Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans-and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino)purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino)purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl-cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3-to the 2-position in goillg from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino)purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino)purine], which was less than one-fifth as active as ciszeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3, 6-dihydro-1, 2-oxazin-2-yl)purine] was about /l4oo as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization.cis-zeatin2 (II, III) (9) both of which have been found as their 9-ribosyl derivatives in tRNA (13,18). Because of the variety of modification of terpenoid structures in nature and in order to extend our knowledge of the influence of side chain geometry on biological activity, we have determined the structure-activity relationship of trans-and cis-isozeatin (V, VI) and their 9-ribosyl derivatives (XIV, XV), together with dihydrozeatin (IV) and its 9-ribonucleoside (XIII), dihydroisozeatin (VII), cis-norzeatin (VIII), and cyclic-norzeatin (IX), all compared in the tobacco callus bioassay with the naturally occurring 6-(3-methyl-2-butenylamino)purine, 2iP (I), and its 9-ribonucleoside, 2iPA (X). MATERIALS AND METHODSBioassay Procedure. The tobacco bioassay (12) was used to determine the cytokinin activity. The medium contained the specified mineral salts (Table 6, part A, of ref. 12) and the following organic constituents: 30 g/liter of sucrose, 10 g/liter of Difco agar, 100 mg/liter of myoinositol, 2 mg/liter of indole-3-acetic acid, and 0.4 mg/liter of thiamine hydrochloride. The chemicals to be tested were dissolved in dimethylsulfoxide and added to the cooling autoclaved media at the uniform rate of 0.05% (v/v), (0.025 ml of (CH3)2SO solution to each flask with 50 ml of medium). The use of (CH3)2SO as specified does not affect growth of the t...

The use of dimethylsulfoxide as a solvent for synthetic purine and urea derivatives in the tobacco callus bioassay for cytokinin activity (10) has simplified the test procedure at two stages. First, the high solubility of these compounds in DMSO' has made it possible to test the less active materials, or those which are only slightly water-soluble, through a wide range of concentrations; secondly, full strength DMSO stock solutions act as sterilizing agents. Test compounds in DMSO solutions can be added directly to the cooling agar medium after autoclaving, thus avoiding destructive heating, and reducing the work, time, and equipment needed for the bioassay.In this laboratory DMSO was first employed as a solvent in the bioassay of diphenylurea. Bruce and Zwar (1) assay of an extensive series of urea derivatives which were added to the culture media as ethanolic solutions before autoclaving.Their preliminary tests indicated no alcohol toxicity (about 1 %c final strength) and no loss of activity as a result of the autoclaving process. However, in our experience there was a marked loss in activity when DPU was added before autoclaving, while its low solubility in water and also in alcohol precluded its assay in a full range of concentrations. DMSO effectively dissolved DPU, which could then be added to the medium in a small volume of solvent, to obtain concentrations in the medium as high as 20 jM for maximum growth, and 200 AzM for bud production.The highest concentration of DMSO which could be added to

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