Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) we re used as explants to conduct in vitro regeneration. Then, Agrobacterium t umefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The f requency of transformation varied among families infected with A. tumefacie ns. The highest frequency (100%) of transient fl-glucuronidase (GUS)-expres sing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hyp ocotyls, and radicles of co-cultivated mature zygotic embryos, as well as i n callus and shoots derived from co-cultivated mature zygotic embryos. Nine ty transgenic plants were regenerated from hygromycin-resistant callus deri ved from families WO3, 8-1082 and 11-1029, and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction, Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-m ediated transformation protocol for stable integration of foreign genes int o loblolly pine has been developed and that this transformation system coul d be useful for future studies on transferring economically important genes to loblolly pine.