Plant Cell, Tissue and Organ Culture 30: 69-75, 1992. t~) 1992 Kluwer Academic Publishers. Printed in the Netherlands. Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook G. Manders, A.V.P. dos Santos 1, F.B. d'Utra Vaz, M.R. Davey* & J.B. Power Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, Nottingham, NG7 2RD, UK; 1Universidade Federal de Alagoas, Rio Largo, 57100 Alagoas, Brazil (* requests for offprints) Accepted in revised form 21 January 1992 Key words': chloramphenicol acetyltransferase, electroporation, Eucalyptus citriodora, protoplasts, transient gene expression Abstract Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm -1 (1000 p.s), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 Ixg ml- 1 ), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation. Abbreviations: BAP-6-benzylaminopurine, CAT-chloramphenicol acetyltransferase, CPW 13M- CPW salts medium with 13% (w/v) mannitol, DC-direct current, FDA-fluorescein diacetate, f.wt.-fresh weight, GUS-/3-glucuronidase, K-Kao (1977), MES-2-N-morpholinoethane sulfonic acid, MS-Murashige & Skoog (1962), NAA-a-naphthaleneacetic acid, PVP-10-polyvinylpyrrolidone (Av MW 10,000), TLC- thin layer chromatography Introduction The use of chemical and/or electrical methods to introduce foreign genes into woody plant proto- plasts has been restricted to a few species. Such studies have mainly involved the evaluation of transient expression of the cat gene (Gorman et al. 1982) and/or gus gene (Jefferson 1987). CAT activity has been reported in white spruce (Bek- kaoui et al. 1988; Wilson et al. 1989), jack pine and black spruce (Tautorus et al. 1989) and larch (Charest & Klimaszewska 1990). The gus gene has been expressed in alder (S6guin & Lalonde 1988), larch (Charest & Klimaszewska 1990), and white spruce (Bekkaoui et al. 1988). More recently, Teuli~res et al. (1991) reported tran- sient expression of the gus and cat genes in protoplasts of Eucalyptus gunnii. Expression of the cat gene in Eucalyptus gunnii protoplasts was obtained following PEG-mediated DNA uptake. However, no significant CAT activity above en- dogenous levels was observed after electropora- tion with the same plasmid. Eucalyptus citriodora is valued in Brazil for its cellulose fibre content for paper pulp production, as a source of timber and essential oils. DNA transfer will provide important approaches for genetic improvement of the Eucalyptus species. Given the recent advances towards the regenera- tion of plants from E. citriodora cotyledon proto-