Báo cáo khoa học: Study of endogenous plant growth Douglas fir I. Cytokinin analysis

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Báo cáo khoa học: "Study of endogenous plant growth Douglas fir I. Cytokinin analysis"Study of endogenous plant growth substances inDouglas fir I. Cytokinin analysis M. Bonnet-Masimbert 2N. Imbault P. Doumas C. Joseph1 Laboratoire des Composes Ph6noliques, Université d’Orl6ans, BP 6769, 45067 Orleans Cedex02, and2 INRA, Station dAm6lioration des Arbres Forestiers, Ardon, 45i60 Clivet, FranceIntroduction Cytokinin isolation extracted with 80% methanol in Cytokinins were buffer (pH 7.2). After concentration, phosphateTo ascertain the part played by a natural the extracts were passed through a diethylami-substance in a biological phenomenon, it is noethyl-cellulose column and purified either onnecessary to follow the endogenous evolu- an immunoaffinity (IA) column (as describedtion of this compound during the induction below) octadecylsilica one. Cytokinins or on anof the process. This is a real problem with then separated by high-performance liquid wereplant growth substances (PGS). Indeed, chromatography (HPLC) using a reverse phase column (MacDonald et al., 1981) and measuredtheir very low concentrations in tissues either by UV absorption or by ELISA or RIA (asmake PGS difficult to quantify. Because of reported below).their sensitivity and specificity, immuno-logical methods have been adapted to theanalysis of PGS and enable, in somecases, measurements at the level of a Immunological methodssingle organ, as reported for principally For IAC and EL procedures, monoclonal SA Jherbaceous species (Weiler, 1984). In this antibodies were raised against cytokinins conju-paper, some of their applications to the gated to bovine serum albumin (MacDonaldwoody plant, Douglas fir (Pseudotsuga and Morris, 198!i). IA columns of 1 ml eachmenziesii Mirb.), are presented: purifica- contained equal amounts of anti-ribosylzeatin ntion by immunoaffinity chromatography (anti-RZ) and anti-isopenteny!adenosine (anti- IPA) antibodies coupled to a cellulose matrix.(IAC) and measurement by an enzyme- With this mixture of antibodies, IAC waslinked immunosorbent assay (ELISA) or a performed according to MacDonald and Morrisradioimmunoassay (RIA). (1985). Thus, the usual cytokinin bases and ribosides were recognized. ELISA was perform- ed as described in Bataille et al. (1987); detec- tion limit and range were 15 pg and 20-5000Materials and Methods pg, respectively. RIA was done according to MacDonald et al. (1981) using polyclonal anti- cytokinin antibodies; detection limit and rangeMaterial here were, 50 pg and 100-5000 pg, respec-The sexual buds of study was performed on tively.Douglas fir.Results plant material. However, the detection limit by UV absorpi:ion (254 nm) after IAC was only 1-5 ng. For small samples, the more 1 shows the HPLC chromatogram ofFig. sensitive ELISA or RIA (15 or 50 pg) could extract from a female bud of Douglasone be used. Thus, despite the inherent diffi-fir subjected to IAC (B) or not (A). IAC culties of the woody material, PGS analy-cleared the extract of UV absorbing com- sis is possible and practical at the organpounds. Further cytokinin quantification level, where physiologicalperformed by RIA on HPLC fractions processes One application of this possibilitydemonstrated no significant losses of occur. was illustrated by the study of Imbault etthese PGS through iAC. Therefore, IAC, al. (1988), which showed the interventionwhich retained only immunologically reac- of IP and IPA in Douglas fir flowering.tive compounds, acted as a selective filterenab ...