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Field Evaluation of a Transgene Containment Strategy for Plant-Made Pharmaceuticals in Tobacco
H.M. Davies, O. Chambers, C. N.Stewart
Department of Plant and Soil Sciences
Plants used to manufacture pharmaceuticals and industrial proteins must not be able to transfer the genes encoding these products to the equivalent conventional crops. This project examines a genetic strategy for obviating, or greatly reducing the potential for, this 'gene flow' when tobacco plants are used as the production system.
2011 Project Description
Genetic constructions were assembled containing the fluorescent-protein marker genes, and whole-plant- and pollen-specific promoters, as planned. Nicotiana tabacum and Nicotiana glauca plants were transformed with these constructs, and lines showing the highest levels of fluorescence were self-pollinated and selected to obtain homozygous material with optimal phenotype. (Considerable, unanticipated optimization of the transformation protocols was required for N. glauca, hence the delay in progress that required no-cost extensions to the overall project.) These transgenic plants represent a project output which will be useful in future studies of transgene flow and containment.
The transgenic N. tabacum and N. glauca lines then served as parents in the production of interspecific hybrids which formed the critical subject-matter of the project. As these hybrids are infertile, segregating populations had to be used in the field experiments. Accordingly, the seedlings of the hybrids having leaf-expressed marker were examined to verify fluorescent phenotype prior to transplanting into the field.
In each of the two successive field seasons transgenic interspecific hybrids, male-sterile TN90 N. tabacum (non-transgenic, pollen recipient), and fertile SN2108 N. tabacum (non-transgenic control pollen source) plants were arranged in the Nelder wheel layout in the field, in two locations, as planned. Data were recorded on pod/seed-set, and all seeds formed on the hybrid plants were collected for analysis of presence of the marker gene, as were a sampling of the seeds set on the TN90 plants.
Data on the presence of the marker and the numbers of seeds comprise the planned outcome of the project. Following the first growing season these data were shared with other researchers through presentations at several conferences and workshop events. Although data are still being obtained from counterpart greenhouse control crosses and the 2011 field experiment at the time of compiling this final report, we now have a good sense of the overall findings.
It appears that the interspecific hybrid plants will not form a complete protection against outcrossing. While there was very little seed-set on the hybrids in the field, a small percentage of those seeds were viable and some of those yielded seedlings expressing the fluorescent marker gene. This suggests that the hybrids could be a source of released transgenic seeds in the field, especially if pollinated from adjacent tobacco crops. However, we did not observe the reciprocal situation, suggesting that the hybrid cannot successfully export a transgene through pollination of a nearby non-transgenic tobacco plant (male-sterile TN90 in this project).
There has been considerable concern about the projected use of transgenic plants for biomanufacturing of medical and industrial materials, especially in regard to the possibilities for inadvertent transfer of the transgene to conventional crops of the same species, resulting in contamination of the food supply with the non-food materials. Tobacco has been suggested as a preferable (non-food) crop for these purposes, but concerns about this transgene flow (outcrossing) aspect still influence the regulatory requirements, and acceptance of the strategy by tobacco farmers and their organizations is uncertain.
Having observed that hybrid plants made by crossing different species of Nicotiana exhibit greatly reduced fertility under greenhouse conditions we wished to examine whether this trait might serve as a containment strategy for transgenes in the plant under field conditions. To our knowledge this research, conducted with the BRAG funding, was the first to examine the use of potentially sterile plants in the field as an optimal way to obviate outcrossing concerns. Thus the results of the project, whether favorable or unfavorable relative to the proposed strategy, add significant and useful knowledge to the ongoing development of biotechnology-based strategies for using plants as renewable, cost-effective biomanufacturing solutions.
At the time of preparing this report our results suggest that the hybrids do not offer absolute genetic containment, in that viable seed containing the transgene may still be formed on the hybrids, enabling them to persist in the field (albeit at low frequency).
Given that fields would likely be monitored for the appearance of volunteer transgenics in subsequent growing seasons as a matter of routine, this is much less important that the prospect of export of transgene via pollen from the hybrids to nearby non-transgenic tobacco. And in that regard our results are very encouraging, suggesting the absence of such transfer (within the scope of experiments at this scale). Thus our findings suggest the utility of the interspecific hybrids as contributors to production strategies for transgenic tobacco making new materials, especially if used in parallel with other anti-geneflow measures (e.g. combined with plastid-based gene expression etc.).
Chambers O, Rice H, Mundell R, Millwood R, Stewart N & Davies HM. FIELD EVALUATION OF AN INTERSPECIFIC HYBRID AS A CONTAINMENT STRATEGY FOR PLANT-MADE PHARMACEUTICAL APPLICATIONS IN TOBACCO. 65th Tobacco Science Research Conference September 18-21, 2011 - Lexington, KY.