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 shoot-bud regeneration was harnessed to establish a highly efficient transformation and regeneration procedure that does not require a pre-selection stage on antibiotics. Aspen stem segments were transformed using wild-type Agrobacterium rhizogenes (LBA9402) with the binary p35SGUSINT plasmid carrying the genes coding for β-glucuronidase (GUS) and neomycin phosphotransferase II. High levels of transient GUS expression 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 found in 75% of the roots. Shoot-bud regeneration in root cultures was very high: 99% of the roots yielded shoot-buds (4.3 buds per root), of which 91% expressed GUS. Southern blot analysis and polymerase chain reaction confirmed the transgenic nature of the plants expressing GUS. Kanamycin resistance of transformants was tested with respect to callus growth and bud regeneration. Callus from transgenic plants exhibited a high growth rate in the presence of up to 100 μg/μl kanamycin, and bud regeneration from transformed roots occurred in the presence of up to 30 μg/μl kanamycin. Callus and buds from control (non-transformed) plants failed to proliferate or regenerate, respectively, in the presence of kanamycin at concentrations above 10 μg/μl. Ninety-four independent clones from different transformation events were established, of which 52 were phenotypically true-to-type.