Transformation of Nicotiana paniculata L., a recalcitrant species, using a T-DNA construct carrying two WUSCHEL-related homeobox genes.

A binary vector carrying two WUSCHEL-related homeobox (WOX) genes, WOX2 and WOX8, under the control of a chemical-inducible expression system, worked in the transformation in N. paniculata, a recalcitrant species of Nicotiana. The resulting transformants exhibited improved culture performance in regeneration from leaf segments and suspended cells. Multicellular masses generated from freely suspended cells showed a specific cell division pattern similar to that of somatic embryo, likely owing to the function of the two WOX genes.

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 ß-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.

Timing of the G1/S transition in tobacco pollen vegetative cells as a primary step towards androgenesis in vitro.

KEY MESSAGE: Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro. In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15-36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2'-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.

Shoot formation from root tip region: a developmental alteration by WUS in transgenic tobacco.

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.

Two promoters conferring active gene expression in vegetative nuclei of tobacco immature pollen undergoing embryogenic dedifferentiation.

In order to visualize the specific state of tobacco pollen undergoing dedifferentiation from immature pollen to embryogenic cells, we established tobacco marker lines transgenic for a vital reporter gene regulated under the transcriptional control of an 840 bp fragment, named A22pro. This fragment was obtained from the 5'-flanking region of a gene corresponding to a cDNA named A22 that was previously isolated through differential screening from a cDNA library prepared from tobacco pollen undergoing dedifferentiation. The reporter gene, named H3sGFP, consisting of synthetic green fluorescent protein gene (sGFP) and tobacco H3 histone gene for nuclear localization, was designed to distinguish the gene expression in the generative cell from that in the vegetative cell in a pollen grain. The marker line produced pollen showing a green fluorescent signal in the generative nuclei (GN) but the expression level of the transgene was low. Pollen after culture for dedifferentiation showed an intense signal transiently in the vegetative nuclei (VN), at a specific developmental stage of pollen, with a rapid increase of expression level of the transgene. Serial observations revealed that all androgenic embryos originated from the pollen grains that had shown the signal in their VN. Thus, A22pro is originally functional in gametogenesis but is activated in VN of pollen undergoing embryogenic dedifferentiation. Additionally, we observed a gene expression pattern identical to that described above, using another 5'-flanking region of a gene for a cDNA, named B27pro, homologous to A22 as a promoter of the reporter gene.