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One of the concerns surrounding the commercial release of genetically modified (GM) crops is the escape of transgenes into agricultural or semi-natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato (Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt-tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives (Solanum hirsutum and Solanum nigrum) and a non-GM tomato variety; (b) bumblebee-mediated crosses between GM and non-GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non-GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee-mediated cross-fertilisation rate between GM and non-GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non-GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross-fertilisation between GM and non-GM tomato and (3) the Cry3Bb1-expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees. © 2012 The Authors. Annals of Applied Biology © 2012 Association of Applied Biologists.

The subject of this thesis is the use of transgenic plant resistance as a method to control the Colorado potato beetle, Leptinotarsa decemlineata Say in eggplant. The gene conferring resistance is coding for a Cry3B toxin and it is a synthetic version of a wild-type gene originally obtained from the soil bacterium Bacillus thuringiensis Berl.Eggplant cultivations are constantly attacked by a number of serious pests (e.g. the fruit and shoot borer, the Colorado potato beetle, soil-borne fungi). In spite of the heavy losses they may cause, breeding for resistance on this crop has been very limited because of the lack of desirable traits in the eggplant genome or sexual incompatibility with the resistant wild related species.The first chapter reviews Colorado potato beetle biology and its control, with a special emphasis on beetle-eggplant relationships. L. decemlineata has become a major problem for eggplant cultivation and sometimes its control on this crop is even more problematic compared to potato cultivation. The longer life cycle of the plant and the intensive regime under which horticultural crops are cultivated have contributed to the increasing importance of this pest.In the second chapter, a review of the source of resistant genes available in both the eggplant gene pool and wild Solanum relatives is presented. Considering the genetic basis of resistant traits, the possible strategies for eggplant breeding are discussed with emphasis on approaches based on the integration of classical breeding methods (crosses and selection) with biotechnological ones (anther culture, genetic transformation, protoplast fusion and marker-assisted selection).In the third chapter, the results of the study in which protein extracts of Escherichia coli expressing the toxin gene from B. thuringiensis were tested for effect on the behavior and development of L. decemlineata larvae using in vitro bioassays are presented. No antifeedant effect due to Cry3B toxin was found, even in concentrations which caused mortality or severely inhibited larval growth.In the fourth chapter, the production of transgenic eggplants and their evaluation is presented. A modified Bt gene of Bacillus thuringiensis var. Tolworthi, encoding a coleopteran insect-specific Cry3B toxin, was transferred via Agrobacterium tumefaciens to the female parent of the commercial F1 hybrid 'Rimina' eggplant (released by the Istituto Sperimentale per l'Orticoltura). A large number of transgenic plants were regenerated and tested by PCR and NPTII expression assays. The presence of the Cry3B toxin in leaf extracts was demonstrated by the DAS-ELISA test in 57 (62.3%) transgenic plants which contained a 74 kDa protein cross-reacting with the serum anti-Cry3B toxin. Twenty-three out of 44 S. melongena plants tested by an insect bioassay showed a significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus, transgenic Bt eggplants may represent a very effective means of CPB pest control.Transgenic potato clones expressing a Cry3B endotoxin were used to study the trophic interactions between newly emerged Colorado potato beetle adults and these resistant clones (Chapter 5). Adult longevity and fitness were studied for the first 3 weeks after emergence. The reproductive biology of the beetle on highly resistant clones, partly resistant clones and control potato plants was monitored by dissecting females after 7-15 days of feeding and by analyzing the haemolymph's protein content after 3 days of feeding. Feeding behavior on either highly toxin expressing or control plants was monitored individually for 36 beetles feeding on leaf-discs. Beetles feeding on transgenic or control clones as the sole source of food had similar longevity. However egg production was completely inhibited on transgenic plants.Dissection studies indicated that adult males living on transgenic plants were still able to mate and produce mobile sperms, but the females were impaired in their reproductive ability since ovarioles were not normally developed. An exam of the haemolymph revealed the protein concentration in females living on transgenic plants to be dramatically reduced (about 50% compared to the control). The feeding behavior of Colorado potato beetle adults was not affected by the different food plants. This shows that transgenic potato plants were readily accepted as host plants by the beetles. The implications of these findings for the use of transgenic plants as a means of L. decemlineata control are discussed.In the sixth chapter the most relevant results of the first year of a field experiment with the transgenic eggplant lines are presented. Two of the 3 transgenic lines used showed a high level of resistance in two separate trials, as indicated by the analyses of L. decemlineata population levels and crop yield. Fruit production was almost doubled in the resistant lines compared to a DR2 untransformed control. Only one transgenic line showed an intermediate level of resistance, giving results more similar to the control under heavy CPB attack, whereas it gave comparable results to the other transgenic lines where natural infestation was milder. No detrimental effects on non-target arthropods (including the chrysomelid beetle Altica spp.) were apparent. Field observations confirmed that Bt-expressing transgenic plants might be able to successfully control Colorado potato beetle infestations in eggplant cultivations, representing a potentially effective and environmentally safe means of pest control.In chapter 7, the relationships between L. decemlineata egg density and Coleomegillamaculata DeGeer predatory behavior is presented. Despite aggregation in areas of the highest prey density by C.maculata , egg consumption was inversely related to egg mass density at the smallest and the largest spatial scales tested. The experimental data on predation rates in high and low density field treatments, were included in a mathematical model to simulate the impact of natural enemies on the rate of L.decemlineata adaptation to Bt-toxin-expressing transgenic potato plants when Bt-expressing plants are mixed at the plot-to-plot level with normal potato plants. Results showed that C.maculata predatory behavior could decrease the rate at which L.decemlineata adapted to Bt-toxins if plot-to-plot mixed plantings were used.Finally, a simulation model to predict the possible adaptation of L. decemlineata to the Cry3 toxin expressed in transgenic eggplant is presented (Chapter 8). The use of mixed fields of transgenic and susceptible isolines at a 90:10 ratio has been simulated. Beetle movement, which is a fundamental parameter when studying plant mixtures, has been addressed with a 'two-stage' hypothesis. The biological and genetic characteristics of the beetles have been set to specifically address their possible interactions with resistant eggplant. The role of gene dominance, migration, and fitness costs associated with the resistant genotype have been examined. Using the hypothesis of partial dominance of the resistant gene, only a high level of migration (very likely, in most agricultural areas) or a considerable reduction of the fitness of resistant beetles, associated with the change in their genome, can guarantee a long-lasting efficacy of the germplasm. The simulations clearly indicate that the effect of resistance in transgenic clones expressing Bacillus thuringiensis -derived toxins can be optimized only in accordance with opportune agricultural practices.