Summary
Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chen WH, Gartland KMA, Davey MR, Sotak R, Gartland JS, Mulligan BJ, Power JB, Cocking EC (1987) Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. Plant Cell Rep 6:297–301
Damm B, Willmitzer L (1988) Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes. Mol Gen Genet 213:15–20
Davey MR, Cocking EC, Freeman J, Pearce N, Tudor I (1980) Transformation of Petunia protoplasts by isolated Agrobacterium plasmids. Plant Sci Lett 18:307–313
Feldmann KA, Marks MD (1987) agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A nontissue culture approach. Mol Gen Genet 208:1–9
Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793
Guerche P, Charbonnier M, Jouanin L, Tourneur C, Paszkowski J, Pelletier G (1987) Direct gene transfer by electroporation in Brassica napus. Plant Sci 52:111–116
Hain R, Stabel P, Czernilosfki AP, Steinbiß HH, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Mol Gen Genet 199:161–168
Hauptmann RM, Vasil V, Ozias-Akins P, Tabaeizadeh Z, Rogers G, Frawley RT, Horsch RB, Vasil IK (1988) Evaluation of selectable markers for obtaining stable transformants in the Gramineae. Plant Physiol 86:602–606
Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Köhler F, Golz C, Eapen S, Kohn H, Schieder O (1987a) Stable transformation of moth bean Vigna acontifolia via direct gene transfer. Plant Cell Rep6:313–317
Köhler F, Golz C, Eapen S, Schieder O (1987b) Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean. Plant Sci 53:87–91
Krens FA, Molendijk L, Wullems GJ, Schilperoort RA (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74
Krüger-Lebus S, Potrykus I (1987) A simple and efficient method for direct gene transfer to Petunia hybrida without electroporation. Plant Mol Biol Rep 5:289–294
Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Frawley RT, Horsch RB (1986) Transformation of arabidopsis thaliana with Agrobacterium tumefaciens. Science 243:464–468
Lörz H, Baker B, Schell J (1985) Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199:178–182
Menczel L, Nagy F, Kiss Z, Maliga P (1981) Streptomycin-resistant and sensitive somatic hybrids of N. tabacum + N. knitiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195
Meyer P, Walgenbach E, Bussmann K, Hombrecher G, Saedler H (1985) Synchronized tobacco protoplasts are efficiently transformed by DNA. Mol Gen Genet 201:513–518
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8:363–373
Paszkowski J, Shillito RD, Saul M, Mandák V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3:2717–2722
Paszkowski J, Pisan B, Shillito RD, Hohn T, Hohn B, Potrykus I (1986) Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome. Plant Mol Biol 6:303–312
Potrykus I, Paszkowski J, Saul MW, Petruska J, Shillito RD (1985) Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer. Mol Gen Genet 199:169–177
Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988) Genetically transformed maize plants from protoplasts. Science 240:204–207
Schmidt R, Willmitzer L (1988) High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants. Plant Cell Rep 7:583–586
Schocher RJ, Shillito RD, Saul MW, Paszkowski J, Potrykus I (1986) Co-transformation of unlinked foreign genes into plants by direct gene transfer. Biotechnology 4:1093–1096
Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8:291–298
Shillito RD, Saul M, Paskowski J, Müller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Biotechnology 3:1099–1103
Uchimiya H, Fushimi T, Hashimoto H, Harada H, Syono K, Sugawara Y (1986a) Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.) Mol Gen Genet 204:204–207
Uchimiya H, Hirochika H, Hashimoto H, Hara A, Masuda T, Kasumimoto T, Harada H, Ikeda J-E, Yoshioka M (1986b) Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts. Mol Gen Genet 205:1–8
Valvekens D, Van Montague M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540
Waldron C, Malcolm SK, Murphy EB, Roberts JL (1985) A method for high-frequency DNA-mediated transformation of plant protoplasts. Plant Mol Biol Rep 3:169–173
Zhang HM, Yang H, Rech EL, Golds TJ, Davis AS, Mulligan BJ, Cocking EC, Davey MR (1988) Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep 7:379–384
Author information
Authors and Affiliations
Additional information
Communicated by H. Saedler
Rights and permissions
About this article
Cite this article
Damm, B., Schmidt, R. & Willmitzer, L. Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet 217, 6–12 (1989). https://doi.org/10.1007/BF00330935
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330935