Agrobacterium rhizogenes-mediated DNA transfer in Pinus halepensis Mill
1996
Tzfira, T. | Yarnitzky, O. | Vainstein, A. | Altman, A.
Agrobacterium rhizogenes strain LBA9402 was used to transform Pinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by beta-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.
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