HIGHLY EFFICIENT TRANSFORMATION AND REGENERATION OF ASPEN PLANTS THROUGH SHOOT-BUD FORMATION IN ROOT CULTURE

The natural capacity of aspen (Populus tremula L.) roots for direct sh oot-bud regeneration was harnessed to establish a highly efficient tra nsformation and regeneration procedure that does not require a pre-sel ection stage on antibiotics. Aspen stem segments were transformed usin g wildtype Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSI NT plasmid carrying the genes coding for beta-glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expressio n were found in the basal cut surface of 87% of the segments, and 98% of these formed well developed adventitious roots. Proliferating root cultures were established in liquid culture, and GUS expression was fo und in 75% of the roots. Shoot-bud regeneration in root cultures was v ery high. 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain r eaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callu s growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 mu g/mu l kanamycin, and bud regeneration from transformed roots occurred in the presence o f up to 30 mu g/mu l kanamycin. Callus and buds from control (non-tran sformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 mu g/mu l. Ninety -four independent clones from different transformation events were est ablished, of which 52 were phenotypically true-to-type.