Abstract
Eleven cytokinins-including bases, ribosides, glucosides, and ribotides-were tested for their retention on C18 cartridges that were washed with 40 mL of water or a dilute acid at pH 3. Cytokinins were then eluted with methanol and analyzed by high performance liquid chromatography (HPLC). All pure cytokinin were well retained when the cartridge was washed with water, but Z and (diH)Z were less well retained at pH 3. The ribotides required 80% methanol for elution. Cotton leaf tissue (500 mg dry wt) was spiked with cytokinins, extracted with 80% methanol, and the extract bulk purified with hexane, insoluble polyvinylpyrrolidone, and minicolumns (strong anion exchange, amino, and C18 cartridges). Ribotides, added to leaf tissue, could not be recovered as ribotides; it was necessary to hydrolyze and purify them as ribosides. The cytokinins were separated and analyzed by HPLC on strong cation exchange and C18 columns. Recoveries through the entire procedure averaged 70%.
Similar content being viewed by others
Abbreviations
- (diH)Z:
-
Dihydrozeatin
- (diH)Z:
-
dihydrozeatin riboside
- (diH)[9R]Z:
-
trans-zeatin
- Z:
-
t-zeatin riboside
- [9R]Z:
-
t-zeatin-O-glucoside
- (OG)Z:
-
t-zeatin riboside-O-glucoside
- (OG)[9R]Z:
-
t-zeatin riboside-5′-monophosphate
- [9R-5′P]Z:
-
N6(Δ2-isopentenyl)adenine
- iP:
-
N6(Δ2-isopentenyl)adenosine
- [9R]iP:
-
N6(Δ2-isopentenyl)adenosine-5‵-monophosphate-[9R-5′P]iP
References
Akiyoshi DE, Morris RO, Hinz R, Mischke BS, Kosuge T, Garfinkel DJ, Gordon MP and Nester EW (1983) Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci 80: 407–411.
Andersen RA and Sowers JA (1968) Optimum conditions for bonding of plant phenols to insoluble polyvinylpyrrolidone. Phytochemistry 7: 293–301.
Biddington NL and Thomas TH (1976) Effect of pH on the elution of cytokinins from polyvinylpyrrolidone columns. J Chromatogr 121: 107–109.
Capelle SC, Mok DWS, Kirchner SC and Mok MC (1983) Effects of Thidiazuron on cytokinin autonomy and the metabolism of 313–4 in callus tissues of Phaseolus lunatus L. Plant Physiol 73: 796–802.
Davies PJ, Horgan R, Heald JK and McGaw BA (1986) Endogenous cytokinins in vegetative shoots of peas. Plant Growth Reg 4: 311–323.
Doumas P, Bonnet-Masimbert M and Zaerr JB (1989) Evidence of cytokinin bases, ribosides and glucosides in roots of Douglas-fir, Pseudotsuga menziesii. Tree Physiol 5: 63–72.
Dunlap JR and Guinn G (1989) A simple purification of indole-3-acetic acid for GC-SIM-MS analysis by microfiltration of aqueous samples through nylon. Plant Physiol 90: 197–201.
Guinn G (1973) Purification of leaf nucleotides and nucleosides on insoluble polyvinylpyrrolidone. Anal Biochem 54: 276–282.
Guinn G and Hendrix DL (1985) Bracketing, a simple loading technique that increases sample recovery, improves resolution, and increases sensitivity in high-performance liquid chromatography. J Chromatogr 348: 123–129.
Holland JA, McKerrell EH, Fuell KJ and Burrows WJ (1978) Separation of cytokinins by reversed-phase high-performance liquid chromatography. J Chromatogr 166: 545–553.
Lejeune P, Kinet J-M and Bernier G (1988) Cytokinin fluxes during floral induction in the long day plant Sinapis alba L. Plant Physiol 86: 1095–1098.
Letham DS and Singh S (1989) Quantification of cytokinin O-glucosides by negative ion mass spectrometry. Plant Physiol 89: 74–77.
Loomis WD and Battaile J (1966) Plant phenolic compounds and the isolation of plant enzymes. Phytochemistry 5: 423–438.
MacDonald EMS and Morris RO (1985) Isolation of cytokinins by immunoaffinity chromatography and analysis by high-performance liquid chromatography radioimmunoassay. In: Methods in Enzymology, vol 110, pp 347–358. New York: Springer-Verlag.
MacDonald DMS, Akiyoshi DE and Morris RO (1981) Combined high-performance liquid chromatography-radioimmunoassay for cytokinins. J Chromatogr 214: 101–109.
Morris RO, Zaerr JB and Chapman RW (1976) Trace enrichment of cytokinins from Douglas-fir xylem extrudate. Planta 131: 271–274.
Muller P and Hilgenberg W (1986) Isomers of zeatin and zeatin riboside in clubroot tissue: evidence for trans-zeatin biosynthesis by Plasmodiophora brassicae. Physiol Plant 66: 245–250.
Palmer MV, Horgan R and Wareing PF (1981) Cytokinin metabolism in Phaseolus vulgaris L. I: Variations in cytokinin levels in leaves of decapitated plants in relation to lateral bud outgrowth. J Exp Bot 32: 1231–1241.
Palni LMS, Summons RE and Letham DS (1983) Mass spectrophotometric analysis of cytokinins in plant tissues V. Identification of the cytokinin complex of Datura innoxia crown gall tissue. Plant Physiol 72: 858–863.
Scott LM, Martin GC, Horgan R and Heald (1982) Mass spectrometric measurement of zeatin glycoside levels in Vinca rosea L crown gall tissue. Planta 154: 273–276.
Summons RE, Entsch B, Letham DS, Gollnow BI and MacLeod JK (1980) Regulators of cell division in plant tissues XXVIII. Metabolites of zeatin in sweet-corn kernels: purifications and identifications using high-performance liquid chromatography and chemical-ionization mass spectrometry. Planta 147: 422–434.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Guinn, G., Brummett, D.L. Solid-phase extraction of cytokinins from aqueous solutions with C18 cartridges and their use in a rapid purification procedure. Plant Growth Regul 9, 305–314 (1990). https://doi.org/10.1007/BF00024916
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00024916