Regeneration and Agrobacterium-mediated transformation of Forsythia×intermedia "Spring Glory"

Abstract

Internode explants of in vitro plants of Forsythia×intermedia "Spring Glory" were transformed with the gus and npt II genes after inoculation with the A. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An averge of 1% of explants regenerated transgenic shoots.

β-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed β-glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.