C. Forsyth

Tuberization of Dioscorea bulbifera stem nodes in culture

Tuberization of Dioscorea bulbifera expiants was induced in culture by media manipulation. This plant, commonly known as the air potato, produces aerial tubers in response to short day conditions in vivo. Nodal stem segments harvested from vigorously growing vines in spring and early summer served as experimental material. Nodal stem expiants of Solarium tuberosum were used in one experiment to ascertain whether the response was similar in other tuberizing species. An increase in sucrose concentration from 2% (w/v) to 8% (w/v) and/or a decrease in cytokinin concentration in the basal culture medium induced tuberization in both these plant species under non-inductive day lengths.

The metabolism and cell division activity of adenine derivatives in soybean callus

Summary The metabolism of radiolabeled adenine and four cytokinins was compared in soybean callus cultures over a 48 h period. Rates of metabolism varied but were rapid regardless of the cytokinin supplied. There appeared to be no metabolite common to all the cytokinins studied. Callus growth on optimal concentrations of the four cytokinins was best when kinetin or benzyladenine were supplied and lower when zeatin or dihydrozeatin were supplied. Although zeatin and dihydrozeatin were metabolised in different ways, thus yielding different metabolites, growth of callus was much the same regardless of which of these two cytokinins was supplied. Thus it seems that each of the cytokinins studied is active per se and that there is no common, active metabolite.

An improved method of in vitro propagation ofDioscorea bulbifera

Nodal stem segments ofDioscorea bulbifera were induced to form plantlets in vitro. Rooted plantlets were obtained on Murashige-Skoog [14] revised medium supplemented initially with 5 mg 1−1 kinetin and subsequently with 5 mg l−1 indole-butyric acid. By increasing the kinetin concentration from 5 mg l−1 to 10 mg l−1, the number of shoots forming per node was increased from five to eight. When kinetin was substituted with 6-benzylaminopurine at only 1 mg l−1, nine shoots formed on each node. Each shoot could be excised from the node and induced to form a new crop of multiple shoots. Rooted plantlets could be successfully transferred to in vivo conditions.

Cytokinin metabolism in tomato plants. II: Metabolites of kinetin and benzyladenine in decapitated roots

Decapitated tomato plants were supplied via the roots with [8-14C]-kinetin or [8-14C]-benzyladenine in a nutrient solution for a period of 24 h. After this time the root material, the root sap produced during the 24 h period and the nutrient solution remaining at the end of the experiments were analysed for cytokinins. HPLC techniques and chemical treatments were used to tentatively identify radioactive metabolites formed. Uptake of kinetin and benzyladenine by the roots was found to be limited but once within the root tissues metabolism was both rapid and extensive.At least 14 metabolites of kinetin were recovered from root tissue and root sap. Many of these appeared to be degradation products. There was, however, some evidence of formation of zeatin-like derivatives. Side-chain cleavage of the original kinetin which occurs rapidly is suggested as a possible route for the eventual production of these endogenous cytokinin forms.The benzyladenine taken up by the roots was apparently both ribosylated and glucosylated. No unmetabolized benzyladenine was detected in the root tissues after 24 h. Only very low levels of radioactivity were associated with the retention time of adenine, suggesting that in the case of benzyladenine side-chain cleavage is of limited importance.The significance of these reactions in relation to the potential use of cytokinins in the regulation of plant growth is discussed.

A radioimmunoassay for dihydrozeatin and dihydrozeatin riboside, and its application to a study of the in vitro metabolism of dihydrozeatin by soybean callus

Summary A radioimmunoassay was developed for the quantitation of DHZ and DHZR, using antibodies raised to a DHZR-BSA conjugate, and [ 3 H]-DHZ as the tracer. The effective measuring range of the assay was 0.11–22.6 pmol for DHZ, and 0.071–14.2 pmol for DHZR. The antiserum showed sufficient cross-reaction with Z and ZR to necessitate their separation from the respective dihydro derivatives prior to RIA. This was adequately achieved by oxidation with KMnO 4 . Cross-reaction with kinetin, BA and HA was also unusually high, and this indicated the potential suitability of this assay system for their quantitation. The in vitro metabolism of DHZ by soybean callus over an incubation period of 48 h was followed by RIA, and by monitoring the degradation of added [ 3 H]-DHZ by HPLC. Both assay systems gave similar results, indicating a conversion of DHZ to DHZR.

