Coexpression of WUSCHEL related homeobox (WOX) 2 with WOX8 or WOX9 promotes regeneration from leaf segments and free cells in Nicotiana tabacum L.

To examine the effect of the ectopic expression of three Arabidopsis genes, including WOX2, WOX8 and WOX9, on the regenerative competency of tissues and cells cultured in vitro, we developed a transgenic variety of Nicotiana tabacum, in which these genes were under the transcriptional control of a chemical-inducible expression system. We designed a two-step culture method to feasibly demonstrate the effect as follows. Leaf segments of approximately 10？mm2 were prepared from transgenic plants and their hybrids and cultured in a liquid medium based on modified Murashige and Skoog medium supplemented with an auxin, 2,4-dichrorophenoxyacetic acid and/or an expression inducer β-estradiol for 10 days in dark. The segments were subsequently cultured on a solidified medium in the absence of both the auxin and inducer in light for 3 weeks. We observed remarkable regeneration of plantlets only in segments derived from the hybrids possessing two transgenes, WOX2 combined with WOX8 or WOX9, but no regeneration in the segments derived from their parental lines. We also observed that free cells released from the hybrid explants in the liquid medium developed into embryo-like structures due to the transient application of the inducer. In a wide range of species including recalcitrants, the effect of the coexpression of these genes may be useful for developing an alternative to conventional protocols that requires cytokinin