StableAgrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

Abstract

An efficient transformation protocol based on kanamycin selection was developed forAgrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which containedneomycin phosphotransferase II(nptII) as a selectable marker gene andβ-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−1kanamycin 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 withAgrobacteriumstrain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for thenptIIanduidAgenes and expression of theuidAgene. 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.