We examined the effect of ectopic expression of WUS on the morphology of tobacco seedlings and the segments in vitro. WUS was amplified from Arabidopsis cDNA and introduced into the tobacco genome under the transcriptional control of the beta-estradiol-inducible expression system. When 1-week-old transgenic seedlings were cultured in the presence of beta-estradiol, only the root tip region developed bulbous tissues followed by shoot formation and plant regeneration, suggesting its applicability for improving the strategy of micropropagation in recalcitrant species. Evident abnormality was not observed in the cotyledons, hypocotyl nor root except for the tip. However, ectopic WUS seemed to be functional in those parts through the observation of gene expression and the behavior of cultured segments. Small root segments with a root tip treated with beta-estradiol also showed bulbing but no shoots unless exogenous cytokinin was supplied. These findings suggest the existence of unknown factors regulating ectopic WUS function in the seedling.

A specific deleted version of ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) lacking the signal receiver domain (1-152 amino acids)-coding sequence, referred to as ARR1DDDK, was amplified using Arabidopsis thaliana cDNA prepared from adult leaves and transferred into the genome of Nicotiana tabacum cv. Samsun under the transcriptional control of a b-estradiolinducible expression system. The ectopic expression of ARR1DDDK affected the morphology of transgenic seedlings and their segments in vitro. In the presence of an inducer, b-estradiol, ectopic expression of ARR1DDDK induced only the formation of soft, pseudo-bulbous tissue in the root tip region of intact seedlings, which appeared similar to callus generated on a hypocotyl segment in the presence of 2,4-D and 6-benzyladenine (BA), both at 1 lM. Those callus tissues on the root tip region could not generate shoots unless 1 lM BA was supplied. In segment culture, ectopic expression of ARR1DDDK induced calluslike tissue around the cut-end of cotyledon and hypocotyl segments with occasional shoot formation, suggesting that the expression of ARR1DDDK could substitute for the effects of cytokinin on these segments. Additionally, treatment with only b-estradiol induced NtWUS, a WUS ortholog in tobacco, which was detected during the process of callus tissue formation in the root tip region and also in cotyledon or hypocotyl segments. These findings suggest that the NtWUS might be associated in the transdifferentiation process caused by the functional regulation of ARR1DDDK in transgenic tobacco seedlings.