Genetic transformation of the actinorhizal tree Allocasuarina verticillata by Agrobacterium tumefaciens

Abstract

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 β-glucuronidase containing an intron. The transformed embryos were cultivated on nutrient medium supplemented with 0.5 μM NAA, 2.5 μM BA, 100 mg l-1 kanamycin and 250 mg l-1 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 transgenes was demonstrated by PCR analysis and by the expression of the β-glucuronidase; integration of the T-DNA was confirmed by Southern hybridization. More than 100 transgenic plants from a total of 23 independent transformation events have been successfully established in soil. The possibility to obtain nitrogen-fixing nodules after inoculation of transgenic A. varticillata plants by the actinomycetal strain of Frankia Allo2 was established.