Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens

We have developed an efficient transformation system for the tropical actinorhizal tree #Allocasuarina verticillata$ using #Agrobacterium tumefaciens$ mediated gene transfer. Mature zygotic embryos were inoculated with the disarmed strain C58C1 carrying, in the binary vector BIN19, the nptll gene, providing kanamycin resistance as a selectable marker, and the reporter gene beta-glucuronidase containing an intron. The transformed embryos were cultivated on nutrient medium supplemented with 0,5 microM NAA, 2,5 microM BA, 100 mg/l kanamycin and 250 mg/l cefotaxime. After 2 months, a 21% transformation frequency was obtained. Within 6-9 months, transgenic plants were recovered from 70% of the transformed calli. The presence of the trangenes was demonstrated by PCR analysis and by the expression of the beta-glucuronidase; integration of the T-DNA was confirmed by Southern hybridization. More than 100 transgenic plants from a total of 23 independent events have been successfully established in soil. The possibility to obtain nitrogen-fixing nodules after inoculation of transgenic #A. verticillata$ plants by the actinomycetal strain of Frankia Allo2 was established. (Résumé d'auteur)