USE OF GREEN FLUORESCENT PROTEIN AS A NON-DESTRUCTIVE MARKER FOR PEANUT GENETIC TRANSFORMATION

• Published in: In vitro cellular & developmental biology. Plant 2005-07, Vol.41 (4), p.437-445
• Main Authors: JOSHI, MADHUMITA, NIU, CHEN, FLEMING, GERALDINE, HAZRA, SULEKHA, CHU, YE, NAIRN, C. JOSEPH, YANG, HONGYU, OZIAS-AKINS, PEGGY
• Format: Article
• Language: English
• Subjects: Arachis hypogaea; biolistics; Biotechnology/Genetic Transformation/Functional Genomics; Callus; Cell lines; chimerism; DNA; embryo culture; Embryos; field crops; Fluorescence; gene expression; genetic engineering; genetic transformation; green fluorescent protein; groundnut; microprojectile bombardment; Peanuts; Plant cells; Plants; Plasmids; promoter regions; regulatory sequences; reporter genes; transgenic; Transgenic plants
• ISSN: 1054-5476; 1475-2689
• DOI: 10.1079/IVP2005676

The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP.1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12 410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.