Abstract
Stable transformation of pearl millet ( Pennisetum glaucum) was achieved by microprojectile bombardment with two plasmids carrying the hygromycin phosphotransferase (hph) selectable marker and the β-glucuronidase (GUS) reporter gene. The calli were selected for their resistance to hygromycin. The presence of the hph gene in the transformants was confirmed by Southern hybridization and hygromycin phosphotransferase enzyme assay. The non-selected GUS marker was expressed in 50% of the resistant lines and was detected by DNA blot analysis in the GUS positive lines. Quantitative determination of transgene expression during long-term callus culture showed a stable expression of the hph gene contrasting with a gradual decrease of the GUS activity. This inactivation was related to a progressive methylation of the GUS gene, as demonstrated by DNA analysis with isoschizomeric enzymes responding differentially to methylation of restriction sites and by gene reactivation after treatment with 5-azacytidine.
Published Version
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