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ARS Home » Northeast Area » Kearneysville, West Virginia » Appalachian Fruit Research Laboratory » Innovative Fruit Production, Improvement, and Protection » Research » Publications at this Location » Publication #217673
Title: A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

Author
item Liu, Zongrang
item Hily, Jean Michel

Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2008
Publication Date: 12/18/2008
Citation: Liu, Z., Hily, J. 2008. A simple and sensitive high-throughput GFP screening in woody and herbaceous plants. Plant Cell Reports. 28:493-501.

Interpretive Summary: Green fluorescent protein (GFP) has been used widely as a visual technique to confirm the addition of genetic material in plants. The existing methods of measuring GFP in tissues or whole plants is inefficient in most species. Here, we report for the first time that a fluorescence scanner can be used as a sensitive means to detect GFP in plant. Three GFP detection methods were tested and compared in plants. Our results indicate that the Typhoon scanner is much more sensitive than two existing detection methods in detecting GFP in herbaceous and woody species. Our results also show that the Typhoon scanning can detect very strong GFP fluorescence in both green and non-green tissues of five-day-old seedlings or even imbibed seeds, qualifying it as a potential high throughput screening tool. Remarkably, Typhoon scanning can screen up to 11,000 seedlings grown in 16 Petri plates within an hour, making the screening process significantly more efficient than any other existing screening method.

Technical Abstract: Green fluorescent protein (GFP) has been used widely as a powerful bioilluminant reporter, but its visualization by existing methods in tissues or whole plants remains challenging in most species. Here, we report for the first time that a fluorescence scanner can be used as a sensitive means to detect GFP in herbaceous and woody species. Three GFP detection methods were tested and compared in Arabidopsis and apple plants expressing eGFP and erGFPINT, respectively. Our results indicate that fluorescence microscopy can detect GFP in Arabidopsis flowers and siliques, but not apple leaves, and UV illumination fails in both species. However, the Typhoon scanner is able to detect GFP in Arabidopsis flowers, siliques and leaves, as well as, apple leaves, demonstrating its superior sensitivity in herbaceous and woody species. Additionally, Typhoon scanning can detect very strong GFP fluorescence in both green and non-green tissues of five-day-old seedlings or even imbibed seeds qualifying it as a potential high throughput screening tool. This was further demonstrated by screening the seedlings of primarily transformed T0 seeds. Of the 25,000 germinating seedlings analyzed, at least 37 eGFP-positive lines were identified, accounting for a transformation efficiency of approximately 0.147%, which is comparable to that routinely achieved in our lab using the kanamycin resistant gene as a selection marker. Remarkably, Typhoon scanning can screen up to 11,000 seedlings grown in 16 Petri plates within an hour, making the screening process significantly more efficient than any other existing screening method.