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

We have used a [H-3] thymidine incorporation assay and microscopic observat ion in order to reassess recently published data dealing with the response of tobacco protoplasts to phytohormones, lipochitooligosaccharides and pept ides (Harling at al., 1997; Hayashi et al., 1992; Miklashevichs et al., 199 6; Miklashevichs et al., 1997; Rohrig et al., 1995; Rohrig at al., 1996; va n de Sande et al., 1996; Walden et al., 1994). These proliferation assays r eveal that, in contrast to published data, isolated cells of the investigat ed mutant plant lines axi159 (Hayashi et al., 1992; Warden et al., 1994), a xi4/1 (Harling et al., 1997) and cyi1 (Miklashevichs et al., 1997), which w ere generated by activation T-DNA tagging, were unable to grow in the absen ce 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 protoplast s as claimed by Rohrig et al. (1995, 1996). The finding of van de Sande er al. (1996) that ENOD40 confers tolerance of high auxin concentration to wil d-type tobacco protoplasts was also reinvestigated, The results of our inve stigations show that we were unable to reproduce the proliferation data pre sented in this study, which were obtained by counting tobacco protoplast-de rived cells undergoing division. In total, none of the published data on ph ytohormone-independent division of tobacco cells could be reproduced.