DsRed reporter-gene based screening for evaluation of Rhizobium rhizogenes transformation efficiency

An optimization of co-cultivation experiments with wild type Rhizobium rhizogenes strains consists of selecting the most optimal explant type, bacterial strain and plant genotype. Since Ri technology utilizes the native root inducing (Ri) plasmid of R. rhizogenes, the resulting hairy roots and hairy root regenerated plants do not contain any selectable marker nor reporter genes and are thus considered as non-GMO. However, phenotypic distinction between spontaneous root formation and true hairy roots is often unreliable, which encumbers the process of hairy root selection and subculture. This, combined with the fact that emergence of hairy roots can occur anywhere from 4 to 10 weeks post-inoculation, can make the development of an efficient hairy root induction protocol a long and arduous process. Here, we report on the opportunities and potential shortcomings of implementing a reporter-gene based strategy by DsRed for optimizing hairy root induction and selection in eight different herbaceous ornamental genera (encoded to keep this information confidential). Our results showed that, overall, the DsRed fluorescence gave an underestimation of the actual transformation efficiency, resulting in method accuracy of 66.7-100%. Except for Osteospermum fruticosum (3 cultivars) and genus E for which the method accuracy was only between 16.7 and 28.6 or 45.5%, respectively. The co-transformation efficiency was at minimum 66.8%. DsRed genes were only present when pRi genes were also monitored.