An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons |
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Authors: | Andrew Wieczorek Hélène Sanfaçon |
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Institution: | (1) Pacific Agriculture Research Centre (Vancouver), 6660 N.W., Marine Drive, V6T 1X2 Vancouver, British Columbia, Canada |
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Abstract: | Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed. |
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Keywords: | Cucumis Cucumber Protoplast Transient expression Tomato ringspot virus |
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