Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation |
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Authors: | Madhumita?Joshi Chen?Niu Geraldine?Fleming Sulekha?Hazra Ye?Chu C?Joseph?Nairn Hongyu?Yang Email author" target="_blank">Peggy?Ozias-AkinsEmail author |
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Institution: | (1) Department of Horticulture, University of Georgia, Tifton Campus, 31793 Tifton, GA;(2) School of Forest Resources, University of Georgia, 30602 Athens, GA |
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Abstract: | Summary 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, 12410 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. |
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Keywords: | Arachis hypogaea genetic engineering groundnut microprojectile bombardment transgenic |
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