Re-evaluation of phytohormone-independent division of tobacco protoplast-derived cells

We have used a [³H] thymidine incorporation assay and microscopic observation in order to reassess recently published data dealing with the response of tobacco protoplasts to phytohormones, lipochitooligosaccharides and peptides (Harling et al., 1997; Hayashi et al., 1992; Miklashevichs et al., 1996; Miklashevichs et al., 1997; Rohrig et al., 1995; Rohrig et al., 1996; van de Sande et al., 1996; Walden et al., 1994). These proliferation assays reveal that, in contrast to published data, isolated cells of the investigated mutant plant lines axi159 (Hayashi et al., 1992; Walden et al., 1994), axi4/1 (Harling et al., 1997) and cyil (Miklashevichs et al., 1997), which were generated by activation T-DNA tagging, were unable to grow in the absence of auxin or cytokinin. Furthermore, lipochitooligosaccharides which play a key role in the induction of nodules on roots of legumes were unable to promote auxin- or cytokinin-independent cell division in tobacco protoplasts as claimed by Rohrig et al. (1995, 1996). The finding of van de Sande et al. (1996) that ENOD40 confers tolerance of high auxin concentration to wild-type tobacco protoplasts was also reinvestigated. The results of our investigations show that we were unable to reproduce the proliferation data presented in this study, which were obtained by counting tobacco protoplast-derived cells undergoing division. In total, none of the published data on phytohormone-independent division of tobacco cells could be reproduced.