Isolation and morphological studies of a variant ofCeratodon purpureus

Abstract

Summary. Morphological variants from protonema of Ceratodon purpureus (Hedw.) were obtained using nitrosoguanidine (NTG). In one of the variants, reduced growth was accompagnied by hyperbranching and inhibition of caulogenesis. The deposition of branches in this strain can be studied by enzymatic analysis 9Light 1'2, biochemical 3 and cellular 4 factors, among Others act on the branching of the protonema of Bryales. Few results have been published concerning genetic studies on the morphogenesis of this system 5-8. These organisms merit further study with the same techniques as are used for fungi. The essential contribution of genetics to the better understanding of mycelium growth and branching does not need to be emphasized 9,10. Bryales mutants are often auxotroph strains. Results obtained with fungi n suggest that only morphological variants can lead us to understand the morphogenesis of filamentous systems. In auxotroph mutants, the block in development is a function of an organic molecule, which restores morphogenesis of the wild type. On the contrary, morphological mutants do not have any new metabolic needs but are characterized by changes in the regulation of metabolic processes in the later stage of development. The attainment of hyperbranching mutants in fungi permitted the partial elucidation of the mechanisms leading to the production of lateral buds. In the case of Bryales protonema, we have not yet obtained morphological mutants which are easy to characterize even though we used parameters which permitted us to define morphogenesis (growth speed, mitotic rate, cell length, branching capacity). We tried therefore to isolate morphological mutants of Ceratodon purpureus and determine the branching mechanism of this filamentous system. Material and methods. Mutagenesis was carried out on aqueous suspensions of spores of Ceratodon purpureus at a concentration of 2 • 103 cells/ml. Nitrosoguanidine (NTG) was added at concentrations varying from 10 to 750 ~tg 9 ml-1 during 30 min, in agitated medium, at room temperature. The spores were collected by centrifugation, then washed and spread in Petri dishes on Kofler A medium 3. The culture development was continued under multilateral light (fluorescent tubes 'Lumirre du jour de luxe' type) at 14,000 erg cm -2 sec-l, at 23 ~ during 8 days. NTG concentrations greater than 500 rtg. ml-I gave the percentage of survival (2%) necessary to obtain the most 1 probable variants. At 750 I.tg. ml-thalli were picked and maintained on medium A. Implants were then taken from the edges of cultures and inoculated in van Tieghem cells according to the usual technique 3 under light of 1500 erg cm -2 see -t. Kofler A medium 3 was used in some cases supplemented with a-D(+) glucose (Ag). Resuhs. Figures 1 and 2 summarize the results obtained with the wild strain and one of the most interesting morphological variants (133,750). In culture chambers the growth rate of this strain was very inferior to that of the wild type ( fig.l, a), and the elongation of filaments was 29% of that i...