title: Plastid transformation in lettuce (Lactuca sativa L.) by polyethylene glycol treatment of protoplasts.
abstract: A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1-2 spectinomycin-resistant cell lines per 10(6) viable protoplasts. Fertile, diploid, homoplasmic, plastid-transformed lines were obtained. Transmission of the plastid-encoded transgene to the T1 generation was demonstrated.
authors.affiliation: Rijk Zwaan Breeding B.V., Fijnaart, The Netherlands.
authors.forename: Cilia L C
authors.initials: CL
authors.lastname: Lelivelt
authors.forename: Kees M P
authors.initials: KM
authors.lastname: van Dun
authors.forename: C Bastiaan
authors.initials: CB
authors.lastname: de Snoo
authors.forename: Matthew S
authors.initials: MS
authors.lastname: McCabe
authors.forename: Bridget V
authors.initials: BV
authors.lastname: Hogg
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Nugent
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D002736:Chloroplasts / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D018545:Lactuca / Q000201:enzymology / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011523:Protoplasts / Q000166:cytology; D000198:Spectinomycin / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D014162:Transfection / Q000379:methods*; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D021901:DNA, Intergenic; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D000198:Spectinomycin; D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase