Joshi, M and et al, . (2005) Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation. In Vitro Cellular & Developmental Biology - Plant , 41 (4). pp. 437-445.
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Abstract
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 mm 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.
| Item Type: | Article |
|---|---|
| Additional Information: | This work was supported by the USDA Multicrop Aflatoxin Elimination Program, the Peanut Foundation, and USDA-CSREES. We thank Anne Bell, Evelyn Perry, and Jenny Kicklighter for technical assistance, Ben Mullinix for statistical analysis, and Tracey Woodward-Vellidis for assistance with graphics. Madhumita Joshi acknowledges C.S.I.R. India for a Ph.D. research fellowship. |
| Author Affiliation: | 1.University of Georgia Department of Horticulture Tifton Campus 31793 Tifton GA |
| Subjects: | Plant Production Crop Improvement |
| Divisions: | Groundnut |
| Depositing User: | Mr. SanatKumar Behera |
| Date Deposited: | 20 Feb 2012 13:21 |
| Last Modified: | 20 Feb 2012 13:21 |
| Official URL: | http://dx.doi.org/10.1079/IVP2005676 |
| URI: | http://eprints.icrisat.ac.in/id/eprint/3061 |
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