Title : The presence of rol genes in nature and the applications of the rol-technology via non-GMO breeding to improve horticultural plant traits
Abstract:
Rhizobium rhizogenes is a gram-negative, soil-borne bacteria capable of infecting plants through horizontal gene transfer of a DNA fragment called T-DNA present on its root inducing (Ri) plasmid. Among other traits, the bacteria infection induces the formation of hairy roots. This is a result of the insertion of different genes in the pathogen’s T-DNA into the plant’s genome. The mostly known genes are the root oncogenic loci (rol-genes), namely rolA, B, C and D, and other open reading frames with less characterized functions. As a consequence, the Ri-phenotype can develop in host plants. Interestingly, sequences matching the T-DNA of R. rhizogenes have been found in different plant genera such as Nicotiana, Linaria and Ipomoea. This is a result of natural transformation involving R. rhizogenes over several million years.
The aim of this study was to find new plant species within different genera that have been naturally transformed. In particular, this method was utilized to investigate the presence of rolB in various plant species belonging to different genera of ornamental value. Plant material from these species, grown both in vivo and in vitro was examined by PCRs targeting rolB. Positive bands were cloned in E. Coli plasmid and later sequences were compared with R. rhizogenes rolB. In a secondary part of this study, Linaria repens was used in transformation with R. rhizogenes to generate Ri lines. This was obtained through the inoculation and infection of the plant material (i.e. leaves and stems) with the bacterium in order to obtain hairy roots as the main phenotypic response of the successful inoculation. Consequently, the hairy root tissue was put through a regeneration process of the plant through the use of different combination of auxin and cytokinin hormones.
Useful breeding traits through the use of this bacterium have been successfully obtained in other ornamental plant species with a high market value, such as Kalanchoe.The use of the rol-technology to obtain relevant traits such as compact phenotype is breakthrough in order to avoid the use of chemical growth retardants with malicious effects for health, production costs and environmental reasons. More recently drought tolerance could be a new relevant trait derived of the Ri phenotype based on the improved root growth also observed in Ri Kalanchoe lines. Moreover, the use of this technology is defined as non-GMO in e.g. the European Union as it uses unmodified bacterial strains, thus making it more accessible for breeders in the near future.
