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 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 polymerase chain reaction 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 to 55.3%.
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Clarke, R. C. (1999) Botany of the Genus Cannabis. In: Advances in Hemp Research (Ranalli, P., ed.), Haworth Press, Binghamton, NY, pp. 1–19.
MacKinnon, L., McDougall, G., Aziz, N., and Millam, S. (dy2001) Progress towards transformation of fibre hemp. In: Annual Report of the Scottish Crop Research Institute, 2000/2001, (Macfarlane Smith, W. H., and Heilbronn, T. D., eds.), SCRI, Invergowrie, Dundee, pp. 84–86.
Fisse, J., Braut, F., Cosson, L., and Paris, M. (1981) Étude in vitro des capacités organogénétiques de tissus de Cannabis sativa L.; effet de différentes substances de croissance. Pl. Méd. Phytoth. 15, 217–223.
Mandolino, G. and Ranalli, P. (1999) Advances in biotechnological approaches for hemp breeding and industry. In: Advances in Hemp Research (Ranalli, P., ed.), Haworth Press, Binghamton, NY, pp. 185–212.
Feeney, M. and Punja, Z. K. (2003) Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.). In Vitro Cell. Dev. Biol-Plant 39, 578–585.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.
Gamborg, O. L., Miller, R. A., and Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158.
Hood, E. E., Helmer, G. L., Fraley, R. T., and Chilton, M.-D. (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–1301.
Negrotto, D., Jolley, M., Beer, S., Wenck, A. R., and Hansen, G. (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep. 19, 798–803.
Joersbo, M. (2001) Advances in the selection of transgenic plants using non-antibiotic marker genes. Physiol. Plant. 111, 269–272.
Wright, M., Dawson, J., Dunder, E., et al. (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep. 20, 429–436.
Kramer, C., DiMaio, J., Carswell, G. K., and Shillito, R. D. (1993) Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red. Planta 190, 454–458.
Schluter, C. and Punja, Z. K. (2002) Genetic diversity among natural and cultivated field populations and seed lots of American ginseng (Panax quinquefolius L.) in Canada. Int. J. Plant Sci. 163, 427–439.
Koetsier, P. A., Schorr, J., and Doerfler, W. (1993) A rapid optimized protocol for downward alkaline Southern blotting of DNA. BioTechniques 15, 260–262.
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© 2006 Humana Press Inc., Totowa, NJ
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Feeney, M., Punja, Z.K. (2006). Hemp (Cannabis sativa L.). In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:373
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DOI: https://doi.org/10.1385/1-59745-131-2:373
Publisher Name: Humana Press
Print ISBN: 978-1-58829-843-0
Online ISBN: 978-1-59745-131-4
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