title: An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons.
abstract: 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.
authors.affiliation: Pacific Agriculture Research Centre (Vancouver), 6660 N.W., Marine Drive, V6T 1X2, Vancouver, British Columbia, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Wieczorek
authors.forename: H
authors.initials: H
authors.lastname: Sanfaçon