In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter
| Autor: | Lucien Ledou, Kouakou Tiecoura, N'nan-Alla Oulo, Abou Bakari Kouassi, Monique Dinant, Sery Gonedele Bi |
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| Rok vydání: | 2015 |
| Předmět: |
biology
fungi Mutant food and beverages GUS reporter system biology.organism_classification Applied Microbiology and Biotechnology Molecular biology Transformation (genetics) Plasmid Botany Genetics Arabidopsis thaliana Agronomy and Crop Science Molecular Biology Pennisetum Gene Pennisetum glaucum stable transformation chlorsulfuron (ALS) β-glucuronidase (GUS) CaMV35S and Emu promoters Biotechnology Southern blot |
| Zdroj: | African Journal of Biotechnology; Vol 14, No 46 (2015); 3112-3123 |
| ISSN: | 1684-5315 |
| Popis: | The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters. |
| Databáze: | OpenAIRE |
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