Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2009
Publication Date: 10/15/2009
Citation: Kamo, K. and Joung, H.Y. 2009. Long-term gus expression from Gladiolus callus lines containing either a bar-uidA fusion gene or bar and uidA delivered on separate plasmids. Plant Cell Tissue Organ Culture. 98:263-272. Interpretive Summary: We are interested in genetic engineering of Gladiolus for virus and fungus resistance. A foreign gene that is put into a plant is frequently silenced rather than expressed in the plant. Silencing of a foreign gene is undesirable when trying to get expression of that gene. In this manuscript the expression of a foreign reporter gene (GUS) was examined in lines of Gladiolus that had been engineered three years ago. Typically silencing of a foreign gene occurs with time in culture. Surprisingly all 7 callus lines that contain the reporter gene fused to a selectable marker gene show gus expression. In comparison only 4 of the 7 callus lines that contain the reporter gene alone show high levels of gus expression, and 2 of the lines show a few cells that continue to express gus. One of the lines did not express gus, and it was shown that this line had lost the reporter gene. It was concluded that expression of a foreign gene continues in Gladiolus, more so than in many other plants.
Technical Abstract: Gus expression was determined for 7 lines of embryogenic Gladiolus callus that contained the 35S-bar-uidA-nos fusion gene and for 7 callus lines that had been cobombarded with the 35S-bar-nos and 35S-uidA-nos plasmid DNAs. All 14 callus lines were selected for further analysis because initially they showed strong gus expression as determined by histochemical staining, and they grew well on selection medium supplemented with 6 mg l-1 phosphinothricin. All callus lines, contained only 1-2 copies of the 35S-bar-uidA-nos fusion gene or 35S-bar-nos contruct, except for one callus line with 3 copies and one with 4 copies. Three years later all 7 callus lines that contained the 35S-bar-uidA-nos fusion gene continued to express gus whereas only 4 of the 7 callus lines that had been cobombarded expressed gus as determined by enzyme assay. One of the lines that no longer expressed gus lacked the 35S-uidA-nos gene although the 35S-bar-nos gene was present. Two callus lines showed gus expression by only a few cells indicating that gus expression was not completely silenced in these lines. Gus expression could not be reversed using 5-azacytidine with these two low level gus expressors, and Southern blot hybridization supported that methylation of the genomic DNA had not occurred. Average levels of gus expression were 5.6X higher in cells with the 35S-bar-uidA-nos fusion gene as compared to the cobombarded callus lines indicating the benefits of using a bar-uidA fusion gene for both higher levels and sustained gus expression in Gladiolus.