CKX Inhibitors: From Shoot Tip Micropropagation to Rooted Plantlet

Cytokinin homeostasis has an indispensable role in plant developmental processes and is regulated, in part, by cytokinin oxidase/dehydrogenases (CKXs), which irreversibly deactivate cytokinins. Recently developed CKX inhibitors offer a promising approach for modulating endogenous cytokinin activity and overcoming limitations in plant regeneration and micropropagation. In this study, we evaluated the effects of two diphenylurea-based CKX inhibitors, compounds 52 and 77, on the in vitro clonal propagation of two Populus alba clones, Villafranca and L-12. The compounds were applied at concentrations of 1 µM and 0.1 µM, either alone or in combination with 1 µM 2-isopentenyladenine (2iP) or 1 µM 6-benzylaminopurine (BAP). After 48 days, we analyzed multiplication parameters, biomass, pigment content, and biochemical parameters. For Villafranca, compound 52 at 0.1 µM yielded the highest multiplication rate (5.57), and media containing only inhibitors had a rooting percentage exceeding 80%. At 0.1 µM, the inhibitors tended to increase total chlorophyll content, but only in Villafranca. For L-12, multiplication rate was lower, showing no major differences across most formulations. L-12 exhibited a higher content of phenolic compounds and flavonoids, as well as greater antioxidant capacity, but exhibited a lower rooting rate compared to Villafranca. Multiplication and rooting rate on the same medium, in the absence of exogenous cytokinins, were achieved differently in L-12 and Villafranca clones, indicating a genotype-dependent response.