Auxin And Cytokinin Influerence In Vitro Rhizoid Formation In Rosa Canina

Organogenesis and somatic embryogenesis are the perquisites of plant regeneration. In vitro, a new protocol of regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration; the phytohormones auxin and cytokinin are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their function in the control of organogenesis in rose is as yet uncharacterized.In order to investigate the role of auxin during rhizoid initiation in Rosa canina, leaf explants were incubated on medium containing different concentration of 2,4-D and NAA, respectively. We found that rhizoids were induced by 0.1μg/mL 2,4-D and only callus could be distinguished at less than 0.1μg/mL, while threshold concentration of rhizoid initiation induced by NAA was 0.5μg/mL. These results suggested that a certain concentration of auxin is required for rhizoid induction.WUSCHEL (WUS)-related Homeobox (WOX) genes have been shown to play an important role in coordinating gene transcriptions, which are involved in both shoot and root meristem function and organogenesis. In order to test whether Rosa canina WOX genes are involved in auxin-induced rhizoid formation, we isolated and characterized a Rosa canina WUSCHEL-related homeobox gene, RcWOX1, which contains a typical homeodomain (HD). Real-time reverse transcription PCR analysis revealed that RcWOX1 was expressed in the whole process of callus formation and in the early stage of rhizoid formation. More importantly, its expression was induced by auxin treatment. By observing the Arabidopsis transformants expressing RcWOXlpro::GUS and 35S::GFP-RcWOXl, we found that this gene was specifically expressed in roots, and was localized in the nucleus. Overexpression of RcWOXl in Arabidopsis increased the density of lateral roots.We found that treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. To further characterize the role of cytokinin in rhizoid formation, we isolated the full-length cDNA of a type-A response regulator (RR) gene of cytokinin signaling, RcRR1, for which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation, and its expression level was rapidly up regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants, and overexpression of RcRR1 resulted in the increasing of primary root length and lateral root density. Moreover, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants.In summary, we suggest that RcWOXl is involved in auxin-triggered rhizoid organogenesis in Rosa canina and cytokinin regulates its output via regulating the distribution of auxin. Our data provide new insights into the mechanism of auxin and cytokinin infraction in organogenesis of rose.