Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene, bar

Authors: Rajasekaran, Kanniah - Rajah; Majumdar, Raj; Sickler, Christine; Wei, Qijian - Mei Mei; Cary, Jeffrey; Bhatnagar, Deepak

Submitted to: Journal of Crop Improvement
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/4/2017
Publication Date: 6/12/2017
Citation: Rajasekaran, K., Majumdar, R., Sickler, C., Wei, Q., Cary, J.W., Bhatnagar, D. 2017. Fidelity of a simple Liberty leaf-painting assay to validate transgenic maize plants expressing the selectable marker gene, bar. Journal of Crop Improvement. 31(4):628-636. https://doi.org/10.1080/15427528.2017.1327913.
DOI: https://doi.org/10.1080/15427528.2017.1327913

Interpretive Summary: Screening of gene manipulation events (transgenic, mutation/genome editing etc.) is a cost/labor intensive and time-consuming process in plant science research. While Polymerase Chain Reaction (PCR) is the most definitive way for screening desired events, the process still requires efficient DNA extraction and subsequent steps for confirmation. In addition, PCR cannot predict how the expression cassette would perform at the integration site in the genome and would require quantifying target gene expression through real-time approach. To screen a larger number of transgenic plants, we have developed a Liberty herbicide leaf painting method in maize plants expressing the herbicide resistance gene, bar. Liberty leaf painting was > 95% accurate and reproducible in identifying transgenic events and corroborated well with the PCR results and gene expression analysis. Further investigation of bar gene expression in Liberty sensitive lines that were PCR positive for bar gene shows very little to no expression of the bar gene. The current method of identifying transgenic events through Liberty painting might also reflect the potential of transgene cassette expression as the selection marker is tightly linked to the expression cassette at the integration site in the genome and would expedite subsequent downstream research using selected lines. The results will be very useful to biotechnologists interested in studying gene expression transgenic maize plants.

Technical Abstract: Screening of gene manipulation events (transgenic, mutation/genome editing etc.) is a cost/labor intensive and time-consuming process in plant science research. While PCR is the most definitive way for screening desired events, the process still requires efficient DNA extraction and subsequent steps for confirmation. In addition, PCR cannot predict how the expression cassette would perform at the integration site in the genome and would require quantifying target gene expression through real-time approach. To screen a larger number of transgenic plants, it would be ideal to develop a quick screening procedure at the plant level. Here we have implemented a Liberty leaf painting method (against bar gene expressed under 4x35S promoter) in maize plants to screen transgenic events and validated the same lines through PCR and quantitative real-time PCR (qRT-PCR) to authenticate this simple and less expensive approach to screen a bigger population size. Liberty leaf painting at 500 mg L-1a.i. was > 95% accurate in identifying transgenic events that were in line with the PCR results. Further investigation of bar gene expression in Liberty sensitive lines that were PCR positive for bar gene shows very little to no expression of the bar gene. The current method of identifying transgenic events through Liberty painting might also reflect the potential of transgene cassette expression as the selection marker is tightly linked to the expression cassette at the integration site in the genome and would expedite subsequent downstream research using selected lines.