Abstract
Key message
Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression.
Abstract
Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.
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Acknowledgements
We sincerely thank numerous former Monsanto colleagues in Middleton, Wisconsin; Davis, California; Mystic, Connecticut and St. Louis, Missouri for transgenic plant production, greenhouse care and molecular analyses. Special thanks to Drs. David Somers and Doug Boyes for supporting this research and Drs. Jenn To, Miguel Vega-Sanchez, Bertho Lieselot and Julie Francois for critical review of the manuscript.
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This work was solely funded by legacy Monsanto Company, now Bayer Crop Science, in the form of salaries for all authors and the supplies for all experiments.
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The authors are employees of Bayer Crop Science, a manufacturer of seeds produced by conventional and biotechnology methods. A relevant US patent application has been submitted and assigned to Bayer Crop Science.
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Ye, X., Vaghchhipawala, Z., Williams, E.J. et al. Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants. Plant Cell Rep 42, 45–55 (2023). https://doi.org/10.1007/s00299-022-02935-1
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DOI: https://doi.org/10.1007/s00299-022-02935-1