title: Modifying fatty acid profiles through a new cytokinin-based plastid transformation system.
abstract: The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3-ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over-expression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines.
authors.affiliation: Plastid AS, Professor Hansteens Gate 7A, 4036, Stavanger, Norway; Department of Biological Sciences, St John's University, New York, NY, 11439, USA.
authors.forename: Aisling
authors.initials: A
authors.lastname: Dunne
authors.forename: Jodi
authors.initials: J
authors.lastname: Maple-Grødem
authors.forename: Daniela
authors.initials: D
authors.lastname: Gargano
authors.forename: Richard P
authors.initials: RP
authors.lastname: Haslam
authors.forename: Johnathan A
authors.initials: JA
authors.lastname: Napier
authors.forename: Nam-Hai
authors.initials: NH
authors.lastname: Chua
authors.forename: Rosalind
authors.initials: R
authors.lastname: Russell
authors.forename: Simon Geir
authors.initials: SG
authors.lastname: Møller
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D003583:Cytokinins / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism; D000198:Spectinomycin / Q000494:pharmacology*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D003583:Cytokinins; D005227:Fatty Acids; D010940:Plant Proteins; D000198:Spectinomycin; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase
keywords: Nicotiana tabacum; cytokinin; fatty acids; plastid transformation; technical advance