title: Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.
abstract: An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.
authors.affiliation: Laboratorio de Biotecnología Agroforestal, Escuela Politécnica de Mieres, Universidad de Oviedo, Calle Gonzalo Gutiérrez Quirós, 33600 Mieres, Spain ; Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7080, 75007 Uppsala, Sweden.
authors.forename: José M
authors.initials: JM
authors.lastname: Alvarez
authors.affiliation: Laboratorio de Biotecnología Agroforestal, Escuela Politécnica de Mieres, Universidad de Oviedo, Calle Gonzalo Gutiérrez Quirós, 33600 Mieres, Spain ; Área de Fisiología Vegetal, Departamento BOS, Universidad de Oviedo, Calle Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain.
authors.forename: Ricardo J
authors.initials: RJ
authors.lastname: Ordás
mesh_terms: D060054:Agrobacterium / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods; D007612:Kanamycin / Q000378:metabolism*; D028223:Pinus / Q000235:genetics* / Q000382:microbiology*; D030821:Plants, Genetically Modified / Q000382:microbiology* / Q000502:physiology*; D014169:Transformation, Bacterial / Q000235:genetics*
chemical_list: D007612:Kanamycin