Factors influencing transient gene expression in electroporated tall fescue (Festuca arundinacea Schreb.) protoplasts
For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase (GUS) gene was introduced by electroporation. In a time-course study of transie...
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| Format: | Others |
| Language: | en |
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Virginia Tech
2014
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| Online Access: | http://hdl.handle.net/10919/44549 http://scholar.lib.vt.edu/theses/available/etd-09052009-040406/ |
| Summary: | For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase (GUS) gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 x 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3 and 5 day old suspensions. These results show that transient expression studies can be used to optimize electroporation parameters rapidly. Results of such rapid assays can be used as a basis for further studies on stable transformation of this important turf-grass species. === Master of Science |
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