Plant Cell Reports (t988) 7:379-384 Plant Cell Reports © Springer-Verlag 1988 Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts H. M. Zhang, H. Yang, E. L. Rech, T. J. Golds, A. S. Davis, B. J. Mulligan, E. C. Cocking, and M. R. Davey Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham, NG7 2RD, UK Received August 10, 1988 - Communicated by I. Potrykus ABSTRACT Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering. ABBREVIATIONS NPTII, neomycin phosphotransferase; SDS, sodium dodecyl sulphate. INTRODUCTION A major constraint in the production of transgenic plants in most of the cereals is the inability to induce plant regeneration from protoplasts treated with DNA. To date, plant regeneration from transformed protoplast-derived tissues has been achieved only in Zea mays (Rhodes et al. 1988). The experimental approach used most extensively involves DNA uptake into protoplasts by treatment with polyethylene glycol or electroporation. This technique has been used to study transient gene expression (Fromm et al. 1985; Ou-Lee et al. 1986; Werr and L~rz 1986; Hauptmann et al. 1987; Junker et al., 1987) and has also resulted in the production of stably transformed tissues of gramineous species, including Lolium multiflorum (Potrykus et al. 1985b}, Oryza sativa (Uchimiya et al. 1986; Yang et al. 1988a,b), Panicum ma×imum (Hauptmann et al. 1988), Saccharum spp. (Chen et al. 1987), Triticum monococcum (LSrz et al. 1985; Hauptmann et al. 1988), and Zea mays (Fromm et al. 1986). Our studies have established a reproducible procedure for the regeneration of fertile plants from rice protoplasts (Oryza sativa L.v Taipei 309) with a regeneration efficiency adequate for assessments of transgenic plant production (Abdullah et al. 1986; 1988). Recently, we have reported that electroporation is the most efficient procedure for the production of kanamycin resistant Offpr~t requests to." M. R. Davey rice tissues following DNA uptake into rice (Taipei 309) protoplasts (Yang et al. 1988b). We have now combined these optimum conditions for transformation using electroporation with those for plant regeneration to produce transgenic rice plants resistant to kanamycin. MATERIALS AND METHODS Protoplast isolation Protoplasts were isolated using a routine procedure (Abdullah et al. 1986) from an established (more than 10 months old) cell suspension of Oryza sativa L. v Taipei 309. The suspension was initiated from leaf base callus and designated line LB3. Some preparations of freshly isolated protoplasts were heat shocked at 45°C for 5 min, followed by 10 sec on ice (Thompson et al. 1986b) prior to plasmid uptake by electroporation. Plasmid constructs E. coli HBIO] was transformed hy the calcium chloride procedure (Mandel and Higa 1970) with pCaMVNEO (Fromm et al. 1986) carrying a chimaeric gene consisting of the CaMV 35S promoter, the neomycin phosphotransferase (NpTII) gene from Tn5 and the nos polyadenylation region, pHP23 was provided in the same E. coli strain. This plasmid was constructed by fusing the 35S promoter of CaMV to the 5' end of the EcoRV fragment of pABDI. The EcoRV fragment carried the NPTII gene and the 19S promoter from gene VI of CaMV (Raszkowski et al. 1986). After construct verification (Holmes and Quigley 1981), large scale plasmid isolation from E. coli was performed using the alkaline lysis method (Birnboim and Doly 1979). Plasmid was sterilised by ethanol precipitation and dissolved in sterile TE buffer (Maniatis et al. 1982) at 1.0 mg/ml. Electroporation of protoplasts Protoplasts were resuspended at 2.5 x I06/mi in electroporation medium which was modified from a published formulation (Fromm et al. 1986) and contained 0.8 g/l NaCl, 0.02 g/l KCI, 0.02 g/l KH2PO 4 and 100 g/l glucose, pH 7.1. One ml volumes of protoplast suspension were mixed with 20 ~g of pCaMVNEO in 20 ~i of TE buffer. Four hundred ~i samples of protoplast/plasmid mixture were transferred to the chamber of an electroporator (DIA-LOG, G.m.b.H., 4 D~sseldorf 13, West Germany). The plexiglass chamber, which had two parallel