| Changium smyrnioides is a endangered perennial herbaceous species that is the sole representative of the monotypic genus Changium in Apiaceae endemic to Eastern China which especially distributes in the northwestern of ZheJiang province, the southwestern of JiangSu province, and the southeastern of AnHui province, and was filed as a third-class state protected plant species in 1984. Its root is one of rare and valuable herbal medicines named Radix Changii. Artificial cultivation is an effective means for protecting the resource and satisfing the social need of Radix Changii, however the quality and productivity reduces of radix changii due to the regression of resource and the limited techniques, then benefit becomes lower and production ceases. This study collected and sorted 10 populations of resources by investigating wild resources in three provinces that are ZheJiang, JiangSu, and AnHui, and systematically analyzed plant morphous, pollen morphous, bionomics, anti-stress physiological phenomena, chemical constitutes, and genetic diversity to integratively evaluate the resources of C. smyrnioides, and analyzed the dynamic state and the distribution of the beneficial and toxic compositions in plant to acquire the mechanism of quality and productivity of C. smyrnioides, and at last, by experiments of germination, fertilization in field and sunshade treatment, researched the seed traits, fertilizer application pattern, and the needed intensity of light to form a reasonable measure of seedling breeding, fertilizer government, and light control. The main contents and results of this study are as follows:1. Basic biological study on the germplasm of C. smyrnioides10 populations of C. smyrnioides were analysed the ecological environment of communities, phenophase, plant morphous traits and investigated the current situation in JiangSu, ZheJiang, and AnHui that include nine wild populations and one cultural population. The results indicate wild resources gradually decrease even to extinction owing to population aging, and the phenopases of wild populations was 20～30d later than this of the cultivar but the annual growing period of wild populations is longer, and morphouses diversify and there exist 3 categories respectively basing on delamination and root shape, which can act as the basis of resource assessment because of these stable traits.By optical microscope and scanning electron microscope, compared the traits of pollens of 10 populations, and discussed the polymorphism on pollens. The results indicate each population pollen has distinct differences from others in size, the biggest pollen is in Hongshan population with 21.99μmx15.51μm, its size index SQRT(P/E) is 18.46, the smallest is in Jiuhuashan population, and each population pollen’s P/E is bigger than 1, especially that of Dalongshan population’s pollen is biggest and long and narrow, and that of Nangaofeng population’s pollen is smallest and broad, and the ornamentation of pollen differs during each population, the similarity of which is not relevant to geographic locations of populations, and the typical feature is perthitic texture, and each population is diverse on whether it has gap or not and whether the texture is deep or shallow, and originally finds that Hongshan population pollen has 2 variants which is tetracolporites and partial wrinkle of surface ornamentation respectively.The physiology and biochemistry and photosynthetic characteristics of 10 populations under the conditions of high temperature and high light was analyzed to evaluation the stress resistance of the germplasm indirectly. The results showed that there were significant differences of the physiological indxs among different populations. By evaluating synthetically, the two populations of Zijinshan and Maoshan showed a stronger stress resistance and stress resistance on the three populations of Qinglongshan, Nangaofeng and Dalongshan was lower.2. Comparison of chemical constituents among populations of C. smyrnioidesTo evaluate inner quality on the germplasm, water-soluble constituent, polysaccharide, mannitol, bilineurine in the roots, and volatile oil in the leaves were analyzed. The results showed that the difference of constituent content and proportion among populations was significant. The content of water-soluble constituent in Juhuashan was highest, which in Dlongshan and Fushan population were lower than Chinese Pharmacopeia standarded. There were obvious differences in the HPLC chromatograms and there were a few common peaks. The water-soluble constituent showed abundant diversities. The main components of the volatile oil in the leaves are fenchyl acetate and aromadendrene by GC-MS. There is much difference among the 10 populations on total number of peaks:Hongshan