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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.

StableAgrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection