摘要
暖地大叶藓(Rhodobryum giganteum (Hook.) Par.)是一种重要的药用苔藓植物,对治疗心血管疾病具有很好的疗效。本论文以暖地大叶藓幼嫩茎尖为外植体,应用组织培养方法,成功地获得了无菌的暖地大叶藓原丝体和植株,在此基础上,研究了培养基类型、机械切割、激素、温度、配子体自身的提取液、培养基组成等对原丝体诱导和生成量的影响,同时比较了组织培养诱导形成的配子体形态、光合和红外光谱特点。主要成果如下:
1、通过培养基的筛选发现改良Knops培养基适合原丝体的扩展生长而且能保持两个月不分化出配子体植株,MS培养基更适合原丝体的分支生长,改良的White培养基中原丝体生长慢同时伴随植株的分化。
2、运用机械切割法和激素诱导法对原丝体的繁殖进行实验,发现机械切割后原丝体繁殖量在一个月内比未切割直接转接的繁殖量增加一倍;低浓度的2.4-D能促进茎段上原丝体的发生。
3、培养基的类型和组成对暖地大叶藓原丝体扩繁有显著的影响。改良Knops培养基的浓度对原丝体生长影响不大,1/10浓度的改良Knops培养基上生长的原丝体跟全量改良Knops培养基上的原丝体生长无显著差异。改良Knops培养基组成成分中硼元素对原丝体生长至关重要,甚至比所有微量元素还重要,从原丝体生长看,不含硼元素的改良Knops培养基上的原丝体比不含微量元素改良Knops培养基上的原丝体更差;改良Knops培养基加适量蔗糖和少量(4g. L-1)琼脂、pH为6.0-7.0的培养基更适合于暖地大叶藓原丝体的生长。
4、原丝体初始转接量对暖地大叶藓原丝体扩繁也有显著影响,本试验中以直径0.8cm左右的原丝体团对原丝体的扩繁生长最为有利。
5、实验表明,暖地大叶藓原丝体生长对温度和水分较为敏感,最适温度是20-22℃。植物激素对原丝体生长有较为明显的作用,6-BA能诱导出愈伤组织团,NAA使原丝体变黑,分支生长少但原丝体纵向生长长,含KT的培养基中原丝体一个月后有较多幼芽分化。
6、组织培养条件下形成的暖地大叶藓植株在形态上有一定程度的分化,论文描述了组培苗的三种类型:大叶顶生型、叶片渐小型和小叶长枝型。不同形态的植株可以从原丝体分化出植株时就有所不同。跟原丝体生长一样,植株分支繁殖形态发生对温度也比较敏感,最适温度也是20℃-22℃。
7、实验室条件下测定了暖地大叶藓生长的光合作用指标,发现组培植株比野生植株有更高的光饱和点,尤其是形态上变异明显的枝长叶小型植株,但光合同化速率不如野生采集的植株高;暖地大叶藓原丝体的光合同化速率较高,与野生植株相近。暖地大叶藓的植株在光强大于饱和点后(≥400μmol m-2 s-1)出现光抑制现象。水分对植株的光合作用起着很大影响,水分丧失较多(VPD>10)时光合作用被明显抑制。植株形态的变异有利于植株适应光照较强(≥350μmol m-2 s-1)下生长,而且变异植株对水分的保持有更好的优势。光合荧光参数反映出野生采集的植株叶片具有更高的PSⅡ的最大光化学量子产量和PSⅡ的潜在活性,但实际光量子效率和光合电子传递速率在两种生长条件下的植株差别不大。
8、应用红外和紫外光谱扫描技术对野生和组培条件下暖地大叶藓植株进行光谱比较,发现虽然两者间的波峰位置、数目和波峰高度均存在较大的差异,说明两者的化学组成和含量可能会存在较大差异。
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.
引文
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