Genetic Variation Of Populations, And Technology Of Domestication In Lycoris

Lycoris radiata is special for its value to contain galanthamine, which is the main component of the cure for the alzheimer's disease. Genetic diversity was evaluated among/within 22 wild L. radiata populations naturally distributed in nine provinces by ISSR (inter-simple sequence repeat) markers. The collected L. radiata plants were domesticated by inter-planting with different tree species. With the help of the Grey Relation Analysis, the degree of association between the galanthamine content and the growth characteristics, physiological indices, biomass, and nutrient components was evaluated, along with the relation of the galanthamine content to the micro-environmental factors. The system of vegetative propagation was established to select and breed high-quality L. radiata germplasm. The main results are as follows:(1) A basic research on wild populations indicated that the annual growth rhythm of populations from the southern part of the subtropical zone was earlier than that from the north-central part. The vegetative propagation coefficient and young bulbs mass of populations from the southern part were similarly better than those from the north-central part. The average vegetative propagation coefficient of populations was 5.08, with the young bulb mass of 1.64 g. Comparison of five biological characters of young bulbs revealed that the number of roots varied from 3.42 to 7.98, the height of bulb from 1.27 to 2.01 cm, the mass of bulb from 1.33 to 1.94 g, and the diameter of bulb from 1.03 to 1.23 cm. Three preliminarily bred populations YL16, YL13, and YL22, whose galanthamine content was as high as, if not more than, populations with the content of galanthamine of 300μg.g^-1, are suggested to be exploited as high quality resources.The stable and reproducible reaction system of ISSR-PCR amplification was established for L. radiata, which was: 20 ng template DNA, 1 U Taq DNA polymerase, 2.0 mmol·L^-1 Mg2+, 200μmol·L^-1 dNTPs and 0.5 mmol·L^-1 primer in the 25μL reaction volume. The amplification program consisted of an initial step of 94℃for 300 s for pre-denaturation, 45 cycles of 94℃for 45 s, 55℃for 50s,and 72℃for 120 s,followed by final 1 cycle of 72℃for 420 s for extension. The genetic diversity of 14 L. radiata populations was analyzed by ISSR markers. The results showed that at the species level, the percentage of polymorphism (P) was 92.31%, Shannon's index (h) was 0.4597, and Nei's gene diversity (I) was 0.3025, indicating a high level of genetic diversity. At the population level, however, they were 49.65%, 0.2620, and 0.1763, respectively, suggesting a low level of genetic diversity. The gene differentiation coefficient (Gst) and gene flow among the populations were 0.5035 and 0.6983, respectively. The analysis of molecular variance (AMOVA) demonstrated that there was a relatively high level of genetic variation (46.12%) among populations and a relatively low genetic variation (53.88%) within populations. The high genetic differentiation among populations and the low genetic diversity within populations could be attributed to the habitat fragmentation and the limited gene flow among populations.(2) The trial of domestication and cultivation of L. radiata and other four tree species, including three deciduous species and one evergreen species, was carried out. The differences, such as illumination intensity, air temperature, humidity, soil moisture, and soil nutrient, were remarkably present in the different interplant patterns. The curve of daily variation of illumination intensity under the different inter species was parabolically shaped, and the illumination intensity of the test points of eastward and westward arrived at the culmination on the 11:00 am and 1:00 pm. The curve of space division of daily illumination intensity under the different test points was in an U shape. The curve of daily variation of air temperature under the different inter species was in singlet parabolic shape, and air temperature arrived at the culmination on 1:00 pm. The curve of space division of daily air temperature was as similar as U shaped. The curve of daily variation of humidity under the different inter species was also similar to be U shape. The differences of soil moisture ranked from high to low by species as Elaeocarpus sylvestris (25.004%), Koelreuteria paniculata (20.997%), Bischofia poiycarpa (19.469%), and Alnus cremastogyne (18.796%). In a way, soil nutrients had been improved after cultivating L. radiata, with a range ranking from high to low as rapidly available phosphorus (-23.53¹00%), hydrolysable nitrogen (2.73⁵6.21%), organic matter (6.59⁴1.07%), and rapidly available potassium (2.37³1.62%).Micro-environment had a complicated impact on the growth of L. radiata. Chlorophyll content ofL. radiata's leaf under different inter-planting species displayed continuous changes in different growth season, but different test points from different inter-planting species kept almost stable (1.84².16mg.g^-1) with the ratio of Chlorophyll a/b ofL. radiata's leaf varying from 1.38 to 2.05. The daily variation of PSâ…¡photochemistry (FV/FM,0.793) ofL. radiata's