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

BMC Research Notes

C Neal Stewart³  H Maelor Davies¹  Laura L Abercrombie³  Orlando D Chambers²  Richard E Mundell²  Reginald J Millwood³  J Hollis Rice³ 

[1] Department of Plant & Soil Sciences, University of Kentucky, 40546 Lexington, KY, USA;Kentucky Tobacco Research & Development Center, University of Kentucky, 40546 Lexington, KY, USA;Department of Plant Sciences, University of Tennessee, 37996, Knoxville, TN, USA

关键词: Orange fluorescent protein (OFP);    Green fluorescent protein (GFP);    Nicotiana;    Bioconfinement;    Plant made pharmaceuticals;    Male-sterility;    Gene flow;   

Others  :  1141539
DOI  :  10.1186/1756-0500-6-383

 received in 2013-04-30, accepted in 2013-09-24,  发布年份 2013

【 摘 要 】

Background

Monitoring 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.

Results

We 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.

Conclusion

The 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.

【 授权许可】

   
2013 Rice et al.; licensee BioMed Central Ltd.

【 图 表 】

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