Tissue Culturing Of Five Bryophytes

The bryophyte tissue culture was established very early. But it goes much more slowly. Recently, the research areas of bryophytes have been broaden, such as the molecular biology, biochemistry, physiology, taxonomy, ecology, and so on. So the bryophyte samples are in great need. However, samples from the wild can not meet the requirement. The bryophyte tissue culture techniques could provide much more clean materials very soon. This work aimed at the basic study of culturing five representative bryophytes. Starting with how to get axenic explants, this work mainly focused on the pattern of sporeling and the development of protonema . In order to preserve and enlarge the bryophytes, and to provide enough materials for later experimental study and practical use, we learned to obtain perfect plants. The entire processes of sporeling and development of protonema of cultured spores were gained through microscopes and photographs. The processes of the developments of oil bodies in one cultured liverwort were achieved too. Those above could provide some data for the bryophyte systematics.The results of this work are reported as follows:1. The spores of Cheilolejeunea ventricosa (Schiffn.) X.L.He and the thallus of Pellia epiphylla (L.) Cord. were cultured on MS media (without acid and vitamin, with 1% agar and 1% sucrose, pH=6.2). The sterilizing treatment with 0.1% Sodium hypochlorite solution for a period of 5 seconds is optimal. The spore germination and sporeling of Cheilolejeunea ventricosa belongs to Lejeunea-type. Oil bodies in primary protonemata are nearly homogeneous, but distinctly compound in the leaf cells of the primary leafy shoots. After a 55-day axenic culture, young plants of C. ventricosa were obtained successfully. The thallus of P. epiphylla gave out fronds directly without callus on MS. It is with that on MS treated with 2,4-D and 6-BA separately. The new thallus stop growing after two months. MS media is not suitable for the thallus growth of P. epiphylla.2. The spores of Brachymenium nepalense Hook. were cultured on MS and 1/2 MS media (without acid and vitamin, with 1% agar and 1% sucrose, pH=6.2), Knopmedia (with 1% agar and 1% sucrose, pH=6.2), and Beneck media (with 1% agar and 1% sucrose, pH=6.2). The sterilizing treatment with 1% Sodium hypochlorite solution for a period of 4 minutes is optimal. The simple media Knop and Beneck promoted the spore germination. Furthermore, plants came out from the spores after 50-day culturing. But MS and 1/2 MS media made the spores deteriorative. The sporeling pattern of B. nepalense belongs to Bryum-type.3. The spores of Physcomitrium courtoisii Par. et Broth were cultured on MS media (without acid and vitamin, pH=6.2, with 1% agar and 1% sucrose), Knop media (with 1% agar and 1% sucrose, pH=6.2), and Beneck media (with 1% agar and 1% sucrose, pH=6.2). The sterilizing treatment with 1% Sodium hypochlorite solution for a period of 8 minutes is optimal. MS media promote the growth of protonema while restrains the differentiation of fronds. Whereas, Knop and Beneck promoted the differentiation and growth of fronds. Protonemata transferred to Knop and Beneck presented juvenile leaves after one month. The sporeling pattern of P. courtoisii belongs to Funaria-type4. The stems of Amblystegium serpens(Hedw.)B.S.G. were cultured on MS and 1/2 MS media (without acid and vitamin, pH=6.2, with 1% agar and 1% sucrose), Knop media (with 1% agar and 1% sucrose, pH=6.2), and Beneck media (with 1% agar and 1% sucrose, pH=6.2). The sterilizing treatment with 0.1% Sodium hypochlorite solution for a period of 20 seconds is optimal. The protonema grows best on 1/2 MS media. The media treated with 2,4-D helped to induce callus from stems. The more 2,4-D you put into in a certain range, the better callus you would obtain. Knop and Beneck promote the differentiation and growth of fronds. Protonema and callus transferred to Knop and Beneck presented juvenile leaves after 20 days.