Differential Cytokinin Structure - Activity Relationships in

The activities of eight cytokinins in promoting callus growth were tested In two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3position of the isoprenoid Nf side chain. In Kingston, trans-zeatin was 3-fold more active than dibydrozeatin and 30-fold more active than ciszeatin. The activities of N6-42-lsopentenyl)adenine and N6-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N6-4sopentyladenine was 100-fold more active than N6-42-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolibm. The structure-activity relationships of cytokinins have been investigated in detail by Skoog and Leonard and their associates using the tobacco callus bioassay system (2, 9, 10). The data for other cytokinin bioassay systems are much less extensive, but it is clear that the relative activities of cytokinins may vary in different bioassays, depending on both the type of biological response examined and the particular plant material selected for study. The causes of such differences in structure-activity relationships are not certain, although differences in cytokinin uptake, metabolism, and possible differences in the structural requirements for cytokinin activity at the site(s) of action in different plant materials may be involved. In an attempt to identify physiological traits of potential value in investigations of the genetic regulation of cytokinin metabolism and function, we are examining cytokinin structureactivity relationships in cytokinin-dependent tissue culture lines derived from a number of Phaseolus genotypes. We report here the results of tests of the activities of eight cytokinins in promoting the growth of callus tissue derived from P. vulgaris var. Great Northern and P. lunatus var. Kingston. MATERIALS AND METHODS Chemicals. Zeatin,2 N6-(A2-isopentenyl)adenine, N6-hexylad1 Research was supported by the College of Agriculture Oregon Agriculture Experiment Station and National Science Foundation Grant BMS 75-02588. Technical paper No. 4639 of the Oregon Agriculture Experiment Station. 2 Abbreviations: zeatin, trans-zeatin; t-(ioh4) 6Ade: 6-(4-hydroxy-3methyl-trans-2-butenylamino)purine; cis-zeatin, c-(ioh4)6Ade: 6-(4-hydroxy-3-methyl-cis-2-butenylamino)purine; dihydrozeatin, (ipnoh4)6Ade: enine, N6-benzyladenine, and kinetin were obtained from Sigma; cis-zeatin and dihydrozeatin were from Calbiochem. N6Isopentyladenine was kindly provided by F. Skoog. Picloram was a gift from Dow Chemical. Plant Materials. The genotypes P. vulgaris L. var. Great Northern and P. lunatus L. var. Kingston were used as experimental materials. Tissue cultures were established from the hypocotyl tissue of 5-day-old seedlings as described previously (5). Tissue Culture Medium. Medium for Phaseolus callus tissues consisted of mineral nutrients as described by Murashige and Skoog (6) with the following organic substances added: sucrose (30 g/1), myo-inositol (100 mg/1), thiamine*HCl (1 mg/1), nicotinic acid (5 mg/1), pyridoxine HCI (0.5 mg/1), and picloram (2.5 uM). Kinetin (5 aM) was included in the medium used for stock cultures. The pH of the medium was adjusted to 5.7 and Difco Bacto-agar (10 g/1) was added. The medium was dispensed into 125-ml Erlenmeyer flasks (50 ml/flask) and autoclaved at 120 C for 15 min. The choice of picloram as an auxin source was based on the broad range of concentrations of this compound that are effective in promoting callus growth of Phaseolus tissues (5). Structure-Activity Tests. For structure-activity tests, appropriate amounts of cytokinins were dissolved in dimethylsulfoxide (7) and added to tissue culture flasks containing autoclaved basal medium (0.025 ml of dimethylsulfoxide solution/flask) prior to solidification of the medium. Four replicate flasks were used for each cytokinin concentration tested. Three pieces of callus weighing close to 25 mg each were planted per flask. Tissues were harvested and weighed after 28 days of growth at 28 C in the dark. The effects of the cytokinins on callus growth were determined in the second passage of the callus. The tests were repeated using newly established cultures. An exception to this was the testing of N6-isopentyladenine, which was not repeated due to the limited quantity of the chemical available.