萱藻（Scytosiphon lomentaria）的种质保存技术研究

发布时间：2018-06-02 05:26

本文选题：萱藻 + 丝状体　；参考：《中国海洋大学》2015年硕士论文

【摘要】：萱藻Scytosiphon lomentaria(Lyngbye.)Link隶属于褐藻门,是一种广泛生长于北起波罗的海南至澳大利亚、智利等国沿海海域的大型经济海藻,在我国主要分布在辽东半岛至广东海陵岛间的广大海域。萱藻不但鲜美可口,还具有较高的营养、保健等价值,是长期以来备受沿海居民钟爱的优质海洋蔬菜。此外,萱藻药用价值较高,具有抗氧化、抗细菌、抗病毒以及抗肿瘤的特性。研究发现,萱藻在除氮磷、抗污等方面也能发挥较好的生态修复作用,是一种具有广阔开发前景的新型经济海藻资源。萱藻的生活史为异形世代交替,由大型的叶状配子体世代以及微小的孢子体世代组成。萱藻丝状体是孢子体的形式之一,其体积小、繁殖快、耐受力强,是实验室扩增以及工厂化大规模育苗的主要材料。萱藻丝状体不仅易于培养和调控,还能通过无性生殖形成单室孢子囊并释放游孢子,游孢子可进一步发育成具有经济价值的叶状体,因而萱藻丝状体是萱藻种质保存的最佳对象。对渔业生产来讲,丰富而优质的种质资源是其最为重要的物质保障和基础,因而保护、开发并利用好萱藻的种质资源有利于实现萱藻的良种化生产。本论文通过研究液体培养法、包埋脱水超低温保存法、包埋脱水-20℃低温保存法以及玻璃化-20℃低温保存法对萱藻丝状体种质保存的影响,探讨了各个种质保存方法中不同因素对萱藻丝状体保存后存活率及后续生长发育的影响,为萱藻种质库的构建提供了理论依据、思路和方法,同时也为萱藻的工厂化育苗提供了技术指导。本文的研究成果如下：1、在液体培养法中：(1)温度对萱藻丝状体保存效果的影响并不显著,当光强为5.4μmol/(m2·s)时,在6-18℃条件下均可实现萱藻丝状体的长期保存；(2)光照强度是影响萱藻丝状体液体保存的主要因素,当光照强度高于16.2μmol/(m2·s)时,萱藻丝状体细胞代谢快,保存60d时细胞因缺乏营养盐变为浅黄色或无色,原生质体收缩,可见较高的光照强度不利于萱藻丝状体的长期保存,5.4μmol/(m2·s)条件下萱藻丝状体细胞长势良好,是萱藻丝状体液体保存的最佳光强条件；(3)在6-18℃,5.4μmol/(m2·s)条件下,萱藻丝状体细胞的代谢及生长速度较慢,用液体培养法保存60d后存活率仍在97%以上,而且萱藻丝状体细胞状态良好,颜色呈褐色,原生质体充盈,可见6-18℃,5.4μol/(m2·s)是萱藻丝状体液体保存最适宜的条件。2、在包埋脱水超低温保存法中,蔗糖的预培养浓度和时间、萱藻丝状体胶球的含水量、化冻温度及萱藻丝状体胶球的恢复时间均对萱藻丝状体的存活率有一定影响。其中：(1)通过蔗糖预培养可显著提高萱藻丝状体细胞的存活率,将萱藻丝状体胶球在浓度为0.4mol/L的蔗糖溶液中预培养6h,冻存后萱藻丝状体的存活率最高；(2)萱藻丝状体胶球冻存的最适含水量相对较低,约为15%,当萱藻丝状体胶球的含水量高于27%时,冻存后萱藻丝状体的存活率均为0%；(3)化冻温度不同,冻存后萱藻丝状体细胞的存活率亦有较大的差别,40℃为冻存后萱藻丝状体胶球的最佳化冻温度；(4)将化冻的萱藻丝状体胶球置于黑暗条件下进行恢复,恢复18h可显著提细胞存活率；(5)在最佳的条件下,萱藻丝状体经包埋脱水超低温保存后的存活率最高可达54.79%,恢复后的萱藻丝状体与冻存前的萱藻丝状体在形态结构等方面并无区别,丝状体经诱导后能够产生正常的孢子囊,由萱藻孢子囊放散的孢子能够发育成健康完整的萱藻叶状体。3、在包埋脱水-20℃低温保存法中,萱藻丝状体胶球的含水量是影响萱藻丝状体冻存后存活率的关键因素,胶球含水量过高或者过低均会导致萱藻丝状体包埋脱水-20℃低温保存后存活率的下降,15%是冻存萱藻丝状体胶球最适宜的含水量,在该条件下,萱藻丝状体低温保存30d后的存活率仍在50%以上。4、在玻璃化-20℃低温保存法中,装载液、装载时间、玻璃化液和脱水时间是影响萱藻丝状体冻存后存活率的主要因素。其中：(1)稀释的玻璃化液不宜用于萱藻丝状体细胞的装载,LS5是萱藻丝状体玻璃化-20℃低温保存的最佳装载液；(2)装载时间过长或过短均会对保存效果产生不利影响,萱藻丝状体在室温下装载30min时,其冻存后的存活率最高；(3)萱藻丝状体用不同的玻璃化液脱水后,细胞冻存后的存活率显著不同,若用VS2对萱藻丝状体脱水,冻存效果最好；(4)在0-60min的脱水过程中,萱藻丝状体细胞冻存后的存活率随脱水时间的延长呈先增大后减小的趋势,当萱藻丝状体在0℃下脱水30min时,其冻存后的存活率大大提高；(5)在最佳的条件下,萱藻丝状体经玻璃化-20℃低温保存后的存活率最高可达38.42%,经恢复,冻存前、后的萱藻丝状体基本没有区别,虽然冻存后的萱藻丝状体在孢子囊枝形成比例及孢子放散量方面略低于未冻存的萱藻丝状体,但是冻存后的丝状体具有正常的生长发育和繁殖能力,可以形成完整的萱藻叶状体。
[Abstract]:Hemerocallis Scytosiphon lomentaria (Lyngbye.) Link belongs to the alga gate. It is a large economic seaweed widely grown in the coastal waters of the northern Baltic Hainan to Australia, Chile and other coastal waters. It is mainly distributed in the vast sea between Liaodong Peninsula and Hailing Island of Guangdong in our country. In addition, the medicinal value of Hemerocallis Hemerocallis is high, and it has the characteristics of antioxidation, anti bacteria, antivirus and antitumor. It has been found that Hemerocallis can also play a better ecological restoration effect in the aspects of nitrogen and phosphorus removal and anti pollution. It is a new economy with broad prospects for development. The life history of Hemerocallis Hemerocallis is a heteromorphic generation, which consists of a large leaf gametophyte generation and a small sporophyte generation. The Hemerocallis Hemerocallis filamentum is one of the forms of the sporophyte. It is small in size, fast in breeding and strong in tolerance. It is the main material for laboratory amplification and large-scale plant breeding. The Hemerocallis Hemerocallis filamentous filaments are not only easy to cultivate. It can also form single chamber spores and release spores through asexual reproduction. The spores can be further developed into the phyllodes with economic value. Therefore, Hemerocallis is the best