Cytokinin analysis by immunoaffinity chromatography (IAC), high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) or enzyme-linked immunosorption assay (ELISA) has been used to study two separate topics: the role of tRNA in bacterial cytokinin biosynthesis and the changes in cytokinin concentration which occur during cereal grain development. Transfer RNA isopentenylation in the gall-forming plant pathogen Agrobacterium tumefaciens is encoded by the chromosomal miaA locus. Mutation of miaA reduces tRNA isopentenylation significantly and preliminary data suggest that turnover of isopentenylated tRNA is responsible for low level secretion of free N6-isopentenyladenine (iP) by the bacteria. However, the major route of cytokinin biosynthesis by gall-forming plant patho- genic bacteria is not via tRNA turnover but by direct biosynthesis mediated by dimethylallylpyro- phosphate: 5'-AMP transferase (DMAPP :AMP transferase) encoded by such genes as ipt, tzs (from A, tumefaciens) or ptz (from Pseudomonas savastanoi). Analysis of cytokinin levels in developing wheat and rice grains in the period immediately following pollination showed large transient increases in zeatin (Z) and zeatin riboside (ZR) which coincided with the period of maximum endosperm cell division reported by others. Detailed analyses of maize kernels, where development can be staged readily, showed that Z and ZR concentrations peaked 9 days after pollination (DAP). During the period 8-10 DAP, cytokinin oxidase underwent a significant increase in specific activity, indicating that cytokinin catabolism was enhanced as endosperm cell division ended.

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Qualitative and quantitative analyses were made of cytokinins secreted into liquid culture by virulent and avirulent strains of Rhodococcus fascians, and of the endogenous cytokinins in pea plants inoculated with the same strains. Both virulent and avirulent strains secreted a range of cytokinins into liquid culture. The nonhydroxylated cytokinins were present in the liquid cultures at levels up to 1000-fold higher than hydroxylated cytokinins, but the virulent strains secreted only slightly higher levels of isopentenyladenine (iP), isopentenyladenosine (iPA) and the tentatively identified methylthio-isopentenyladenosine (ms-iPA), than the avirulent strains. Pisum sativum cv. Novella plants that were inoculated with virulent strains contained only slightly higher levels of iP and ms-iPA but had lower levels of the cytokinin nucleotides than those plants that had been inoculated with the avirulent strains. The origin of the cytokinins secreted by avirulent and virulent strains is discussed, as is the significance of the cytokinin content of fasciated plants.

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