The Preservation Technology Of Germplasm Of Scytosiphon Lomentaria

Scytosiphon lomentaria (Lyngbye.) Link belongs to Phaeophyta, which is widely grown in the coastal waters from the Baltic Sea to Australia and chile. S. lomentaria is an economic macroalgae that mainly distributes from the north of Liaodong Peninsula to the south of Hailing Island in China. S. lomentaria is decelious in taste, rich in nutrition and health care value, and it has always been enjoyed by coastal residents as a precious marine vegetable. Besides, S. lomentaria has a high value in medicine with the properties of antioxidant, antibacterial, antivirus and antitumor. S. lomentaria is a new kind of economic algae resource with a broad development prospects since it has been proved that S. lomentaria can play a good role on ecological restoration in the aspects of antifouling and removing the excess nutrient elements in seawater.S. lomentaria has a heteromorphic life history which consists of macro gametophytes and tiny sporophytes. As one type of sporophytes, filaments of S. lomentaria are the primary materials to amplify in the laboratory because of the quality of small size, fast breeding and strong tolerance. Filaments of S. lomentaria are the best object for germplasm preservation, because filaments are not only easy to culture and control but able to form unilocular sporangia that release swimming spores, which finally develop into gametophytes of high economic values. In this paper, through the research of liquid preservation, encapsulation-dehydration -20℃ cryopreservation, encapsulation-dehydration cryopreservation and vitrification -20℃ cryopreservation, the effects of various factors in the four preservation methods on the viability and development of filamnets of S. lomentaria were studied. The research will provide theoretical foundation, idea and method for the construction of germplasm bank of S. lomentaria as well as the technique support to the industrial breeding of S. lomentaria.The results of this research were as follows:1. In liquid preservation:(1) temperature was not the key factor to the preservation of filaments of S. lomentaria, the filaments of S. lomentaria could achieve long-term preservation under the condition of 5.4 u mol/(m2·s),6-18℃; (2) The illumination intensity had a significant impact on the liquid preservation of the filaments of 5. lomentaria, the cells of filaments of 5. lomentaria preserved for 60d under any illumination intensity higher than 16.2 u mol/(m2·s) were not in good condition, so high intensity was not conducive to the preservation of the germplasm, 5.4 μmol/(m2·s) was the most helpful condition; (3) After being preserved under 6-12℃,5.4 μol/(m2·s) for 60 days, the cells of the filaments of S. lomentaria were still in good condition and the survival rates were higher than 97%.2. In encapsulation-dehydration cryopreservation, the sucrose concentration, preculture time, water content of the beads, thawing temperature and the recovery time of the beads all had a certain impact on the survival rate of the filaments of S. lomentaria. The results indicated that:(1) The survival rate was highly elevated when filaments of S. lomentaria were precultured with sucrose, the beads of S. lomentaria had highest survival rate after being precultured in 0.4mol/L sucrose for 6 hours; (2) The optimum water content of the beads was relatively low, only about 15%, the survival rate was 0% when the water content was higher than 27%; (3) The survival rates of filaments of S. lomentaria varied with different thawing temperature,40℃ was the most suitable temperature for thawing the beads; (4) After being thawed, the survival rate of S. lomentaria could be improved significantly after recovering in dark for 18 hours; (5) Under the optimal conditions, the survival rate of the filaments of S. lomentaria after being thawed could reach as high as 54.79%, and the recovered filaments were as same as those before being cryopreserved, the induced filaments could produce normal sporangia, and the spores released from the sporangia were able to grow into the thalli.3. In encapsulation-dehydration cryopreservation (-20℃), water content of the beads was the main factor to the survival rates of cryopreserved filaments of S. lomentaria, the viabilities of the filaments of S. lomentaria after encapsulation cryopreservation were reduced whether the water content of the beads was too high or too low,15% was the optimal water content under which the survival rate of the filaments of S. lomentaria preserved one month still could be higher than 50%.4. In vitrification preservation (-20℃), the viabilities of the cryopreserved filaments of S. lomentaria were mainly affected by the loading solutions, loading times, vitrification solutions and vatrification times. (1) Diluted vitrification solutions could not be used as loading solutions for filaments of S. lomentaria, LS5 was the best loading solutions in the vitrification preservation of filaments of S. lomentaria. (2) The highest viability of filaments of S. lomentaria was achieved when filaments were loaded for 30min at room terperature. (3) Different vitrification sulutions had different preserving effects, VS2 was the most suitable vitrification solution for filaments of S. lomentaria in vitrification cryopreservation. (4) The survival rate could be improved greatly when filaments of S. lomentaria were dehydrated for 30min at 0℃. (5) Under the optimal conditions, the survival rate of the cryopreserved filaments of S. lomentaria reached as high as 38.42%, compared with non-cryopreserved filaments, the cryopreserved filaments of S. lomentaria produced fewer sporangial branchlets and fewer spores. However, the spores released by cryopreserved filaments of S. lomentaria showed strong vitality and were able to grow into normal, healthy thalli.