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 β-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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Communicated by B.E. Ellis
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Wieczorek, A., Sanfaçon, H. An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons. Plant Cell Reports 14, 603–610 (1995). https://doi.org/10.1007/BF00231947
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DOI: https://doi.org/10.1007/BF00231947