Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

Abstract

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding.

Modulation of the anatomical and physiological responses of in vitro grown Alcantarea imperialis induced by NAA and residual effects of BAP

“…Satisfactory rates of induction of adventitious roots of bromeliads from in vitro shoots are usually promoted by AUX supplementation in the culture medium (Martins et al, 2018a). The ratio of endogenous AUX to CK concentrations plays as an important key for adventitious root formation (Bernula et al, 2020). This study demonstrates that alterations in hormone homeostasis induced by earlier exposure to BAP and then direct exposure to NAA can significantly change the rooting response.…”

Polyamine Metabolism Is Involved in the Direct Regeneration of Shoots from Arabidopsis Lateral Root Primordia

“…Based on the recognition that high auxin to cytokinin ratios promote root while high cytokinin to auxin ratios shoot formation, the establishment of in vitro plant regeneration systems were elaborated for hundreds of plant species. In the model plant, Arabidopsis thaliana , shoot regeneration is usually achieved via indirect organogenesis from root explants [ 2 , 3 , 4 ]. In recent years, it has been recognized that callus formation is not required for de novo shoot formation from root tissues and the direct conversion of lateral root primordia (LRP) to shoot meristem can take place in response to cytokinin application [ 5 , 6 , 7 ].…”

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

“…The gradient of endogenous auxin and polar auxin transport influenced by PINFORMED1(PIN1) were identified as the key factors for WUSCHEL (WUS)-induced somatic embryogenesis (Fehér 2019). In Arabidopsis, a short-term low concentration of cytokinin can promote somatic embryogenesis, but if the concentration of cytokinin is too high, it can inhibit polar auxin transport, thus inhibiting somatic embryogenesis (Bernula et al 2020). Similarly, in our study, when the concentration of BA in the medium was increased, callus differentiation of F. mandshurica was inhibited.…”

“…Similarly, in our study, when the concentration of BA in the medium was increased, callus differentiation of F. mandshurica was inhibited. Cytokinin inhibits the expression of LEC2 and FUSCA 3 (FUS3), the key transcription factors in somatic embryogenesis (Horstman et al 2017;Bernula et al 2020).…”