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.