Morphogenesis in the moss Physcomitrella patens
A method was developed for recording the development of moss protonema using time-lapse video microscopy. This has provided a detailed record of the time-course of development from spore germination to the production of gametophores. Detailed records of the growth of primary and secondary chloronema, the transition of primary chloronema to caulonema, and the development of side-branches were obtained. Filaments were found to undergo the transition to caulonema earlier than previously thought. The majority of caulonemas ide-branches were found to begin as chloronema and switch to caulonema after one or two cell cycles. The early cell divisions of bud formation were found to follow a distinct pattern, which was upset by high concentrations of cytokinin and lanthanum. The response of caulonema apical cells to polarotropic light was recorded and compared to the gravitropic response. The time-lapse studies provided the basis for the further development of the quantitative analysis of protonemal branching patterns to include second and third side-branches of a sub-apical cell, and transitional caulonema. Analysing side-branch patterns should allow the detection of developmental mechanisms underlying the determination of side-branch fate. The potential of this method for assessing the effect of hormone treatments and for analysing more precisely mutant phenotypes was explored. An analysis of bud spacing was carried out to determine if the formation of a bud on a filament was inhibitory to other buds forming on the same filament. It was found, to the contrary, that buds tended to form in clusters. The hypothesis that the primary mode of action of cytokinin is an enhanced influx of calcium ions into the cell was investigated. Classical electrophysiology was used in order to detect any change in membrane potential suggestive of ionic fluxes in response to cytokinin treatment. No definitive changes in membrane potential were detected in response to cytokinin. This appeared to rule out the involvement of voltage-regulated channels in cytokinin action. The effects of some inhibitors used in studies of calcium on the moss protonemal system were examined. It is suggested that the concentrations commonly used had toxic effects that were not specific to calcium channels. The ionophore A23187 was used to characterise the protonemal response to a sustained influx of calcium. Some mutant strains were found to have a differential response to the ionophore. This may mean that they have mutations affecting their calcium regulatory system. Two new techniques of imaging calcium were used in order to detect changes in intracellular calcium in response to cytokinin. A method was developed for loading the dual wavelength fluorescent dye Indo-1 into moss protonema using iontophoretic microinjection, and intracellular calcium was imaged using ratio-image technology. Wild-type moss and some mutant strains were also successfully transformed with the gene for apoaequorin, and calcium luminescence measured in response to cold-shock and plant hormones. Some different responsesto temperatures hock were apparent in one of the transformed mutants. Preliminary experiments did not reveal any aequor independent calcium luminescence in response to cytokinin.