The Establishment Of Several Mosses Gametophyte Regeneration System

The bryophyte tissue culture had already experienced more than a century.But its development was very slow.The main reason was that the bryophyte were little and plants were mostly composed by single-cell.To disinfect the bryophyte plants moderately was very crucial. To obtain the bryophyte axenic gametophyte abundant was still very difficulty.To explore bryophyte tissue culture technology further and establish the gamephytes regenation systems had significance for carrying out the bryophyte molecular biology,physiology,phytochemistry and so on.In this paper,the explants and spores were used as experimental material.We established gamephytes regenation systems for six mosses species,e.g.Grimmia pilifera P.Beauv., Pogonatum inflexum(Lindb.) Par,Weisia controversa Hedw.,Pseudoleskeopsis zippelli(Dozy et Molk.),Rhynchostegium pallidifolium(Mitt.) Jaeg and Atrichum undulatum P.Beauv.We dealt with the effects of culture conditions such as hormone on the gametophyte differentiation and growth.The results were listed as follows:1 Gametophyte regeneration system of Grimmia pilifera was initial established through the explant culture and spore culture.Each method has its own advantages.It was easy to obtain the gametophyte material that was plentiful collected directly outside and be used to do the explant culture.But the critical step of this method was to sterilize the material.The best way to sterilize the Grimmia apical explant:First,sterilized the shoot tips of Grimmia with 0.05% concentration of Mercuric Chloride for 60 to 70 second.Then washed them with axenic distilled water for 7 to 8 times.We found that 6-BA hormone had an obvious effect on filaments expansion.It was faster to get the gametophyte through the spore culture,but it was only useful when we collect the fresh and mature spores.Optimal medium of the spore germination of Grimmia pilifera was the improved MS medium.We transferred the germinating spores to the Beneck medium without the glucose.When the buds were differentiated from the protonema and grew into the gametophyte,we transferred the tender gametophytes into the Beneck medium with the glucose(10g/L),then we got the purified regenerative gametophytes.2 We reserched the effects of medium PH,the component of medium and the different hormone on the gametophyte regeneration system of Grimmia pilifera.The results indicated:(1) The medium PH had little effects on filamentous growth of Grimmia pilifera P.Beauv.in the range of PH using in our experiment.(2) 0.25mg/L 6-BA had the significant effects on the filamentous growth.(3) Glucose could promote the growth of protonema in the range of 0～10g/L. However the higher concentrations of glucose had little effects on the growth of protonema.(4) The BCD medium with ammonium tartrate could induce the formation of the caulonema.(5) The medium without glucose and containing low concentration of inorganic salt was of advantage to the germination of spores.The concentration of hormone in the medium had some effects on filamentous growth.The medium with 10^-4 M 6-BA and 10^-6M IAA had the most significant effect on the filamentous growth.3 The experiment covered spore germination and protonema development and gameto-phyte regeneration system of 5 species of mosses:Weisia controversa Hedw.,Pseudoleskeopsis zippelli(Dozy et Molk.)Broth.,Rhynchostegium pallidifolium(Mitt.) Jaeg.,Pogonatum inflexum (Lindb.) Par.and Atrichum undulatum P.Beauv.The results showed that all of 5 moss species spores germinate outside the ruptured exospore.Pogonatum inflexum and Atrichum undulatum belong to Funaria-type,and the others belong to Bryum-type according to Nishida's classification of spore germination types of mosses.Different moss protonema development were related to the growing environment.The growing environments of Weisia controversa and Grimmia pilifera were the arid surface of rock.The shape of cells of chloronema was cylindric, and the branch were short and swarm.The protonema germinated from one spore formed a ball. The growing environments of Rhynchostegium pallidifolium was the wet surface of rock.4 We can easily get a large amount of mature spores of Weisia controversa Hedw., Pseudoleskeopsis zippelli(Dozy et Molk.)Broth.,Rhynchostegium pallidifolium(Mitt.)Jaeg., Pogonatum inflexum(Lindb.)Par.and Atrichum undulatum P.Beauv.So it had more advantages in establishing gametophyte regeneration system through spore culture.But different mosses need different method of spore sterilizing.In the same situation of tissue culture,gametophytes came out from the spores of Rhynchostegium pallidifolium after 60-day culturing.Protonema of Weisia controversa transferred to Beneck medium with 6-BA could presented the gametophytes more quickly.