Agrobacterium rhizogenes-mediated DNA transfer in Pinus halepensis Mill

Agrobacterium rhizogenes strain LBA9402 was used to transform Pinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by β-glucuronidase (GUS) expression, with more than 85% showing considerable transient GUS expression in the radicle. GUS expression was also observed in cotyledons, but at a lower rate of about 24% of the embryos (1-5 spots/embryo). Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infected P. halepensis seedlings. Inoculum injections into intact seedling hypocotyls induced callus and root formation at the wound sites in 64% of the seedlings. Dipping seedling cuttings in a bacterial suspension resulted in adventitious root formation in 71% of the seedling cuttings, all of which expressed GUS activity. Adventitious shoots, that were induced on 2.5-year-old seedlings by pruning and spraying with 6-benzylaminopurine, were infected by injecting of bacterial suspension into their basal side. Two months later, adventitious roots and root primordia regenerated in 74% and 40% of 2- and 5-month-old shoots, respectively. Non-transformed shoots, either without or with auxin application, failed to form roots. Polymerase chain reaction and Southern blot analyses confirmed the uidA-transgenic nature of the root and callus, as well as the presence of rolC and rolB genes in roots from infected P. halepensis seedlings.