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.

Petals and leaves share common evolutionary origins but have different phenotypic characteristics, such as the absence of stomata in the petals of most angiosperm species. Plant NAC transcription factor, NAP, is involved in ABA responses and regulates senescence-associated genes, and especially those that affect stomatal movement. However, the regulatory mechanisms and significance of NAP action in senescing astomatous petals is unclear. A major limiting factor is failure of flower opening and accelerated senescence. Our goal is to understand the finely regulatory mechanism of dehydration tolerance and aging in rose flowers. We functionally characterized RhNAP, an AtNAP-like transcription factor gene that is induced by dehydration and aging in astomatous rose petals. Cytokinins (CKs) are known to delay petal senescence and we found that a cytokinin oxidase/dehydrogenase gene 6 (RhCKX6) shares similar expression patterns with RhNAP. Silencing of RhNAP or RhCKX6 expression in rose petals by virus induced gene silencing markedly reduced petal dehydration tolerance and delayed petal senescence. Endogenous CK levels in RhNAP- or RhCKX6-silenced petals were significantly higher than those of the control. Moreover, RhCKX6 expression was reduced in RhNAP-silenced petals. This suggests that the expression of RhCKX6 is regulated by RhNAP. Yeast one-hybrid experiments and electrophoresis mobility shift assays showed that RhNAP binds to the RhCKX6 promoter in heterologous in vivo system and in vitro, respectively. Furthermore, the expression of putative signal transduction and downstream genes of ABA-signaling pathways were also reduced due to the repression of PP2C homolog genes by RhNAP in rose petals. Taken together, our study indicates that the RhNAP/RhCKX6 interaction represents a regulatory step enhancing dehydration tolerance in young rose petals and accelerating senescence in mature petals in a stomata-independent manner.