本文選題：萱藻 + 絲狀體��； 參考：《中國海洋大學(xué)》2015年碩士論文

【摘要】：萱藻Scytosiphon lomentaria(Lyngbye.)Link隸屬于褐藻門,是一種廣泛生長于北起波羅的海南至澳大利亞、智利等國沿海海域的大型經(jīng)濟海藻,在我國主要分布在遼東半島至廣東海陵島間的廣大海域。萱藻不但鮮美可口,還具有較高的營養(yǎng)、保健等價值,是長期以來備受沿海居民鐘愛的優(yōu)質(zhì)海洋蔬菜。此外,萱藻藥用價值較高,具有抗氧化、抗細(xì)菌、抗病毒以及抗腫瘤的特性。研究發(fā)現(xiàn),萱藻在除氮磷、抗污等方面也能發(fā)揮較好的生態(tài)修復(fù)作用,是一種具有廣闊開發(fā)前景的新型經(jīng)濟海藻資源。萱藻的生活史為異形世代交替,由大型的葉狀配子體世代以及微小的孢子體世代組成。萱藻絲狀體是孢子體的形式之一,其體積小、繁殖快、耐受力強,是實驗室擴增以及工廠化大規(guī)模育苗的主要材料。萱藻絲狀體不僅易于培養(yǎng)和調(diào)控,還能通過無性生殖形成單室孢子囊并釋放游孢子,游孢子可進一步發(fā)育成具有經(jīng)濟價值的葉狀體,因而萱藻絲狀體是萱藻種質(zhì)保存的最佳對象。對漁業(yè)生產(chǎn)來講,豐富而優(yōu)質(zhì)的種質(zhì)資源是其最為重要的物質(zhì)保障和基礎(chǔ),因而保護、開發(fā)并利用好萱藻的種質(zhì)資源有利于實現(xiàn)萱藻的良種化生產(chǎn)。本論文通過研究液體培養(yǎng)法、包埋脫水超低溫保存法、包埋脫水-20℃低溫保存法以及玻璃化-20℃低溫保存法對萱藻絲狀體種質(zhì)保存的影響,探討了各個種質(zhì)保存方法中不同因素對萱藻絲狀體保存后存活率及后續(xù)生長發(fā)育的影響,為萱藻種質(zhì)庫的構(gòu)建提供了理論依據(jù)、思路和方法,同時也為萱藻的工廠化育苗提供了技術(shù)指導(dǎo)。本文的研究成果如下：1、在液體培養(yǎng)法中：(1)溫度對萱藻絲狀體保存效果的影響并不顯著,當(dāng)光強為5.4μmol/(m2·s)時,在6-18℃條件下均可實現(xiàn)萱藻絲狀體的長期保存；(2)光照強度是影響萱藻絲狀體液體保存的主要因素,當(dāng)光照強度高于16.2μmol/(m2·s)時,萱藻絲狀體細(xì)胞代謝快,保存60d時細(xì)胞因缺乏營養(yǎng)鹽變?yōu)闇\黃色或無色,原生質(zhì)體收縮,可見較高的光照強度不利于萱藻絲狀體的長期保存,5.4μmol/(m2·s)條件下萱藻絲狀體細(xì)胞長勢良好,是萱藻絲狀體液體保存的最佳光強條件；(3)在6-18℃,5.4μmol/(m2·s)條件下,萱藻絲狀體細(xì)胞的代謝及生長速度較慢,用液體培養(yǎng)法保存60d后存活率仍在97%以上,而且萱藻絲狀體細(xì)胞狀態(tài)良好,顏色呈褐色,原生質(zhì)體充盈,可見6-18℃,5.4μol/(m2·s)是萱藻絲狀體液體保存最適宜的條件。2、在包埋脫水超低溫保存法中,蔗糖的預(yù)培養(yǎng)濃度和時間、萱藻絲狀體膠球的含水量、化凍溫度及萱藻絲狀體膠球的恢復(fù)時間均對萱藻絲狀體的存活率有一定影響。其中：(1)通過蔗糖預(yù)培養(yǎng)可顯著提高萱藻絲狀體細(xì)胞的存活率,將萱藻絲狀體膠球在濃度為0.4mol/L的蔗糖溶液中預(yù)培養(yǎng)6h,凍存后萱藻絲狀體的存活率最高；(2)萱藻絲狀體膠球凍存的最適含水量相對較低,約為15%,當(dāng)萱藻絲狀體膠球的含水量高于27%時,凍存后萱藻絲狀體的存活率均為0%；(3)化凍溫度不同,凍存后萱藻絲狀體細(xì)胞的存活率亦有較大的差別,40℃為凍存后萱藻絲狀體膠球的最佳化凍溫度；(4)將化凍的萱藻絲狀體膠球置于黑暗條件下進行恢復(fù),恢復(fù)18h可顯著提細(xì)胞存活率；(5)在最佳的條件下,萱藻絲狀體經(jīng)包埋脫水超低溫保存后的存活率最高可達54.79%,恢復(fù)后的萱藻絲狀體與凍存前的萱藻絲狀體在形態(tài)結(jié)構(gòu)等方面并無區(qū)別,絲狀體經(jīng)誘導(dǎo)后能夠產(chǎn)生正常的孢子囊,由萱藻孢子囊放散的孢子能夠發(fā)育成健康完整的萱藻葉狀體。3、在包埋脫水-20℃低溫保存法中,萱藻絲狀體膠球的含水量是影響萱藻絲狀體凍存后存活率的關(guān)鍵因素,膠球含水量過高或者過低均會導(dǎo)致萱藻絲狀體包埋脫水-20℃低溫保存后存活率的下降,15%是凍存萱藻絲狀體膠球最適宜的含水量,在該條件下,萱藻絲狀體低溫保存30d后的存活率仍在50%以上。4、在玻璃化-20℃低溫保存法中,裝載液、裝載時間、玻璃化液和脫水時間是影響萱藻絲狀體凍存后存活率的主要因素。其中：(1)稀釋的玻璃化液不宜用于萱藻絲狀體細(xì)胞的裝載,LS5是萱藻絲狀體玻璃化-20℃低溫保存的最佳裝載液；(2)裝載時間過長或過短均會對保存效果產(chǎn)生不利影響,萱藻絲狀體在室溫下裝載30min時,其凍存后的存活率最高；(3)萱藻絲狀體用不同的玻璃化液脫水后,細(xì)胞凍存后的存活率顯著不同,若用VS2對萱藻絲狀體脫水,凍存效果最好；(4)在0-60min的脫水過程中,萱藻絲狀體細(xì)胞凍存后的存活率隨脫水時間的延長呈先增大后減小的趨勢,當(dāng)萱藻絲狀體在0℃下脫水30min時,其凍存后的存活率大大提高；(5)在最佳的條件下,萱藻絲狀體經(jīng)玻璃化-20℃低溫保存后的存活率最高可達38.42%,經(jīng)恢復(fù),凍存前、后的萱藻絲狀體基本沒有區(qū)別,雖然凍存后的萱藻絲狀體在孢子囊枝形成比例及孢子放散量方面略低于未凍存的萱藻絲狀體,但是凍存后的絲狀體具有正常的生長發(fā)育和繁殖能力,可以形成完整的萱藻葉狀體。
[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.
【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q949.2

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