Eighteen nutritional mutants have been isolated in the haploid, monoecious moss, Physcomitrella patens: five nicotinic acid auxotrophs, four p-aminobenzoic acid auxotrophs, four adenine auxotrophs, two amino acid requiring mutants and three nitrate non-utilising mutants. Seventeen of them were obtained using total isolation; one was isolated selectively. Strains resistant to the amino acid analogues, D-serine and p-fluorophenyl-alanine, and the purine analogue, 8-azaguanine, have been selected. Many of the auxotrophs are self-sterile. Crosses between auxotrophic strains have been effected and the progeny analysed. No linkage has been detected. Nicotinic acid auxotrophy has resulted from mutation in at least two genes. Self-sterility segregates as a pleiotropic effect of four mutations which produce nutritional dependence. A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.

The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.

Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants

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The first descriptions of sex chromosomes in plants, of the continuity of chromosomes during the mitotic cycle and of non-Mendelian inheritance as well as the introduction of UV-mutagenesis to genetic research are landmarks in biological sciences first achieved by scientists working on bryophytes. Haploidy of the tissue facilitates mutant isolation and many developmental moss mutants have been isolated. Early moss development is triggered by auxin, by cytokinin and by light, mainly acting via phytochrome and a blue-light receptor. Due to the simplicity of the plants, development can be pinpointed to the differentiation of a single cell and be analysed in living tissue, making mosses ideal candidates for the analysis of development in an integrated approach of cell and molecular biology. Molecular genetic techniques have been applied mainly to Physcomitrella patens (Hedw.) B.S.G., where efficient protocols for transformation of nuclear DNA have been established and several nuclear, chloroplast and mitochondrial genes have been analysed. These studies reveal that Physcomitrella may be an appropriate model to study plant development in molecular terms. Recently, it has been shown that, in this species, nuclear genes can be targeted very efficiently by homologous recombination, now opening the door to reverse genetics for plant biologists.

Development, Genetics and Molecular Biology of Mosses

The bud-inducing effect of the cytokinin N6-(Δ2-isopentenyl)-adenine (i6-Ade) was examined in the moss Physcomitrella patens growing in liquid culture. Under these conditions, buds could be induced on chloronemata as well as on caulonemata. By application of i6-Ade, bud-formation was accelerated in both types of tissue. The number of buds, their size and their site of development were dependent on the concentration of the cytokinin in the range of 10-7 M to 10-5 M. Moreover, the percentage of caulonema cells increased with a cytokinin concentration of 10-5 M. These results indicate that chloronema cells may also function as target cells for exogenous cytokinins. The composition of proteins from caulonemata and chloronemata of two different species (P. patens and Funaria hygrometrica), grown on solid medium were compared. No differences could be detected between the protein patterns of caulonemata and chloronemata of the same species while between the two species the differences were obvious.

Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine

The moss Physcomitrella patens has recently emerged as a powerful genetically tractable model plant system. As a member of the bryophytes, P. patens provides a unique opportunity to study the evolution of a myriad of plant traits, such as polarized cell growth, gametophyte-to-sporophyte transitions, and sperm-to-pollen transition. The availability of a complete genome sequence, together with the ability to perform gene targeting efficiently in P. patens has spurred a flurry of elegant reverse genetic studies in this plant model that address a variety of key questions in plant developmental biology.

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Martin Bopp

Papers

Development of the protonema and bud formation in mosses