leaf under different inter-planting species remarkably surpassed the data(FV/FM,0.779) under full-exposure. PSâ…¡photochemistry(FV/FM ) ofL. radiata's leaf under cultivating Bischofia poiycarpa, Koelreuteria paniculata, Alnus cremastogyne and Elaeocarpus sylvestris was respectively, 101.54%, 101.71%, 101.71%, and 102.48% higher than that under full-exposure. The new and dry biomass of L. radiata under cultivating Koelreuteria paniculata were the heaviest. The galanthamine content ofL. radiata's bulbs coming from inter-planting species was higher than that coming from full-exposure, and the range of galanthamine content of L. radiata's bulbs from high to low was Elaeocarpus sylvestri(s21.39μg·g^-1), Alnus cremastogyne (16.61μg·g^-1), Bischofia poiycarpa (14.80μg·g^-1), and Koelreuteria paniculata (13.53μg·g^-1). Galanthamine content ofL. radiata's bulbs usually demonstrated two peak periods (97.536μg·g^-1 in July of the dormancy stage, and 51.6343μg·g^-1 in November of the leaf growth stage) and two bottom periods (6.3277μg·g^-1 in April of the initial dormancy stage and 22.9554μg·g^-1 in August after flowering). The galanthamine content ofL. radiata's bulbs ascended from the first year (5.6475μg·g^-1) to the fifth year (116.2253μg·g^-1), with a rapid and stable phase from the third year to the fifth year.By analyzing the grey relation between growth characteristics, physiological index, biomass modular, nutrient component, galanthamine content, and environmental factors, it was found that difference was present in the principal environmental factors affecting these indices, and humidity and soil moisture were the key environmental factors restraining the bulbs growth. Regression analysis indicated that there was a significant linear correlation between leaf area, bulbs'length, root fresh weight, bulbs'fresh weight, bulbs'dry weight, protein, soluble sugar, starch, reducing sugar, and environmental factors at the 5% level. There was a significant linear correlation between leaf fresh weight, leaf dry weight, root dry weight, and galanthamine content in the third year at the 1% level. All regression equations are available. But significant linear correlation between bulb-width, and galanthamine content in the second year could not be constructed at the 5% level. Through analyzing regression among growth characters, it was found that one character usually related to other characters, from which other characters could be speculated.(3) Three field-experiment-schemes on fertilization could increase bulbs fresh and dry biomass. Different kinds of fertilizers could increase the bulb size and affect the bulbs biomass increment, with an effect ranging from good to poor: potassium fertilizer, compound fertilizer, and nitrogen fertilizer; potassium fertilizer, nitrogen fertilizer, and compound fertilizer. An increase in the natural tiller number of bulbs, in addition, was observed after continuously applying potassium fertilizer.In the potting experiment, potassium fertilizer increased root activities, root CEC, and galanthamine content of bulbs, and these showed similar daily variation curves in shape. Compared with control, root activities, root CEC, and galanthamine content of bulbs were improved via increasing potassium application by 0.3 g·kg^-1 or 0.6 g·kg^-1. Root activities, root CEC, and galanthamine content of bulbs rose to a peak of 97.35 ug·g^-1h ^-1, 9.67 cmol·kg^-1, and 276.4054μg·g^-1 when potassium application was increased to 0.9 g·kg^-1. However, these indices mentioned above started to decline when potassium was applied by the dosage of 1.2 g·kg^-1. (4) The natural propagation coefficient of L.radiata bulbs ranged from 2.495 to 2.656. The curve of vegetative propagation coefficient took on two peaks under different development phases, varying from 0.80 to 6.8, In order to get high vegetative propagation coefficient, it is suggested that vegetative propagation should be carried out in June, July, and September. In order to get more massive young bulbs, vegetative propagation should be carried out in September and April. In order to get larger young bulbs, vegetative propagation should be carried out in June, July, September, and April. In order to get good root activities, vegetative propagation should be carried out from March to July, and in September. The number of young bulbs produced varied from in L. chinensis, with the average of vegetative propagation coefficient being 7.09.Embryogenic callus (EC) was induced and differentiated by tissue culture in L. aurea. Embryogenic callus inductivity reached 100% in three months with the treatment of improved MS+BA 5 mg·L^-1+NAA 3 mg·L^-1.Improved MS+BA 1.8 mg·L^-1+KT 0.7 mg·L^-1+NAA 2.5 mg·L^-1+peptone 100 mg·L^-1 was favorable for multiplication of embryogenic callus and for promoting differentiation of somatic embryo. Under this medium the embryogenic callus could be induced from sprouts again. MS+BA 1.5 mg.L^-1+KT 0.5 mg.L^-1+NAA 2 mg.L^-1 +YE 100 mg·L^-1+Sugar 8%, a better medium, could effectively alleviate the problem of seriously vitrifiation as well as sustaining a relatively stable speed of multiplication of embryogenic callus and promotion differentiation of somatic embryo.