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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%. [ABSTRACT FROM AUTHOR]

Plants are susceptible to infection by a broad range of fungal pathogens. Many horticulturally important crop species lack adequate genetic resistance to disease. Studies on potential mechanisms of disease resistance in plants have revealed the importance of a range of pathogenesis-related (PR) proteins with antifungal activity in reducing colonization of plant tissues by pathogens. We are evaluating a range of PR-proteins, through heterologous expression in transgenic carrot tissues, for their effects on fungal disease development. The protocols for carrot transformation with a thaumatin-like protein are described. In addition, the use of herbicide resistance as a selectable marker in carrot transformation is illustrated. In this protocol, petiole segments from carrot seedlings are exposed to Agrobacterium for 10-30 min and co-cultivated for 3 d, after which herbicide selection is imposed until embryogenic calli are produced after 8-12 wk. The transfer of the embryogenic calli to hormone-free medium yields transgenic plantlets. This genetic transformation protocol has supported the generation of transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern positive independent events out of 100. [ABSTRACT FROM AUTHOR]