| Dendrobium offlcinale Kimura et Migo,a perennial epiphytic herb fromDendrobium, Orchidaceae, mainly distributes in Yunnan, Guangxi, Guangdong, Guizhou, Zhejiang, Sichuan, Jiangxi, Hunan and Taiwan provinces.D. officinaleis one of the famous and precious Chinese medicines, first recorded in Shennong’s herbal as top grade, with the effect of nourishing yin and clearing heat, tonifying stomach and promoting fluid. On account of the polysaccharide, mannitol, alkaloid and other effective components in D. officinale, it has the effects of anti-aging, antioxidant and immune regulation.Since the low rate of seed germination, long growth cycle, coupled with long-term excessive excavation and other reasons, D. officinaleisfacing a situation ofsharp decrease and even extinction.The wide distribution, different growth conditions and complex ofmutation type of D. officinaleleading to various quality and production. In addition, adulterants or counterfeitof D. officinale could be frequently found in the market. Thus, identification and evaluation of D. officinale has become a key issue to ensure its efficacy.Currently, there are a number of studies reported in the identification and evaluation of the quality of D. officinale, but most studies were lacked of representative sample sources and research methods, not enough indexes and many other reasons, a more reliable identification and quality evaluation system is still remain undergoing.In thepresent studies, different variation types of D. officinale wereauthenticatedby using the methods of morphology, microstructure and molecular identification, while analyzing and evaluatingit through the aspects of the biological characteristics, effective ingredients, nutritional ingredients and endophyte abduction efficiency.The main results were as follows:(1) According to the variationsof morphological characteristics,9variation types were separated, which arethe spindle-crude-stem type, purple-rod-red-leave type, green-rod-crude-stem type, long-rod-black-section type, dwarf-rod-black-section type, dwarf-rod-bending-stem type, red-rod-red-leave type and dwarf-rod-crude-stem type. These types were different in stem color, shape, size, node and internode characteristics, leaf color, leaf shape and leaf size, and flower color and so on.(2) The results of macroscopical characteristics analysis revealed thatstem shape, stem color, stem length, stem diameter, internode length, section significant or not, leaf shape, leaf color, leaf length and width, leaf spot, flower color and lip plaque, were shown significantly different among9variation types of D. officinale. Among them, stem shape, stem color, stem size, leaf shape and leaf color, section significant or not were relatively stable features to distinguish different variation types of D. officinale.(3) The microscopic identification results showed that no significant differences were detected between the stem cuticle and the epidermic cells in the variant types, while the skin layer cell diameter, cell layer of subcutaneous layer, fiber diameter, fiber bundle diameter, number, unit area of vascular bundles number, phloem and xylem diameter were quite different. Therefore, the microscopic characteristics were useful for identification.There are some characteristicsdifferences were observed between the two-and three-year-old D. officinale. For example, the biennial ones had less subcutaneous cell layers, less fiber, smaller fiber diameter and smaller vascular diameter compared with three-year-old ones, which indicated that the mechanical tissue and conducting tissue decreased in ratio, while the parenchyma increased in ratio, as the growth of D. officinale.In addition, those variation types with shortand crude stem,purple spotsspread throughoutfleshed and surface of stem (such as TP1, TP8, TP9, etc.)have larger polysaccharide particles diameter than those withvimineous and less purple spots stem (such as TP3, TP5etc.), which are the main characteristics reflecting qualityof D. officinalegermplasm.(4)The analysis of ITS sequences of D. officinale showed that those9variation types could be classified into four categories. The first category included variation type TP3, TP4, TP5, TP6, TP8and TP9, the second category only contained TPl, the third type was TP2, while the fourth type contained only TP7. The ITS sequence BLAST (GenBank) results showed that the homology level of ITS sequence from different variance types was91%-99%. The highly homology ITS sequences can be used as the identification and the index of different variation types of D. officinale.The polymorphism analysis of ITS sequence showed high level of genetic polymorphisms between these different variation types.32variable sites were detected in the ITS1zone,30sites in5.8S zone, and33sites in zone2. This may be closely related to the diversity of natural growth conditions of D. officinale. The results provide an important basis for establishment of the core collection resources of D. officinale in china.(5) From the analysis results of characteristics of D. officinale, the biennial stem was better than three-year-old one, indicating the best harvest time should be the second year and TP7was priority to promote among all the variation types. In addition, the polysaccharide content in the biennial stem was usually higher than that in the three-year-old one, and TP5has the highestpolysaccharide content (45.14%). The mannose content in the biennial stem was usually higher than that in the three-year-old one, and TP6was the highest (30.42%). The dendrobine content in the biennial stem was usually higher than that in the three-year-old one, and TP3was the highest (0.087%). The crude fiber content in the biennial stem was usually higher than that in the three-year-old one, and TP1was the highest (15.00%). The protein content in the biennial stem was usually higher than that in the three-year-old one, and TP3was the highest (1.12g·100g-1). The amino acid content in the biennial stem was usually higher than that in the three-year-old one, and TP1was the highest (34.15mg·g-1). In all, the best harvest time should be the second year, the variation types of TP1, TP3, TP5, TP6and TP7had better developing prospects.(6)580strains of27types of endophytic fungi were isolated and purified from9variation types. The morphological and molecular identification results indicated that these27types of endophytic fungi belonged to9genus, mainly attributable to the Deuteromycotina and Ascomycotina.18strains were selected and induced, and the same endophytic fungi were re-isolated from roots and leaves of the test-tube plantlet, while no fungus was isolated from the control group. The results indicated a close symbiotic relationship between the18strains. Only5species of fungi had effect on the growth and development of the herb and the synthesis and accumulation of the effective components in the18strains, the L6-226strain promote effectively the grouth and polysaccharide content of test-tube plantlet of D. officinale.The polysaccharide and endophytic fungi species have not been found to have significant correlation, while the polysaccharide, mannose and amino acid has certain relevance. So synthesis or accumulation of polysaccharide is likely related to the amino acids.In conclusion, this study through a variety of evaluation methods of comprehensive application, optimize the integration of the advantages of various identification methods, make up the lack of a single identification method, and set up the comprehensive identification method of different variation types of D. officinale. Meanwhile, The quality evaluationsystem of the germplasm resources of D. officinalewere estabalished based on the index of biomass, polysaccharide, mannose, crude fiber, alkaloids, proteins and amino acids for the quality control ofD. officinale.
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