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


CONCLUSIONS
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.
Department of Plant Sciences, University of Tennessee, 37996, Knoxville, TN, USA. nealstewart@utk.edu.
J Hollis
JH
Rice
Reginald J
RJ
Millwood
Richard E
RE
Mundell
Orlando D
OD
Chambers
Laura L
LL
Abercrombie
H Maelor
HM
Davies
C Neal
CN
Stewart
D005298:Fertility; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006824:Hybridization, Genetic; D008164:Luminescent Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011058:Pollen / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins