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

Abstract

To examine the effect of the ectopic expression of three <i>Arabidopsis</i> genes, including <i>WOX2</i>, <i>WOX8</i> and <i>WOX9</i>, on the regenerative competency of tissues and cells cultured in vitro, we developed a transgenic variety of <i>Nicotiana tabacum</i>, 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, <i>WOX2</i> combined with <i>WOX8</i> or <i>WOX9</i>, 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.