AN IMPROVED METHOD FOR THE GENERATION AND TRANSFECTION OF PROTOPLASTSFROM CUCUMIS-SATIVUS COTYLEDONS

A protocol was developed for the preparation of Cucumis sativus var St raight 8 protoplasts that incorporates a two-step Ficoll(R) gradient a nd results in a high percentage of viable, debris-free protoplasts sui table for the transient expression of foreign genes. Polyethylene glyc ol and electroporation were compared for their effect on protoplast tr ansfection with commonly used reporter genes. Using a polyethylene gly col method, cucumber protoplasts transfected with a plasmid containing the beta-glucuronidase gene showed high expression levels, while prot oplasts transfected with a plasmid containing the chloramphenicol acet yl transferase gene showed levels of activity that were barely disting uishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of vi ral particles was confirmed.