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. verticillata plants by the actinomycetal strain of Frankia Allo2 was established.

Stem nodules of the legume Sesbania rostrata are ovoids, contain chlorophyll and have determinate growth. They possess a large central mass of infected cells. Stem mamillae are regularly arranged in vertical files along the stem and develop into nodules when they are infected by a specific Rhizobium. Each nodule arises from the development of an infected region of the incipient root cortex. The infection in S. rostrata has been shown to proceed in four sequential stages. Some of them have never been shown to occur in other legumes: (i) bacterial penetration takes place in degenerated (dead) cortical cells; (ii) proliferation of the bacteria occurs in the intercellular cavities and initiates a meristematic nodule; (iii) protusion of infection threads at first occurs intercellularly and then intracellularly from the cavities; (iv) finally there is an intracellular release of Rhizobia by an endocytotic process.