The Role of Adenine and Adenosine in the Synthesis of Cytokinins by Excised Maize Roots

Summary While cytokinin-like activity was detected in aseptically cultured excised roots of Zea mays no evidence could be found that (8- 14 C)adenine or (U- 14 C)adenosine was incorporated into the biologically active compounds over a 42 day culturing period. This information raises serious doubts as to whether adenine and/or adenosine serve as primary precursors for cytokinin biosynthesis. The possibility does however, exist that the excised roots lacked a shoot produced precursor necessary for cytokinin production.

The role of adenine in the synthesis of cytokinins in tomato plants and in cell-free root extracts

The incorporation of labelled adenine into cytokinin-like compounds was investigated in intact tomato plants, decapitated tomato roots and cell-free root extracts. In all three cases evidence was found for the incorporation of adenine into endogenous cytokinins. In intact plants and decapitated root systems no evidence was found for the incorporation into cytokinin nucleotides. Cytokinin bases and nucleosides were however, labelled. In the cell-free root extract there was some evidence for the incorporation of labelled adenine into cytokinin nucleotides. This suggests that the biosynthetic process may be strictly compartmentalized. The present results provide no evidence for the relative importance of cytokinin nucleotides in the biosynthetic process. What is clear is that the rate of adenine incorporation into cytokinins is extremely low and that only a small proportion of the cytokinins which became labelled were exported to the shoot via the root exudate.

Metabolism of kinetin by soybean callus

Soybean callus metabolised applied 6-furfurylamino (8-14C) purine very rapidly to polar compounds, some of which were retained on a Dowex 50 cation exchange resin, and were unaffected by alkaline phosphatase; while others were apparently phosphorylated and were detected in the aqueous fraction. Adenine was detected as an intermediate and it can be concluded that it was formed as a result of the rapid and efficient removal of the furfuryl side chain. The formed adenine was rapidly incorporated into the polar metabolites. Exactly how the presence of this cytokinin stimulates cell division is not apparent from the results.

The Metabolism of Kinetin and Benzyladenine in Soybean Callus: Determination by Radioimmunoassay and Radioassay

Summary Previous investigations indicated the potential for the use of antisera produced against dihydroribosylzeatin for the quantification of kinetin (K) and benzyladenine (BA). Assays were developed for these synthetic cytokinins and their respective ribosides. Assay measuring range for K and BA was 0.12–46.5 pmol and 0.22–44.4 pmol, respectively. Similar measuring ranges were obtained for both K and BA riboside (0.07–28 pmol). The assays were applied to a study of the metabolism of K and BA in soybean callus cultures. Extract purification was achieved with ion exchange chromatography, and the bases and ribosides separated using thin layer chromatography. The results were in good agreement with those obtained using the more conventional methods of Sephadex LH-20 and HPLC, and indicated rapid metabolism of both K and BA during the 48 h experimental period. No metabolites apart from BA riboside were detected by RIA. HPLC however indicated the presence of other metabolites, and these are reported in a concurrent paper.

Adenine Incorporation into Cytokinins in Aseptically Cultured Tomato Roots

Excised tomato roots cultured in a liquid medium were used to investigate the fate of applied (8-(14)C) adenine over a 12 day period. Root material and the staled culture medium were analysed separately for cytokinin-like activity. In both cases the major radioactive compound recovered co-chromatographed with authentic adenine. Root material also yielded low levels of radioactive compounds which after Sephadex LH-20 fractionation and HPLC analysis cochromatographed with zeatin, ribosylzeatin, adenosine, and glucosylzeatin. Both the root material and the culture medium contained a larger, unidentified, polar peak which did not respond to alkaline phosphatase or β-glucosidase treatments.