A ��FW3 < 2 was considered as growth inhibition.An the following site evaluation of maturation was determined by counting the number of mature somatic embryos per gram fresh tissue after 3 months on maturation medium. A mature somatic embryo was a white to yellowish embryo on which cotyledons are visible. This corresponds to stage 3 of somatic embryo development in P. pinaster as defined by Ramarosandratana et al. [44]. Data are presented as mean �� standard error of three independent replicates per experiment. Each experiment was performed at least twice at different times and assayed using a completely randomized design. The statistical analysis of categorical variables was carried out with the ��2 test for overall and pairwise comparisons, except where indicated.
Quantitative data were analyzed by analysis of variance (ANOVA) using the Holm-Sidak test for post hoc comparisons. Differences were considered significant at the 5 percent level. All statistical tests were performed with SigmaPlot v11.0 (Systat Software, Inc., San Jose, CA, USA).3. Results3.1. Embryogenic Line Sensitivity to the Selective AgentsTo determine the sensitivity of embryogenic cultures to the selective agents, untransformed lines (L01, L05, L13, L15, and L26) were cultured on maintenance medium supplemented with 500mgL?1 cefotaxime plus the selective agent at different concentrations. The selective agents tested inhibited the growth of the EM. Kanamycin and hygromycin showed a similar behaviour since a concentration of 15mgL?1 was enough to inhibit growth in all lines tested (Figure 1), and after three subcultures, the tissue necrotized (Online Resource 3).
Figure 1EM sensitivity to the selective agents. Kan: kanamycin, Hyg: hygromycin. Data were collected after 3 subcultures (6 weeks) and the values for the 5 embryogenic lines were averaged.3.2. Agrobacterium Strains and Transformation ProtocolThe susceptibility of maritime pine embryogenic cultures to infection with three different A. tumefaciens strains (EHA105, LBA4404, and AGL1) harboring the pBINUbiGUSint plasmid was also studied. Cefotaxime was successfully used to suppress the growth of the three Agrobacterium strains tested. No transformation events were obtained for lines L13, L15, and L26. Line L05 showed transformation events only with the AGL1 strain at OD600nm 0.3 and a 48h cocultivation (6.3 �� 0.7 transformation events per gram of fresh mass).
Line L01 showed transformation events with AGL1 and EHA105 strains, but none were obtained with the LBA4404 strain. After 120 days of culture, the number of transformation events per gram of fresh mass varied significantly, depending on the strain used (Table Drug_discovery 1). Table 1Number of transformation events per gram of fresh mass in line L01 after 120 days on selective medium. LBA4404 strain did not show transformation events.