An efficient hairy root transformation system for protein interaction and gene editing analysis in roses

Roses (Rosa hybrida) are widely distributed ornamental crops with excellent economic and academic value. The Agrobacterium rhizogenes-mediated hairy root transformation system (HRTS) provides a powerful tool for investigating gene function in species with low stable genetic transformation efficiency. The genetic transformation of roses is well-known to be cultivar-dependent, unstable, and low efficiency. Here, we established a rose HRTS with two reporting markers of green fluorescent protein and RUBY genes. The rose HRTS method was optimized to shorten the protocol period of approximately 30 d in the system that maintains explants at 75% relative humidity and uses A. rhizogenes strain K599. The rose HRTS achieved a maximum transformation efficiency of 74.1%. Both stem and leaf explants were used to induce hairy roots in rose HRTS, which could be used for all tested diploid roses, namely R. chinensis ‘Old Blush’ and ‘Slater's Crimson China’ and tetraploid R. hybrida ‘Samantha’. Moreover, we confirmed that the rose HRTS could be effectively used to analyze protein interactions with a bimolecular fluorescence complementation assay and to evaluate sgRNA efficiency while conducting CRISPR/Cas9-mediated genome editing in rose. We established a rapid, effective, and multi-variety-adapted HRTS method with a high root regeneration and genetic transformation rate and explored the potential application for gene function analysis in roses.