Abstract
Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.
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References
DeGreef W, Jacobs M:In vitro culture of the sugar beet — description of a cell line with high regeneration capacity. Plant Sci Lett 17: 55–61 (1979).
Frearson EM, Power JB, Cocking EC: The isolation, culture and regeneration ofPetunia leaf protoplasts. Dev Biol 33: 130–137 (1973).
Fromm ME, Taylor LP, Walbot V: Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82: 5824–5828 (1985).
Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2: 1044–1051 (1982).
Hauptmann RM, Ozias-Akins P, Vasil V, Tabaeizadeh Z, Rogers SG, Horsch RB, Vasil IK, Fraley RT: Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species. Plant Cell Rep 6: 265–270 (1987).
Herrera-Estrella L, Depicker A, vanMontagu M, Schell J: Expression of chimaeric genes into plant cells using a Tiplasmid-derived vector. Nature 303: 209–213 (1983).
Howard EA, Walker JC, Dennis ES, Peacock WJ: Regulated expression of an alcohol dehydrogenase 1 chimeric gene introduced into maize protoplasts. Planta 170: 535–540 (1987).
Lindsey K, Jones MGK: The permeability of electroporated cells and protoplasts of sugar beet. Planta (1987, in press).
Meyer P, Walgenbach E, Bussman K, Hombrecher G, Saedler H: Synchronised tobacco protoplasts are efficiently transformed by DNA. Mol Gen Genet 201: 513–518 (1985).
Morikawa H, Iida A, Matsui C, Ikegami M, Yamada Y: Gene transfer into plant cells by electroinjection through cell walls and membranes. Gene 41: 121–124 (1986).
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F: Hybrid genes in the analysis of transformation conditions. 1. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8: 363–373 (1987).
Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1: 841–845 (1982).
Okada K, Takebe I, Nagata T: Expression and integration of genes introduced into highly synchronised protoplasts. Mol Gen Genet 205: 398–403 (1986).
Ou-Lee T-M, Turgeon R, Wu R: Expression of a foreign gene linked to either a plant-virus or aDrosophila promoter, after electroporation of protoplasts of rice, wheat andSorghum. Proc Natl Acad Sci USA 83: 6815–6819 (1986).
Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I: High efficiency direct gene transfer to plants. Bio/Technology 3: 1099–1103 (1985).
Werr W, Lorz H: Transient gene expression in a Gramineae cell line: a rapid procedure for studying plant promoters. Mol Gen Genet 202: 471–485 (1986).
Widholm JM: The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47: 189–194 (1972).
Zimmermann U: Electrical breakdown, electropermeabilization and electrofusion. Rev Physiol Biochem Pharmacol 105: 175–256 (1986).
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Lindsey, K., Jones, M.G.K. Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol Biol 10, 43–52 (1987). https://doi.org/10.1007/BF00014185
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DOI: https://doi.org/10.1007/BF00014185