Submitted to: Plant Breeding and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2015
Publication Date: 9/1/2015
Citation: Cheong, E.J., Pooler, M.R. 2015. Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene. Plant Breeding and Biotechnology. 3:238-243.
Interpretive Summary: The flowering cherry (Prunus species) is a popular ornamental plant in many temperate parts of the world. The breeding program at the U.S. National Arboretum focuses on broadening the genetic base of ornamental Prunus and developing new cultivars with superior disease and pest resistance and tolerance to environmental stresses. Due to quarantine restrictions, it has become increasingly difficult to bring new Prunus germplasm into the U.S. for breeding; hence, genetic engineering may be one of the few options to introduce genes for resistance to microbial and insect pests into Prunus. In this study, we tested a visible marker, Green Fluorescent Protein (GFP), for its potential use in selecting transformed cells of Prunus incisa, an ornamental flowering cherry species. We found that unlike other reporter genes, cells containing GFP can be visualized non-destructively at an early stage of culture, which also allows for early selection of transgenic cells. By optimizing bacterial strain, regeneration system, and selectable marker, we were able to obtain confirmed transgenic plants of Prunus incisa.
Technical Abstract: An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were transformed with a NPTII/GFP fusion gene, and selected visually using fluorescence microscopy. Transformed cells were then induced to undergo embryogenesis and reselected by growing on media containing kanamycin. The presence of the GFP/NPTII fusion gene in all parts of transgenic plants grown in the greenhouse for one year was confirmed by PCR and Southern blot analysis. This transformation and selection system will be useful in future work to introduce genes for pathogen resistance and ornamental traits into flowering cherry germplasm.