In vitro cultivation of bryophytes: a review of practicalities, problems, progress and promise

Abstract In vitro cultivation is not only essential for the use of bryophytes in cellular, developmental and molecular research but is also vital to the elucidation of the roles of juvenile stages in reproductive biology. Other important uses include the discovery of new characters for systematics, the conservation of rare taxa and understanding the functional significance of fungal and cyanobacterial association in hepatics and hornworts. This article, based on the culturing of over 300 mosses and nearly 50 hepatics sets out the practicalities of in vitro cultivation and reviews the potential rewards. Cultures, initiated from surface-sterilized spores, gemmae or vegetative fragments, are most easily handled in Petri dishes on media containing inorganic salts. Solidification of the medium with Phytogel or Gelrite is preferred to traditional agar because of toxic impurities in the latter. Dilution of the nutrients can be beneficial to the growth of some taxa. Prior knowledge of the seasonality of sporophyte production is vital for establishing cultures from spores, especially those of hepatics. The period of spore availability can be increased by storing ripe capsules at 4°C. Irradiances in culture are usually much lower than in nature, but are still sufficient for good growth, whereas temperatures above 25°C are damaging. Cultured materials can be weaned in vitro onto their natural substrata including rocks. These and protonemal gemmae suspensions are ideal inocula for reintroducing plants into the wild. Cryopreservation techniques are being developed for the long-term preservation of endangered taxa. Specimens grown in vitro are closely similar to their wild counterparts but sometimes produce propagules previously unknown in nature. Striking examples include the first reports of protonemal gemmae in Ditrichum cornubicum, Saelania glaucescens, Seligeria carniolica and Zygodon gracilis. By contrast, the absence of rhizoidal and protonemal propagules in all the British species of the Splachnaceae indicate that an earlier account of protonemal gemmae in Tayloria was probably based on misidentified material. The first description of a highly distinctive protonema in Buxbaumia viridis opens the way to identify this endangered species in the absence of sporophytes. Protonemata are providing an increasing number of new characters useful at all levels in the taxonomic hierarchy. The massive juvenile stages in Andreaea are impossible to equate with the filaments found in the Bryales. Rhizoidal ropes are a characteristic of the Polytrichales. Thalloid protonemata link Tetraphis and Oedipodium, a finding reinforced by molecular sequence data. Pointed chloronemata suggest the reassignment of Ephemerum to the Pottiales, an order characterized by asexual propagules lacking Tmema (abscission) cells. Highly differentiated gemmae with Tmema cells and hyaline sticky acumina support molecular data that Schistostega, Syrrhopodon and Calymperes are closely related members of the Dicranales. At the infraspecific level, distinctive protonemal morphology suggests that Weissia controversa var. densifolia may merit elevation to specific status. In vitro cultivation of hepatics and hornworts is complicated by the presence of fungal and cyanobacterial endophytes. Ascomycetes and basidiomycetes (and Nostoc) can be isolated from their hosts and the associations re-established in culture, thus fulfilling Koch's postulates. Glomus spores or mycorrhizal higher plants may be used as inocula to re-establish zygmycetous infections. Cross-infection studies are revealing a wide host range for the ascomycetes including many members of the Lepidoziaceae and Cephaloziaceae and extending to the Ericaceae, but the fungus Mniacea jungermanniae, that forms blue-green apothecia on the stems of Cephalozia bicuspidata, does not colonize the rhizoids of its hepatic hosts. Basidiomycetes isolated from a range of Jungermanniales are host specific and the fungus from Cryptothallus does not invade Aneura pinguis. The identification of two fungi, distinct both morphologically and in their host range, from alpine and lowland populations of A. pinguis, is in line with the varietal status recently assigned to these in Scandinavian specimens.