pHairyRed: A Novel Binary Vector Containing the DsRed2 Reporter Gene for Visual Selection of Transgenic Hairy Roots

We developed a new plant transformation vector, pHairyRed, for enabling high throughput, non-destructive selection of Agrobacterium rhizogenes-mediated 'hairy-root' transformation. pHairyRed allows easy in planta visualization of transgenic tissue with minimal disturbance to the plant. The DsRed2 reporter gene, encoding a red fluorescent protein, was cloned to yield pHairyRed (harbouring a multiple cloning site), which was used with the highly efficient K599 A. rhizogenes strain to infect soybean (Glycine max L. Merrill) plants. DsRed2 fluorescence was easily detected in planta for the duration of a 5-week study with negligible levels of background autofluorescence. This enabled visual selection of transformed roots and subsequent excission of non-transformed roots. pHairyRed-transformed roots nodulated normally when inoculated with Bradyrhizobium japonicum. Within the nodule, DsRed2 fluorescence was plant-specific, being absent in the bacteroid-dominated nodule infected zone. To test the reliability of pHairyRed as a high-fidelity binary vector reporter system, the gene encoding the soybean Nod factor receptor, GmNFR1 alpha, was cloned into the vector for use in a complementation study with a non-nodulating nfr1 alpha mutant of soybean. Complementation was achieved and, without exception, DsRed2 fluorescence was detected in all hairy roots that successfully formed nodules (100%, n = 34). We anticipate broad application of this reporter system for the further analysis of root-related events in soybean and related legumes.