population has the least, less than half of the population of Nangaofeng and Maoshan. The content of polysaccharide and bilineurine in Qinglongshan is the highest. The content of Mannitol in Dalongshan is the highest. The amount of three active components are relatively high in the population of Zijinshan, Qinglongshan, Dalongshan and Nangaofeng, so they can be used as Germplasm Resources of relatively good quality in combination with other indicators of germplasm resources to carry out comprehensive evaluation. The constituent proportion of water-soluble constituent in the roots and volatile oil in the leaves of Nangaofeng population was most equilibrium, and can be used as reference population to evaluate germplasm by chromatography. By evaluating synthetically, inner quality was better that was of the three populations of Zijinshan, Qinglongshan and Nangaofeng.3. Molecular markers for genetic diversity of C. smyrnioidesAnalysed genetic diversity of C. smyrnioides by RAPD、ISSR and SRAP. The result showed that has much polymorphism among different populations, germplasm has abundant materiel basis to choose, genetic relationship and geographical distribution have some correlation in different populations. PPL of RAPD is 83.01%, Ht is 0.2858, similarity coefficient in different populations is from 0.5026 to 0.8301, Zijingshan and Qinglongshan have higher similar. PPL of ISSR is 83.03%, Ht is 0.2806, similarity coefficient in different populations is from 0.5494 to 0.8150, Fushan and Hongshan have higher similar. PPL of SRAP is 86.50%, Ht is 0.3254, similarity coefficient in different populations is from 0.4959 to 0.8282, Zijingshan and Qinglongshan have higher similar. The location of Maoshan population is instable in three clusting map. By Mantel testing, the correlation between each other for the similarity coefficient of the three molecule marker technology are all over 0.8, the matching ability is good, the results have higher conformance.4. Study on the formation of quality and output in C. smyrnioidesTo analyze the rule of accumulation and distribution of mineral elements in C. smyrnioides, content of 22 different mineral elements in the soil and the plant was detected by ICP-AES. The results show that soil trace elements in main distribution area were high, but two constant elements P and K were on the little side; the enrichment capacity to Se, P and k of root and to Se, P, Mg, Ca and K of stem and leaf were strong, the bioaccumulation capacity of stem and leaf was better than root, the mineral elements contents in different parts of root have significant difference, the content had creasing trends from inner to outer in horizontal position and from upper to lower in vertical position, the content difference of horizontal positions was more than that of vertical positions. As was significant correlation to Pb, Mn, Na. K was significant correlation to Cd, Ni, V, Ti. Cu was significant correlation to Se and Hg. Zn was significant correlation to Cr. Ba was respectively significant correlation to Ca, K. Mo was respectively significant correlation to Ni. Fe was respectively significant correlation to Cu.To explore formation mechanism of quality and output, analyzed dynamic changes of dry matter accumulation and active ingredients in the root that was in different growth phase of C. smyrnioides by HPLC, GC-MS and spectrophotometer. The results showed that the content of active ingredients in the root were significantly different from each other. The content of mannitol, mineral elements and polysaccharides in adult strain was higher than young one, but the content of water-soluble compounds and bilineurine in young strain were higher than adult one. The kinds of water-soluble compounds in young strain were higher than adult one. The main components were increasingly prominent with growth of strain, the total amount and kinds of volatile oil in the leaf declined in growth. The contents of dry matter accumulation and polysaccharides in entire production cycle raised by "S" curve. Yield mainly formed in the second year after transplanting. The amount of dry matter and polysaccharides was highest in June and lowest in December within one year. The quality and output were mainly formed by photosynthesis that was before weathering in June. The optimal harvest time was the later of June.Detected the distribution discipline of active component in C. smyrnioides by HPLC and spectrophotometry. The result showed that the active component was significantly different among different parts. The content of polysaccharide and bilineurine in the root was more than those in the leaf, particularly, the content of bilineurine in the root was 2.01 times than those in the leaf. However, the content of water-soluble extracts and mineral elements