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

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• Monitoring gene flow is crucial for transgenic crops, especially for plant-made-pharmaceuticals (PMPs) in open-field production.
• A Nicotiana hybrid (Nicotiana tabacum × Nicotiana glauca) offers a potential bioconfined PMP platform due to limited male fertility.
• Fluorescent proteins aid in identifying transgenic pollen for effective gene flow assessment.

Purpose of the Study:

• To develop a pollen tagging system using fluorescent proteins to monitor gene flow.
• To create a tagged Nicotiana hybrid for assessing the incidence of gene flow.
• To visually assess transgene zygosity in parent plants.

Main Methods:

• Transformation of Nicotiana tabacum and Nicotiana glauca with tdTomato-ER (orange fluorescent protein) for pollen and mgfp5-er (green fluorescent protein) for vegetative structures.
• Creation of Nicotiana hybrids utilizing fluorescent proteins for pollen movement and gene flow monitoring.
• Manual greenhouse crosses to assess hybrid sexual compatibility and seed formation.

Main Results:

• Successful transformation and expression of fluorescent proteins in parent lines and hybrids.
• Limited seed formation (2%) from hybrid pollination with N. tabacum, yielding non-viable seeds.
• Pollen transfer to the hybrid resulted in seed formation in 19% of crosses, with 10 out of 12 viable progeny showing GFP expression.

Conclusions:

• Orange fluorescent protein is detectable in pollen of parent lines and the Nicotiana hybrid.
• The hybrid exhibits limited outcrossing potential, suitable for bioconfinement with detectable pollen.
• Fluorescent protein tagging is a valuable tool for plant breeding and in vivo ecological monitoring.