Developing a direct organogenesis protocol to obtain compact Ri petunias

Abstract Ri plants characteristically exhibit a compact architectural growth habit, enhancing the economic value of ornamental varieties. These plants are obtained through the natural transformation of plant cells using wild strains of Rhizobium rhizogenes , which means they are not classified as genetically modified organisms (GMOs) in some countries, as USA or Japan. In the standard transformation protocol, rol genes are transferred to plant cells, resulting in the formation of hairy roots from which complete Ri plants can be regenerated. However, there are plant species, such as petunias, in which the regeneration of complete plantlets from hairy roots is not possible. In our research, we have developed a natural transformation protocol that enables the production of Ri petunias through direct organogenesis from leaves, bypassing the hairy root stage. The presence of rolB and rolC genes in these petunias was confirmed by PCR. The phenotypic characterization, both in vitro (laboratory-based experiments) and ex vitro (outside artificial tissue culture), showed that all Ri petunias had a more compact growth, with lower height due to reduced elongation of the internodes. Moreover, two of nine Ri petunia lines exhibited enhanced branching, resulting in an increased number of internodes, leaves, and flowers. These phenotypic changes, especially the increase in the number of flowers, represent an additional increase in their commercial value. However, there were no observable changes in the specific morphology of leaves or flowers of Ri petunias. The transformation protocol developed in this work could be adapted to other species or genotypes in which shoot regeneration from hairy roots is not possible.