title: D-glufosinate as a male sterility agent for hybrid seed production.
abstract: A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F₁ hybrid seed production.
authors.affiliation: Syngenta, Jealott's Hill Research Centre, Bracknell, Berks, UK. tim.hawkes@syngenta.co
authors.forename: Tim
authors.initials: T
authors.lastname: Hawkes
authors.forename: Wendy
authors.initials: W
authors.lastname: Pline-Srnic
authors.forename: Richard
authors.initials: R
authors.lastname: Dale
authors.forename: Emma
authors.initials: E
authors.lastname: Friend
authors.forename: Tricia
authors.initials: T
authors.lastname: Hollinshead
authors.forename: Peter
authors.initials: P
authors.lastname: Howe
authors.forename: Paul
authors.initials: P
authors.lastname: Thompson
authors.forename: Russell
authors.initials: R
authors.lastname: Viner
authors.forename: Andy
authors.initials: A
authors.lastname: Greenland
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000613:Aminobutyrates / Q000737:chemistry / Q000494:pharmacology*; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D006540:Herbicides / Q000737:chemistry / Q000494:pharmacology; D006824:Hybridization, Genetic / Q000187:drug effects*; D016297:Mutagenesis, Site-Directed; D051479:Plant Infertility / Q000187:drug effects*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011058:Pollen / Q000187:drug effects / Q000254:growth & development; D012639:Seeds / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics
chemical_list: D000613:Aminobutyrates; D006540:Herbicides; D010940:Plant Proteins; C003121:phosphinothricin; D003605:D-Amino-Acid Oxidase