Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow

Production of transgenic crops in open field environments is an ongoing concern of due to the potential for gene flow. New transgenic crops, such as plant-made-pharmaceuticals may generate additional concerns about effects of adventitious transgenes. Use of a bioconfinement strategy may alleviate any consequences by preventing gene flow. The following chapters discuss previous and current research on gene flow, testing of a Nicotiana hybrid system for bioconfinement efficiency, and development of methods for transgene detection. The candidate ‘platform plant’ that was tested is a Nicotiana hybrid (Nicotiana tabacum ‘TN 90’ × Nicotiana glauca) previously identified to be sexually sterile. To quantify gene flow from hybrids, the mGFP5ER gene encoding for green fluorescent protein (GFP) was inserted into the paternal lines, which were crossed to form the hybrid. The DNA content and male fertility of these lines were used to characterize GFP-tagged hybrid lines. There were no differences in DNA content but significant differences in male fertility, in which pollen germination was observed at low rates. Two field gene flow studies revealed GFP-hybrids were not totally sterile since hybrids outcrossed and were pollinated by N. tabacum pollen, but they produced few viable seed. These results were confirmed with manual greenhouse crosses. Biomass studies revealed that the GFP-hybrids were comparable in productivity to an N. tabacum cultivar typically used in field production. An additional tagging strategy was created to produce the orange fluorescent protein (OFP), tdTomato-ER, in pollen by using pollen specific promoters in addition to the whole plant GFP cassettes. N. tabacum ‘TN 90’ and N. glauca were transformed, bred and hybridized to generate a hybrid that produced pollen tagged with orange fluorescent protein. Manual crosses were performed in a greenhouse and partially similar results were obtained compared with previous GFP-tagged hybrid crosses. OFP-tagged hybrid outcrossing produced totally non-viable seed and when non-transgenic N. tabacum was supplied as a pollen donor to the OFP-tagged hybrids some non-tagged progeny were observed. The results suggest the Nicotiana hybrid could be a productive biomanufacturing platform and could provide total bioconfinement if grown in physical isolation from N. tabacum and N. glauca.