pest, Ostrinia nubilalis. Overall, similar results were found in all three chapters. The cry1Ab transgene outcrossed effectively into all different genetic backgrounds, and even into a wild/weedy teosinte plants (from Spain). In all resulting Cry1Ab positive crosses, the transgene was stably expressed when compared to the respective GM parental maize. Regardless whether the crossing partners were generated in a commercial maize breeding program or OPVs selected and maintained by small-scale farmers, or even related to wild/weed teosinte from Spain. The resultant Cry1Ab toxin was produced in all crosses generated, in a bioactive form, inducing high mortality rates in the tested insect pest species. Transgene introgression led to consistent, though highly variable, concentrations of Cry1Ab toxins that were similar to those observed in the respective GM parental maize. In most of these GM crosses with non-GM maize, the segregation patterns of the cry1Ab transgene followed Mendelian rules, but not in all. Based on chapter 2, when the Brazilian and South African crosses were exposed to herbivore stress, the testing results of these triple-relationships were diverse and unpredictable. While in the South African crosses, the herbivore damage inflicted by S. littoralis did not affect the entire triple-relationship, the herbivore damage by H. armigera led to higher production of Cry1Ab protein in the Brazilian crosses. Under herbivore stress conditions, the variability in concentration levels of the Cry1Ab protein in the Brazilian crosses was significantly higher than under non-stress conditions, and this led subsequently to a higher variability of the mortality rates among the H. armigera larvae. This relationship was not observed with the South African crosses. Overall, another important result found in all three chapters was that no correlations were observed between the transgene transcription levels and the resultant Cry1Ab protein concentrations, nor between the Cry1Ab protein concentrations and insect mortality rates. Surprisingly, also no correlations were found in the GM parental maize plants. This suggests that while transcription of the cry1Ab transgene reliably determines the presence of Cry1Ab protein, mRNA levels (i.e. the transgene expression) do not reflect, by themselves, the final Cry1Ab protein concentrations found in the plant leaves. Therefore, we conclude that other metabolic plant processes are influencing this process, that can possibly be induced or modulated by herbivore stress (if present). Finally, this research documented that the Cry1Ab toxins produced in GM teosinte hybrids were bioactive and induced mortality rates equal to or above 95% in neonate O. nubilalis after a period of only 4 days, reflecting mortality rates of O. nubilalis similar to those on GM maize plants. Therefore, based on this result, we suggest the potential increase the weediness of the Spanish wild/weedy teosinte plants by acquired defense against the target pest specie, here Cry1Ab toxin. These outcomes are important for environmental risk assessments and various aspects of ecological safety where GM maize plants and wild/weedy teosinte form sympatric populations