Long-term gus expression from Gladiolus callus lines containing either a bar-uidA fusion gene or bar and uidA delivered on separate plasmids |
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Authors: | Kathryn Kamo and Hyang Young Joung |
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Institution: | (1) Floral and Nursery Plants Research Unit, 10300 Baltimore Avenue, Bldg. 010A Room 126 BARC West, USDA, National Arboretum, Beltsville, MD 20705-2350, USA;(2) National Horticultural Research Institute, Floriculture Research Division, Suwon, 441-440, Korea |
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Abstract: | Gus expression was determined for 19 lines of embryogenic Gladiolus callus that contained the 35S-bar-uidA-nos fusion gene and for 21 callus lines that had been cobombarded with the 35S-bar-nos and 35S-uidA-nos plasmid DNAs. These lines were selected for analysis because they grew vigorously on Murashige and Skoog’s medium supplemented
with 6 mg l−1 phosphinothricin. All 19 lines that contained the 35S-bar-uidA-nos fusion gene expressed gus compared to only 15 (71%) of the lines that had been cobombarded as determined by enzyme assay.
The level of gus expression was significantly higher the first year for 12 callus lines containing the bar-uidA fusion gene as compared to 2 years later in culture. Southern hybridization confirmed integration of the uidA gene in all callus lines that had been bombarded with the 35S-bar-uidA-nos fusion gene. Two of the callus lines that had been cobombarded lacked the uidA gene, and another cobombarded line that did not express gus contained a truncated uidA gene. Two callus lines resulting from cobombardment showed gus expression in only a few cells indicating that gus expression
was not completely silenced in these lines. Gus expression could not be reversed using 5-azacytidine in these two low-expressing
lines, and Southern hybridization supported that methylation of the genomic DNA had not occurred. Average levels of gus expression
were significantly higher, 8.9× , in cells with the 35S-bar-uidA-nos fusion gene compared to the cobombarded callus lines indicating the advantage of using a bar-uidA fusion gene for obtaining higher levels of gus expression in Gladiolus. |
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Keywords: | Bulb crops Floral monocot Reporter gene |
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