|Cheong, Eun Ju - VISITING SCIENTIST, FNPRU|
Submitted to: In Vitro Biology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2005
Publication Date: June 7, 2005
Citation: Cheong, E., Pooler, M.R. 2005. Use of the green fluorescent protein (GFP) gene in Prunus incisa 'February Pink' as a selectable marker. InVitro. 41:46-A. Technical Abstract: The flowering cherry (Prunus species) is a popular ornamental plant in the United States and other countries. The shrub breeding program at the U.S. National Arboretum involves development of new cultivars of ornamental Prunus using both traditional breeding methodologies and biotechnology. 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. We have developed an in-vitro regeneration system for one flowering cherry cultivar, P. incisa 'February Pink,' through somatic embryogenesis from in-vitro tissues, and transformed this tissue using the Green Fluorescent Protein (GFP) as a selectable marker. The GFP/NPTII fusion gene construct was introduced into somatic embryos derived from root or leaf tissue by Agrobacterium tumefaciens. Transformed cells expressing the GFP gene were isolated using a stereo microscope set up for GFP detection. Embryogenic callus and somatic embryos were obtained from GFP-expressing callus cultivated on MS medium containing 10 uM 2,4-D plus 50 mg/l kanamycin. These embryos were then germinated on MS medium containing 1 uM BA and 2 uM GA3. Transformed plantlets were propagated by shoot cultures. GFP expression in transgenic plantlets was difficult to detect due to the presence of chlorophyll, but callus induced from transgenic leaves expressed the green fluorescent protein. The presence of the GFP/NPTII fusion gene was also detected by PCR analysis using primers inside this region to amplify a 600bp PCR product. The use of the GFP gene as a selectable marker was successful. This system will be useful in future work to introduce genes for pathogen resistance and ornamental traits into flowering cherry germplasm.