| Cistanche deserticola, a traditional Chinese medicinal plant is known as "deserticolous ginseng". It is a perennial phanerogamic plant of family Orobanchaceae, genus Cistanche Hoffmgg. Et Link, which is obligate-parasitic in the roots of perennial plant Haloxylon ammodendron (C.A.Mey.) Bunge. Endophytic fungi is found universal within the plant, and exists widely. It plays a crucial role in the growth and development of the host plant as well as the whole evolution progress, and it has attracted great attentions of the investigators all over the world. The systematic research on C. deserticola is still blank now. This thesis is focusing on C. deserticola, concerning the difference of localities, seasons, growth years and application portions in order to explore the composition and the diversity of C. deserticola endophyte by traditional morphology and modern molecular bio-technology. At the same time, a series of researches on the seed germination pre-conditioning of C. deserticola were carried out, aiming at providing basis for the selection of high activity strains and the conservation of C. deserticola theoretically and scientificly. The main novel points and conclusions are as follows:1. It is a pioneering investigation on the endophytic fungi of C. deserticola conducted basing on morphological methods and molecular biological technique. A total of 618 isolates were obtained from 2393 pieces of C. deserticola tissues,453 isolates of them sporulated and were identified into 22 genus. The other fungi are asporulated.2. It is a research directly against the diversity of the endophyte of C. deserticola. In the total tissue pieces,570 of 2393 have been found with fungi, from which we got 618 isolates, the total colonization rate is 23.8% and the total isolation rate is 25.8%. According to localities, seasons, growth years and application portions, we explored the diversity of C. deserticola endophtic fungi.Through analysis of these different samples, there is the same colonization rate and isolation rate if the application portion is consensus. There's no apparent diversity among these portions except for epidermis of sub-stem. We got the least C. deserticola endophtic fungi isolated from blossom and seed. The colonization rate and isolation rate of samples from different regions have no apparent difference yet the sorts and amounts of endophytic fungi are more abundant in YongNing, NingXia province than in DengKou, Inner Mongolia province. On the other hand, the autumn samples get the higher colonization rate, higher isolation rate and more varieties compared with the other samples. There's no apparent difference among the samples of different growth years.3. Amplified by PCR and sequenced with ITS, based on sequence comparability and phylogenetic analysis,26 of those fungi which asporulated were identified. The results showed that 4 strains were identified to 4 species level,14 strains to 7 generic level,6 strains to 2 family level,1 strains to order level and the last to class level. We also identified 26 isolates that can't exactly distinguished by morphological method. The results show that 20 isolates were identified consistent with morphological result,12 of them were identified to 9 species level, and the remaining 8 were to 6 genus level.4. This article studies the effects of pre-conditioning to C. deserticola seed in a systematic way and focuses on the difference of C. deserticola seed with different year unprecedentedly. The results show that the variety of preconditioning substance and preconditioning medium concentration impact C. deserticola seed a lot, however, preconditioning time, preconditioning temperature and pH don't have apparent impact upon seed germination. The germination rate is very different among different aged seeds. Analyzing the relativity of morphology and germination rate suggest that there is a certain degree of positive correlation between germination rate and the dry weight of seed. Those seeds of which the dry weight is 0.030 g-0.039 g have the highest germination rate.5. Describing the culture conditions of C. deserticola seed germination is also a part of this thesis. Seed without stratification were used in experiments, several factors were considered in this segment as seed coat, plant seed germination stimulator, plant growth regulators, PH and the sterilization way. The result indicated that seed coat have a notable impact on seed germination; among various plant growth regulators GA3 have a appreciable influence on seed germination; PH have a appreciable effect on seed germination, in accordance with preconditioning experiment,7.0 is the optimum PH value; through the sterilization experiments, the seeds dealing with NaClO at the time of 5min have the best result. |
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