title: Transgenic rice cell lines and plants: expression of transferred chimeric genes.
abstract: Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake. PEG-mediated transformation was more efficient than electroporation. Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a beta-D-glucuronidase (GUS) gene under control of the 1', 2' double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts. Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact. Both genes were expressed in transgenic cell suspensions. GUS activity was detected by histochemical staining of the cells and by enzyme assays. During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again. Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine. Transcripts of the GUS gene could not be detected, in contrast with the HPT gene. Plantlets were regenerated from one transgenic cell line. GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles. A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A. tumefaciens were also introduced into rice protoplasts. Stable integration of both genes was confirmed by Southern analysis. Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts. Expression of the NOS gene at enzyme or RNA level was not detected. Southern analysis suggests that this gene was probably either methylated or scrambled in these lines.
authors.affiliation: Dept. of Plant Molecular Biology, Clusius Laboratory, Leiden University, Netherlands.
authors.forename: E G
authors.initials: EG
authors.lastname: Meijer
authors.forename: R A
authors.initials: RA
authors.lastname: Schilperoort
authors.forename: S
authors.initials: S
authors.lastname: Rueb
authors.forename: P E
authors.initials: PE
authors.lastname: van Os-Ruygrok
authors.forename: L A
authors.initials: LA
authors.lastname: Hensgens
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004558:Electric Stimulation; D005819:Genetic Markers; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012275:Oryza / Q000235:genetics*; D010770:Phosphotransferases / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D012313:RNA / Q000302:isolation & purification; D012038:Regeneration; D012231:Rhizobium / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D005819:Genetic Markers; D012313:RNA; D004247:DNA; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D013762:Tetrahydrofolate Dehydrogenase; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D005966:Glucuronidase