GUS staining in mature embryos (stage 3) sellckchem was often located in the hypocotyl. Young needles also showed GUS staining. No chimeric tissue or escapes were observed (Figure 3).Figure 3GUS activity after 16h incubation in GUS solution. Embryos in different stages after 15 (stage 1 embryo; (a)), 45 (stage 2 embryo; (b)), and 90 days (stage 3 embryo; (c)) on maturation medium. Young needle (d). Bar and divisions = 1mm. …The GUS fluorometric assay revealed significant differences in activity levels (from 550.0 �� 22.1 to 17,831.2 �� 4,501.3pmolMUmin?1mg?1 of total protein) for the 10 transgenic lines during EM proliferation (Figure 2). No significant correlations were found between GUS activity levels and transgene copy number. Both the highest and the lowest expression levels were found in single-copy lines.

4. DiscussionSomatic embryogenesis in P. pinaster has been improved in recent years [1, 26, 45] providing a source of competent cells for genetic transformation. Genetic engineering can facilitate the introduction of economically important genes that may otherwise be difficult to integrate into elite genotypes [5], especially in forest tree species with long reproductive periods where conventional breeding can pose a long-term challenge. Furthermore, genetic transformation is a very attractive alternative for studying candidate gene function. To produce stably transformed plants, the desired DNA has to be introduced into plant cells and integrated into the cell genome. These transgenic cells must then be selected, multiplied, and finally regenerated into a plant.

Therefore, development of efficient gene delivery systems based on efficient in vitro plant regeneration protocols is a prerequisite for the application of genetic transformation in any species. In this work, we report obtaining transformed plantlets from P. pinaster EM based on kanamycin selection. Various factors influencing the efficiency of T-DNA delivery into maritime pine embryogenic cells via A. tumefaciens were evaluated. Cefotaxime, a decontamination agent used to inhibit Agrobacterium growth following infection [23], was successfully used to suppress the growth of the three Agrobacterium strains tested. Explants showed vigorous growth after 6 weeks of culture in the presence of cefotaxime (Online resource 3).

AGL1 strain was confirmed as the superior tested strain and was efficiently used, thereby broadening the range of Agrobacterium strains for maritime pine transformation. AGL1 is a disarmed derivative of C58, a Entinostat hypervirulent strain that has been successfully used to infect various plant species [46, 47]. However, this strain has been rarely employed in pine. Trontin et al. [5] reported very low transformation frequencies with AGL1 as compared with LBA4404 and C58pMP90 strains in a French genotype. This suggests a genotype-dependent compatibility.