Ectopic expression of LONELY GUY7 in epidermis of internodal segments for de novo shoot regeneration without phytohormone treatment in ipecac

Abstract

In many plant species, the application of exogenous phytohormones is crucial for initiating de novo shoot regeneration. However, ipecac [Carapichea ipecacuanha (Brot) L. Andersson] has a unique ability to develop adventitious shoots on the epidermis of internodal segments without phytohormone treatment. This characteristic allows us to evaluate the effects of endogenous phytohormones in this species. Here, we showed that the presence of the pith, including vascular bundles in the internodal segment, is required to activate both endogenous cytokinin (CK) biosynthesis and adventitious shoot formation. Adventitious shoots were mainly formed in the apical region of internodal segments, where the CK biosynthesis genes ISOPENTENYL TRANSFERASE 3 (CiIPT3) and LONELY GUY 7 (CiLOG7) were spontaneously upregulated in the early culture stage on phytohormone-free medium. In addition, CiIPT3 and CiLOG7 were respectively expressed in the pith and the epidermis of the internodal segments. The expression of CiLOG7 was localized as several spots on the epidermis. These findings suggest that CK precursors are generated in the pith, transferred to the epidermis, and then converted into active CKs, facilitating adventitious shoot formation on the epidermis. Conversely, auxin levels rapidly decreased during culture and remained low in the region of shoot formation. Auxin is transferred to the basal region of internodal segments, and strongly suppressed the CiLOG7 expression and decreased the CK levels. Thus, we conclude that the ectopic expression of CiLOG7 in the epidermis of internodal segments contributes to de novo shoot regeneration in ipecac.