object for the preservation of Hemerocallis Hemerocallis. For fishery production, rich and high quality germplasm resources are the most important material guarantee and basis for the fishery. To protect, develop and utilize the germplasm resources of Hemerocallis Hemerocallis, the effect of liquid culture, entrapment dehydration, cryopreservation, cryopreservation and cryopreservation of -20 C, and cryopreservation of vitreous -20 C on the preservation of the seed quality of Hemerocallis hyalina were discussed. The effect of the same factors on the survival rate and subsequent growth of Hemerocallis Hemerocallis filamentus provided a theoretical basis for the construction of Hemerocallis Hemerocallis germ plasm bank, and also provided technical guidance for the plant cultivation of Hemerocallis Hemerocallis. The results of this paper are as follows: 1, in liquid culture method: (1) the preservation effect of temperature on Hemerocallis Hemerocallis filamentous filaments The effect was not significant. When the light intensity was 5.4 mol/ (m2. S), the long-term preservation of the filamentous filamentous body of Hemerocallis Hemerocallis was achieved at 6-18. (2) the intensity of light was the main factor affecting the liquid preservation of the filamentous body of Hemerocallis Hemerocallis. When the light intensity was higher than 16.2 mu (m2. S), the metabolism of the silk cells of the Hemerocallis Hemerocallis was fast, and the cell was changed to the deficiency of the nutrient salt when the 60d was preserved. Light yellow or colorless, protoplast contraction, higher light intensity is not favorable for long term preservation of Hemerocallis filamentum. Under the condition of 5.4 mol/ (m2. S), the filamentous cell of Hemerocallis Hemerocallis is the best light intensity condition. (3) the metabolism and growth of Hemerocallis Hemerocallis filamentous somatic cells under the condition of 6-18 and 5.4 mol/ (m2. S). The survival rate of 60d is still more than 97% after the liquid culture method is used to preserve the filamentous body of Hemerocallis Hemerocallis, the color is brown, the protoplast is filled, the 6-18 C is 6-18, and 5.4 mu (m2. S) is the most suitable condition for the liquid preservation of Hemerocallis Hemerocallis filamentum.2. In the cryopreservation method, the pre culture concentration and time of sucrose are the day, Hemerocallis Hemerocallis The water content of the filamentous filamentous ball, the freezing temperature and the recovery time of the Hemerocallis filamentous colloid have a certain effect on the survival rate of the Hemerocallis Hemerocallis filamentous filamentous body. (1) the survival rate of Hemerocallis Hemerocallis filamentous cells can be significantly increased by sucrose pre culture, and the Hemerocallis Hemerocallis filamentous colloid was pre cultured for 6h in the sucrose solution of 0.4mol/L, and after the freezing of Hemerocallis Hemerocallis The survival rate of the algal filamentous body was the highest; (2) the optimum water content in the freezing of Hemerocallis filamentous colloid was relatively low, about 15%. The survival rate of Hemerocallis Hemerocallis filamentous filaments was 0% when the water content of Hemerocallis Hemerocallis filamentous colloid was higher than 27%. (3) the freezing temperature was different, and the survival rate of the Hemerocallis Hemerocallis filamentous cells was also larger than that of the frozen Hemerocallis Hemerocallis. 