Abstract:
The effect of cytokinins was studied in three systems: the alga Chlorella, callus cultures and etiolated cucumber cotyledons. In Chlorella cultures: 1) A range of concentrations of 6BA and IP had no effect on growth; 2) Low concentrations of an anticytokinin had no effect on growth, whereas higher concentrations appeared to be inhibitory. 3) Characterisation of the Chlorella species suggested that it was surrounded by an impermeable sporopollenin layer which hindered the uptake of cytokinin. 4) The uptake of radioactive adenine occurred readily, whereas the uptake of radioactive 6BA was very slow in both growing and saturated cultures of Chlorella. 5) Extracts isolated from Chlorella and the medium in which Chlorella was growing contained cytokinin-like activity in two bioassays. 6) HPLC analyses of these extracts showed that there were fractions which eluted at the positions of IP and IPA. In callus cultures: A.1) A carrot callus was grown from the secondary phloem of the storage root of carrot. 2) This callus, which was grown on 2,4-D and kinetin, produced roots and shoots when subcultured onto IAA and kinetin. 3) Growth on 2,4-D alone was independent of the presence of kinetin. 4) Growth was inhibited in the presence of an anticytokinin, suggesting that the callus produced a cytokinin. B.1) A tobacco callus was grown from a young leaf of tobacco. 2) This callus habituated to cytokinin independence following subculture onto lower concentrations of kinetin. 3) Subculture of the habituated callus onto a higher concentration of kinetin resulted in the production of roots and shoots. C.1) Cytokinin-dependent soybean and tobacco callus cultures were obtained from the Botany Department, University of Otago. 2) Analysis of the total proteins from suspension and callus cultures of soybean by 1-D polyacrylamide gel electrophoresis showed one small 6BA-induced change in the proteins from the suspension cultures. In etiolated cucumber cotyledons: 1) 6BA caused the expansion of excised etiolated cucumber cotyledons after a 24h-hour incubation in the dark in a solution containing 6BA, in comparison to cotyledons incubated in water only. 2) The cotyledons curved upwards and in the light microscope the cells of the vascular bundles and the lower epidermis exhibited greater expansion than the upper epidermis. 3) Electron microscopic examination showed that the central vacuole of palisade cells from cotyledons treated with 6BA had expanded and that the cytoplasm had probably lost water and was compressed by the vacuole against the cell wall. 4) In contrast to other research, there was no apparent increase in polysome formation in 6BA-treated cotyledons in comparison to untreated cotyledons examined in the electron microscope. 5) A number of protein extraction methods were tried before a method was found which produced a protein extract suitable for both 1-D and 2-D polyacrylamide gel electrophoresis analyses. 6) 1-D and 2-D polyacrylamide gel electrophoresis showed that a number of proteins either increased or decreased following the treatment of cotyledons with 6BA. 7) A number of RNA extraction methods were tried, to obtain RNA suitable for translation in vitro. Only one method produced RNA which appeared to be free of contaminating substances. Weak translation of this RNA was obtained in vitro and it might be possible to develop conditions for optimal translation of the RNA given an adequate supply of an in vitro translation system.