Studies On Tissue Culture Of Rhodobryum Giganteum

Rhodobryum giganteum provides important value as a Chinese traditional medicinal herb in relation to cardiovascular diseases. In the present studies, we took the young shoots of the wild plants as explants and obtained axenic protonemata and gametophytes of Rhodobryum giganteum in virto. We investigated the effects of the type of cultural media, mechanical cutting of the protonemata, hormones, temperature on the proliferation of the protonemata, and also compared the morphological differences between wild gametophytes and those in vitro. The main results are as follows:1. We Compared the different culture media in their influences on the proliferation of the protonemata of R. giganteum, and concluded that modified Knops medium is able to facilitate the elongation of the protonemata and prolong their growth time in vitro, whereas MS medium is able to facilitate the branching of the protonemata, and the protonemata grew slowly in modified White medium, accompanied by the development of gametophores.2. We tested the influences of cutting of protonemata and the application of hormones on the proliferation of protonemata, and got twice dry weight of protonemata after cutting and discovered that 2.4-D in low concentration could promote the protonemata growth.3. Rhodobryum giganteum has a simple growing condition without adding organic materials. Through basic media selection, we concluded that modified Knops medium is able to facilitate the elongation of the protonemata and prolong their growth time in vitro. Unlike the media type, the concentration of Knops medium has less effect on the protonemata growth, which showed no significant difference even the concentration of the medium decreased to 1 to 10. Boron, one of the trace elements in Knops medium, showed greater importance to the protonema growth than all the trace elements in the medium, the protonemata grew even worse in Knops media without boron. The experiments revealed that the best medium for the protonema of Rhodobryum giganteum is modified Knops medium with suitable sucrose (about 10 g.L-1) and agar (4g.L-1), and neutral pH about 6.0-7.0.4. The protonemal mass in their original inoculation posed on distinctly effects on their proliferation. Our test showed that the protonemal colony in a 0.8 cm diameter resulted in the fastest growth.5. The protonemal appeared to be sensitive to temperature and moisture, and its favorite temperature is 20-22℃. The phytohormone has an obvious impact on the protonemal growth. The medium with 6-BA could induce callus tissue of R. giganteum protonema, while the medium with NAA could turn the protonemata brown and reduced their branch number, and the medium with KT may increase the budding of protonema.6. The gametophytes formed in vitro would morphologically changed to some extent. In the present work, we could identified three morphological types from the gametophytes in vitro: A: the gametophytes with larger top leaves on the stem, B: the gametophytes with leaves thinning down along the stem from the base, and C: those with small leaves along the longer stem. Moreover, we observed the difference occur since its differentiation from the protonemata. Resemble to the protonemata, the morphosis of the gametophytes is sensitive to the temperature, and its favorite scope is 20℃-22℃.7. We determined the photosynthesis indices of the wild plants of R. giganteum, and compared with those in vitro. The gametophytes in vitro has a higher light saturation point than that of the wild, especially for those with longer stem and small leaves, but in assimilation rate of photosynthesis, the wild plants as well as protonemata showed higher values. The light saturation point is about 400μmol m-2 s-1, and also the photosynthesis inhibition occur when the vapor pressure deficit value above 10, which demonstrated the importance of the water content in the plants. Through photosynthesis-physiological measurement, we concluded that the morphological change of the plants suit to the radiation in vitro (≥350μmol m-2 s-1) and helped to retain water content. The result of photosynthesis chlorophyll fluorescence parameter of the plant both in vitro and wild showed plants growing in wild had a high maximal quantum yield of PSⅡand PSⅡpotential activity, but for the actual photons efficiency and the photo-electron transferring speed, plants growing in different condition exhibit no significant difference. Moreover, the parameter showed coherency in wild growing plant but discrepancy in vitro, which announced the variability and plasticity of the plants in vitro.8.Through comparing the scanned spectra of FTIR spectroscopy and UV-spectroscopy between plants in vitro and in wild growing condition we concluded that there was prominent differences in the locations and number of the absorb peaks as well as the peak value, which indicated the possible notable differences in the chemical composition and their component proportion.