Expression of genes transferred into monocot and dicot plant cells by electroporation.

Abstract

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts. Protoplast Isolation. Protoplasts were isolated from rapidly growing carrot suspension cells by incubating 12 ml ofpacked cells in 80 ml of 1% cellulase (Cellulysin, CalbiochemBehring) and 0.5% hemicellulase (Rhozyme, Genencor) in protoplast isolation medium (PIM: 0.2 M mannitol/50 mM CaCl2/10 mM sodium acetate, pH 5.8) for 2 hr at 26°C.Protoplasts were separated from large debris by filtration through 60-gm-mesh nylon screen, centrifuged at 200 x g for 4 min, and washed once either in the same solution without enzymes or in Hepes-buffered saline (10 mM Hepes, pH 7.2/150 mM NaCl/5 mM CaCl2) containing 0.2 M mannitol.Electroporation. Each sample of 3 x 106 protoplasts was resuspended in 1 ml of the desired concentration of phosphate-buffered saline (1 x contains 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 1.15 g of Na2HPO4 per liter) or of Hepes-buffered saline, containing 0.2 M mannitol with or without plasmid DNA (10 ,g/ml). Electroporation then was carried out according to published procedures (16). The electrical pulse was supplied by an ISCO model 494 power supply set at 2000 V. The solution containing the protoplasts was held at 0°C for 10 min after the electric pulse. The protoplasts then were diluted into MS medium (29) containing 2% sucrose, 0.3 M mannitol, and 2,4-dichlorophenoxyacetic acid (0.1 jig/ml) and incubated at 26°C in a 10-cm plastic Petri dish. Protoplasts subjected to electroporation and then assayed in duplicate produced CAT activities that varied by Abbreviations: NOS, nopaline synthase; CAT, chloramphenicol acetyltransferase; CaMV, cauliflower mosaic virus; kb, kilobase(s). 5824The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Efficient Transfection of Insect Cells with Baculovirus DNA Using Electroporation

“…Our observation on the relationship between cell viabilities and optimum DNA uptake have shown that, as is the case for mammalian (Chu et al, 1987) and plant cells (Fromm et al, 1985), a certain percentage of cell death is required. In the case of the Spodoptera cells it was found that a cell survival rate of 40 to 609/00 was required for optimal extracellular virus production.…”

“…Several reports have shown that for optimum cell transfection by electroporation some cell death must occur and the optimal cell survival rates have been estimated at between 20 and 50 ~, depending on the cells being tested (Fromm et al, 1985 ;Chu et al, 1987;Rabussay et al, 1987). Initial experiments, therefore, measured cell viabilities over four different field strengths and pulse lengths varying from 0.2 to 37-1 ms.…”

“…However, it is widely appreciated that calcium phosphate-mediated transfection is a relatively inefficient method and that many cell lines remain refractile to transfection using the technique (Gorman, 1984). Zimmermann (1982) described an efficient electric shock or electroporation method for introducing drugs into mammalian red blood cells and, more recently, this technique has been extended to include the electroporation of many mammalian, plant and microbial cells with DNA and RNA (Fromm et al, 1985;Hashimoto et al, 1985;Chu et al, 1987;Rabussay et al, 1987). During electroporation, cell membranes are rapidly polarized at high voltages for a very short time leading to physical breakdown and pore formation.…”

Plantas transgênicas resistentes aos herbicidas: situação e perspectivas

“…A eletroporação consiste na aplicação de um pulso de alta voltagem a uma solução contendo DNA e protoplastos em suspensão, induzindo poros reversíveis nas membranas celulares, resultando em fluxos de íons e moléculas através da membrana deformada (CHANG, 1989). FROMM et al (1985) utilizaram a técnica de eletroporação em protoplastos de milho e fumo, oferecendo assim uma alternativa de transformação de plantas, já que o sistema Agrobacterium era considerado ineficiente em monocotiledôneas.…”

This article has been retracted

“…[1][2][3] Electroporation (EP) is a well-established laboratory technique owing to the transfer of plasmid DNA in vitro and is one of the most efficient non-viral methods for introducing exogenous molecules into cells by high-voltage electric pulses. [4][5][6] Several studies demonstrated that in vivo EP procedures could transfer DNA into skin, muscle, liver and tumor cells. [7][8][9][10][11][12] However, there is little information on the application of in vivo EP procedures to the urinary tract.…”