in the leaf was more than that in the root, and the mineral elements in the leaf was 3.09 times than that in the root. The content of mineral elements in the epidermis of root was 4.91 times than that in vascular cylinder. The total peak area of HPLC of water-soluble constituents in the leaf was 2.81 times than that in the root, but the number of total peaks in the root was significantly larger than that in the leaf. The total peak area of water-soluble constituents in vascular cylinder was 2.74 times than that in the root and 2.30 times than that in the epidermis. The content of active component had a growing decrease trends form upper to lower in the longitudinal of root. Meanwhile, the content of mannitol, polysaccharide and water-soluble extracts had a growing increase trends and the mineral elements had a totally oppositive trends form outer to inner in the horizontal of root. Furthermore, the difference among the departments in horizontality was more than that among the departments in longitudinal. The kinds of water-soluble components in vascular cylinder and cortex were similar, but the proportion of components in cortex and epidermis were similar, total peak area descended from inner to outer.To analyze the effect on growth of C. smyrnioides that was in different light transmissivity, detected the physiology and biochemistry and photosynthetic characteristics by photosynthesis-instrument and spectrophotometry. The results showed that protective enzyme and PAL activities were increased with light transmissivity. SOD activity in the leaf that was in total sunlight was 3.01 times than that in 10% sunlight. The content of so lube sugar, praline, vitamin C and MDA also increased with light transmissivity. The content of praline in the leaf that was in total sunlight was 3.25 times than that in 10% sunlight. The content of chlorophyll in the leaf that was in total sunlight was lowest, but the value of Chla/Chlb was highest. Pn in the leaf that was in 60% sunlight was highest, the capacity of photosynthesis was best, that in the leaf that was in 10% and total sunlight were worse. The best light transmissivity for growth was about 60% sunlight.To analyze the accumulation rule of pollution matter, residuals of pesticides and heavy metals in C. smyrnioides cultivated and its base soil were detected by GC, ICP and AFS. The results showed organochlorine residuals in Radix Changii was existed in the form of y-HCH, and the content of pollution matter in the epidermis was highest.5. Study on cultivation control of quality and yield of C. smyrnioidesStudied seed harvest, seed storage, pre-treatment of germination, germination condiontion, seeding selection and other propagation techniques of C. smyrnioides and bulit a standard for the seeding quality. The result showed that the appropriate storage temperature ranged from 4 to 15℃, and the storage period is about 5 months. The optimum temperature of wet sand stratification pretreatment was 5℃ and stratification time was 45 days. The optimum germination temperature ranged from 10 to 15℃. The thousand-seed weight of high quality seed ranged from 3.8 to 5.3 g. fresh weight of root on high quality seeding should be more than 0.3 g.To make suitable plan of fertilizer application, studied mixture ratio of fertilizer, fertilization time, quantity of fertilizer, fertilization depth and fertilization times. The results showed the best fertilizer element is phosphorus. The strategy of balanced fertilization on C. smyrnioides is attach importance to phosphorus, make-up potassium, controlling nitrogen, the best fertilization time is between the last third of November and the last third of January in the winter, the optimum quantity of fertilizer were 20～30 kg N(total),20-30 kg K2O, 20～35 kg P2O5 per 667m2, separating 2 or 3 times for application, and the suitable depth was 10～15cm.To analyze effect on quality and yield of C. smyrnioides with different light transmissivity, active constituent in the roots was examined By HPLC, and morphology and weight of root were detected. The results showed the shaded was benefit for thickening vascular cylinder. Both quality and yield of Radix Changii were increased, the best light transmissivity was 60% of total, and the yield increased almost doubled. Furthermore, the proportion of active constituent in the roots were the most equable. The content of active constituent was highest that is in the root of C. smyrnioides with the condition of 30% light transmissivity. With the increase in light intensity, the content and sort of water-soluble components decreases. The most suitable light intensity was 30% ～ 60% light transmissivity so that it was indicated to make a reasonable farming system. |
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