In situ localization of endogenous cytokinins during shooty tumor development on Eucalyptus globulus Labill.

Our previous results demonstrated that endogenous cytokinins are involved i n the shooty potential of tumors initiated on Eucalyptus globulus plantlets inoculated with Agrobacterium tumefaciens strain 82.139 [A. Azmi et al. (1 997a) Plant Sci 127: 81-90]. In order to investigate whether or not these h ormones are distributed homogeneously in the tumors prior to the onset of b ud regeneration, decapitated hypocotyls were inoculated with the strain C58 pMP90/T139 GUS-INT harboring the wild transferred DNA (T-DNA) of strain 82. 139 tagged with the beta -glucuronidase (gus)-reporter gene. In situ immuno localization of zeatin, dihydrozeatin and isopentenyladenine was performed in the developing tumors and combined with the histo-enzymological beta -gl ucuronidase assay. It was found that the expression of the T-DNA was restri cted to only some small areas located deeply in the tumors. These sites wer e also provided with a high cytokinin signal while the untransformed parts of the turners displayed a weaker signal, except in the early differentiati ng tracheary elements. The regenerated buds were untransformed and originat ed from superficial parts of the tumors provided with a moderate signal for cytokinins. The method of colocalization of both cytokinins and gus expres sion developed here might be helpful for further studies concerning the rol e of these hormones in controlling gene expression at cell and tissue level s.