A simple and sensitive high-throughput GFP screening in woody and herbaceous plants.
Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T(0) seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.
Hily
Jean-Michel
JM
USDA-ARS Appalachian Fruit Research Station, Kearneysville, WV 25430, USA.
Liu
Zongrang
Z
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D027845:Malus / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D049452:Green Fluorescent Proteins