Abstract
Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23 % ± 0.14 was obtained for all transformation experiments, with a range of 15.1–55.3 %.
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Acknowledgments
We thank S. Clemens for providing technical assistance with Southern hybridizations. This research was funded by the Natural Sciences and Engineering Research Council of Canada to ZKP and the John Yorston Scholarship in Pest Management to MF. In accordance with Health Canada regulations, all hemp cultures used in this research were subjected to regular analyses for THC content over the duration of the study to ensure they did not exceed the legal allowable limit of 0.3 % THC. The cultures were grown under permit No. 00-F0041-R-01 and disposed of according to the requirements.
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Feeney, M., Punja, Z.K. (2015). Hemp (Cannabis sativa L.). In: Wang, K. (eds) Agrobacterium Protocols. Methods in Molecular Biology, vol 1224. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1658-0_25
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DOI: https://doi.org/10.1007/978-1-4939-1658-0_25
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