Factors affecting transient gene expression in electroporatedGlycine maxprotoplasts

✍ Scribed by Sarwan K. Dhir; Seema Dhir; Angus Hepburn; Jack M. Widholm

• Publisher: Springer
• Year: 1991
• Tongue: English
• Weight: 932 KB
• Volume: 10
• Category: Article
• ISSN: 0721-7714
• DOI: 10.1007/bf00236468

✦ Synopsis

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 μF and a protoplast concentration of 1-3 × 10(6)/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.