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 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 mm 2 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.

Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants

“…In a recent publication using the estradiol-inducible system to control WOX gene expression, two combinations of Arabidopsis-derived WOX genes ( WOX2 + WOX8 or WOX2 + WOX9 ) were evaluated in the presence of 1 μM 2,4-D for 10 days. Both combinations resulted in substantial plantlet regeneration from Nicotiana tabacum leaf pieces, in contrast to the wild-type control where no plantlet regeneration was observed [ 54 ].…”

Integrating the Roles for Cytokinin and Auxin in De Novo Shoot Organogenesis: From Hormone Uptake to Signaling Outputs

“…WOX2 plays also other roles in the regulation of shoot development, such as lateral organ formation and separation [113]. Similarly to its role in embryogenic development, WOX2 seems to play an important role in establishing the SAM during DNSO, as either WOX2/WOX8 or WOX2/WOX9 co-expression could result in successful shoot organogenesis from transgenic tobacco leaves on media containing auxin, without the need for exogenous CK [114]. The WOX-mediated promotion of CK signalization occurs on multiple levels, as the WOX2/PHB/PHV/REV network likely involves the upregulation of IPT1 during stem cell initiation in Arabidopsis [112].…”

Ectopic Expression of &lt;i&gt;&lt;/i&gt;WUSCHEL (AtWUS) Gene Alters Plant Growth and Development in Rice

“…Meanwhile, Mookan et al [42] also described that the combined expression of maize BBM and WUS2 genes using cre-lox technology increased the monocot transformation in recalcitrant sorghum and maize inbred lines and obtained fertile plants. Coexpression of WOX genes (WOX2 with WOX8/WOX9) also induced the genes involved in the regeneration of tobacco and increased the shoot like structures in tissue culture without the external supply of cytokinin [43].…”