An orange fluorescent protein tagging system for real-time pollen tracking

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.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field

“…Many researchers have utilized GFP-tagged pollen in transgenic plants to study gene flow [ 51 , 67 , 68 ]. In the present study, OFP was selected as the marker of choice since its expression can be visualized without sample destruction as has been shown with a different OFP variant in tobacco species [ 69 ]. The OFP-tagged pollen method is more rapid and convenient than real-time PCR and other such DNA-based screening methods in which DNA extraction is a prerequisite.…”

“…The blot was probed with 32 P-labeled pporRFP gene fragment. To select the best OFP fluorescent events, visual analysis of OFP fluorescent levels was performed by epifluorescent microscopy for roots and sheaths as described by Burris et al [ 48 ] and pollen (Additional file 2 ) epifluorescence microscopy [ 69 , 75 ].…”