Abstract
BackgroundMonitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification.ResultsWe report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum ‘TN 90’ and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression.ConclusionThe orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.
Highlights
Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production
green fluorescent protein (GFP) was visible in leaves, stems, and roots and Orange fluorescent protein (OFP) was visible in pollen under a microscope (Figure 5) with the aforementioned filter set
Fluorescent individual plants from the most fluorescent N. glauca TD-GFP-K lines were crossed with highly fluorescent TN 90 TD-GFP-H lines to ensure hybrids had both antibiotic resistance genes and would fluoresce brightly, thereby facilitating detection
Summary
Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. These studies have shown that green fluorescent protein (GFP) is an effective tool for the purpose of gene flow tracking and can be targeted to various organs and tissues within plants, including pollen This technology, in effect, could be used in an environmental monitoring system, one of the many uses of FPs in plants [5]. Long-range pollen tracking was conducted in canola species to assay pollen movement in real time (e.g. immediate detection of tagged pollen) using traps at various distances within field and greenhouse experiments This method is quicker and less laborious for determining pollen flow than analyzing progeny from recipient plants (e.g. antibiotic screening, PCR, FP screening) [8]. Drawing upon this previous body of work, it is logical to conceptualize a method to determine bioconfinement efficacy using FP tagging utilizing an improved fluorescent protein for plants
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