Plant Cell Reports (1995) 14:221-226 Plant Cell Reports 9 Springer-Verlag1995 A simple method for isolation, fiquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts Jaideep Mathur 1,2,3, Csaba Koncz 1,2, and Lfiszl6 Szabados 1 1 Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6726 Szeged, P.O. Box 521, Temesv~iri krt. 62, Hungary z Max-Planck Institut f/ir Ziichtungsforschung, D-50829 K61n, Carl-von-Linn6-Weg 10, Germany 3 Department of Botany, University of Gorakhpur, Gorakhpur 273 009, India Received 14 January 1994/Revised version received 13 April 1994 - Communicated by K. Hahlbrock Summary. An efficient technique was developed for the isolation, culture, transformation and regeneration of protoplasts derived from auxin conditioned Arabidopsis root cultures. On an average 30 % of root protoplasts underwent cell division in liquid culture and formed somatic embryo- like structures which regenerated to plants without embedding in Ca2+-alginate. The protoplast protocol was applicable to different landraces of Arabidopsis thaliana (L.) Heynh., such as RLD, Columbia or C24. PEG-mediated DNA uptake into protoplasts using different uidA reporter gene constructs yielded transient gene expression in over 25 % of treated cells indicating that root-derived protoplasts are suitable recipients for transformation. Key words: Arabidopsis thaliana - Root derived protoplasts - Somatic embryogenesis - DNA transformation - Transient uidA gene expression Abbreviations: BA, 6-benzylaminopurine; 2,4D, 2,4- dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3- butyric acid; IPAR, 6-(%'y-dimethylallylamino)purine riboside; NAA, ~- naphthaleneacetic acid; uidA, i3-gincuronidase gene; GUS, i3-glucuronidase enzyme; CaMV, Cauliflower Mosaic Vires; nos, nopaline synthase; MES, 2[N-morpholino]ethane-sulfonicacid; PEG, polyethylene glycol; X-gluc, 5- bromo-4-chloro-3-indolyl glucuronide; MUG, 4-methyl umbelliferyl glucuronide; MU, 4-methylumbelliferone. Introduction A lack of techniques allowing efficient manipulation of single plant cells hampers the application of powerful molecular approaches, such as the complementation of mutations by transformation with cDNA expression libraries, or the induction of gene mutations by homologous recombination, in Arabidopsis thaliana. Despite numerous reports on the isolation of protoplasts from cell suspensions (Axelos et al. 1992, Doelling and Pikaard 1993, Ford 1990, Correspondence to: C. Koncz Gleba et al. 1978, Xuan and Menczel 1980) and leaf mesophyll tissues (Damm and Willmitzer 1988, Karesch et at. 1991a, Masson and Paszkowski 1992, Park and Wernicke 1993), the application of protoplast techniques for regeneration and transformation of Arabidopsis often yields variable results. A practical bottleneck of available methods is that the induction of cell division is achieved by embedding the protoplasts into Ca2+-alginate gel matrix. Although immobilization of cells simplifies the plating (Altmann et at. 1992, Damm et at. 1989), the embedding hinders the removal of contaminating bacteria during co- cultivation with Agrobacterigm. Current techniques are also confronted with low regeneration capability and unstable ploidy of callus cultures (Negrutiu et at. 1975, Negrutiu and Jacobs 1978, Ford 1990) that is partly caused by a systemic endopolyploidy of Arabidopsis tissues (Galbraith et at. 19911). A protocol given below for isolation of protoplasts from proliferating root tissues offers an improved single cell system for PEG-mediated DNA transformation, assay of transient gene expression (Altmann et al. 1992, Axelos et at. 1992, Datum et at. 1989, Doelling and Pikaard 1993) and development of. Agrobacterium co-cultivation technology. Materials and methods Culture media and solutions. 0.5MS: MS medium (Murashige and Skoog 1962) containinghalf concentrationof macrosalts and microelements, and 3% sucrose. Protoplast medium: 0.5 MS medium containing 0.4 M glucose. Enzyme solution for protoplast isolation: protoplast medium containing cellulase "OnozukaR-10" (Serva), with or without macerozyme R-10 (Serva), as outlined in Table l. Ca-agar: 20 mM CaCI 2 and 0.4 M glucose solidified by 1.0% agar. Alginate solution: 1% sodium alginate (Sigma) and 0.4 M glucose (pH 5.8). Gelrite solution: 0.2% gelrite (Phytagel, Sigma) and 0.4M glucose (pH 5.8). Glucose-Magnesium solution: 5 mM MES (pH 5.8), 15 mM MgCI z and 0.4 M .glucose. PEG solution: 40% PEG 4000 dissolved in a solution of 0.1 M Ca(NO3) 2 (pH 5.8) and 0.4 M glucose (Negrutiu et al. 1975). MSAR1, callus inducing