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

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.

098 High Regeneration Ability of Benzyladenine and Naphthalene Acetic Acid Induced Callus of Eastern Hibiscus

The ability of Hibiscus syriacus explants to produce regenerable callus was investigated. Fragments of cotyledons, hypocotyl, and roots were cultured on MS media supplemented with two different auxins (2,4-D and NAA, both 0.3 mg/L) and three different cytokinin (BA, 2iP and kinetin, all 0.1 mg/L). Plants were regenerated on McCown media with three different cytokinins at two different concentrations (0.1 and 1.0 mg/L). The biggest volume of callus was produced on medium containing 2,4-D/2iP (2,821 mm3/explant). The smallest mass of callus was induced on medium with NAA/kinetin (120 mm3/explant). On BA-supplemented media, the auxin type had no significant influence on the amount of callus produced. The highest number of shoots and leaves was produced on callus induced on NAA/BA supplemented media and regenerated on medium with 0.1 mg/L BA (4.2 shoots and 20 leaves/explant). Callus cultured on medium with 1.0 mg/L of BA produced significantly less shoots (2.4 shoots/explant). The lowest number of shoots was observed on callus originating from NAA/kinetin and NAA/2iP callus media and grown on medium with kinetin or 2ip (both 1.0 mg/L). The highest number of roots was produced by cultures originating from NAA/BA callus medium grown on the BA regeneration medium, irrespective of the cytokinin level. The longest shoots were observed on medium supplemented with 1.0 mg/L BA (18 mm). In this study, the best plant regeneration results were obtained for callus initiated on NAA/BA supplemented medium and regenerated on medium with 0.1 mg/L BA, however, the highest production of callus was observed on medium with 2,4-D/2iP.