40 degrees centigrade was frozen. The best cryopreservation temperature of Hemerocallis Hemerocallis filamentous colloid; (4) to restore the frozen Hemerocallis filamentous colloid under dark conditions, and to restore the survival rate of 18h significantly. (5) under the optimum conditions, the survival rate of Hemerocallis Hemerocallis filamentous filamentous is up to 54.79% after the storage and dehydration at ultra-low temperature. After the recovery of Hemerocallis Hemerocallis, the filamentous body and freezing of Hemerocallis Hemerocallis There is no difference in morphological structure between the pre stored Hemerocallis Hemerocallis filamentum, and the filamentous body can produce normal spores after induction. The spores scattered by the spores of Hemerocallis Hemerocallis can develop into a healthy and complete Hemerocallis leaf body.3. The water content of Hemerocallis Hemerocallis filamentous filamentous ball is the influence of the freezing of the filamentous body of Hemerocallis Hemerocallis in the storage and dehydration -20 C cryopreservation. The key factor of post survival rate, the high or low water content of the colloid ball will lead to the decrease of the survival rate after the cryopreservation of Hemerocallis Hemerocallis filamentous dehydration at -20 C. 15% is the most suitable water content of the frozen Hemerocallis filamentous filamentous colloid ball. Under this condition, the survival rate of the Hemerocallis Hemerocallis filamentous filamentous body is still above 50%.4 after the cryopreservation of the filamentous body at low temperature, and is low temperature at -20 C in vitrification. In the preservation method, the main factors affecting the survival rate after the cryopreservation of Hemerocallis Hemerocallis were loading liquid, loading time, vitrification liquid and dehydration time. (1) the diluted vitreous solution was not suitable for the loading of Hemerocallis Hemerocallis filamentous cells, and LS5 was the best loading liquid at -20 C for the vitrification of Hemerocallis, and (2) long or too short loading time The survival rate of Hemerocallis Hemerocallis filamentous body after storage at room temperature is the highest. (3) after dehydration of Hemerocallis Hemerocallis filamentous filaments after dehydration with different vitreous fluids, the survival rate of cells after cryopreservation is significantly different. If VS2 is dehydrated with Hemerocallis Hemerocallis filamentous body, the effect is best; (4) in the process of dehydration of 0-60min, Hemerocallis Hemerocallis is the process of Hemerocallis Hemerocallis. The survival rate of the filamentous cells increased first and then decreased with the prolongation of the dehydration time. When the filamentous filamentous body was dehydrated at 0, the survival rate was greatly improved. (5) under the optimum conditions, the survival rate of the filamentous filamentous filamentous body was up to 38.42% after the cryopreservation of the vitreous -20 centigrade, which was restored and stored before the cryopreservation. The filamentous filamentous body of Hemerocallis Hemerocallis was basically no different, although the proportion of the spore bursa branch formation and the spore release of the Hemerocallis Hemerocallis were slightly lower than the unfrozen Hemerocallis filamentum, but the frozen filamentous filamentous body had normal growth and reproductive ability, which could form a complete leaf like body of Hemerocallis.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q949.2

【相似文献】