Effect and optimization of cryopreservation on vegetative cells and akinete of Haematococcus pluvialis Flotow FACHB-712
-
摘要: 以高产虾青素的雨生红球藻（Haematococcus pluvialis Flotow）FACHB-712藻株为材料，研究2种细胞形态（营养细胞和厚壁孢子）在低温保藏下的复苏率及其差异原因。结果显示，采用两步法（先预冻降温后再投入液氮中）冻存其营养细胞，在不同冻存条件下，其存活率均低于5%，以10%甘油作为保护剂、冻存速率为0.5℃/min、预冻温度为-40℃、保留30 min，然后再投入液氮罐（-196℃）中保藏，其存活率可达到13.3%。采用两步法冻存厚壁孢子，其复苏存活率高达66.13%，复苏萌发后细胞的生长特性、虾青素含量与液氮保藏前无明显差异（P > 0.05）。对液氮保藏前后藻细胞形态和超微结构观察结果表明，超低温保藏后，营养细胞的结构受到较大损伤，而厚壁孢子受到的损伤相对较小。当添加不同保护剂后，直接将厚壁孢子分别冻存在-20℃、-80℃低温及液氮中，发现-80℃低温冻存处理组的复苏存活率相对较高，可达27%。研究表明采用两步法先预冻降温后再投入液氮中冻存厚壁孢子，是长期保藏雨生红球藻FACHB-712的最佳方法，也可采用一步法将厚壁孢子冻存于-80℃冰箱中。Abstract: The survival rate of cryopreservation based on vegetative cells and akinetes of Haematococcus pluvialis Flotow FACHB-712 were compared. Results showed that in most cases the survival rate of the vegetative cells was lower than 5% in liquid nitrogen with two-step cryopreservation, but the rate increased to 13.3% if the protocol was modified as follows:cell suspensions were added with glycerol as a cryoprotectant (10%), cooled at a rate of 0.5℃/min until -40℃, and then maintained for 30 min before being placed to liquid nitrogen. The survival rate of the akinetes reached 66.13% in liquid nitrogen with the two-step protocol. There were no obvious differences (P > 0.05) in the growth characteristics and the astaxanthin content of akinetes before and after preservation in liquid nitrogen. Comparatively, the ultrastructure of the vegetative cells was seriously damaged, whereas that of the akinetes was much less damaged after two-step cryopreservation treatment. If directly placed in -20℃, -80℃, or liquid nitrogen, respectively, the highest survival rate of the samples was about 27% in the treatment under -80℃. In conclusion, the two-step cryopreservation protocol in liquid nitrogen using akinete-stage cells was preferable for the long-term preservation of H. pluvialis FACHB-712, with the one-step protocol at -80℃ also applicable as an alternative approach.
-
Keywords:
- Haematococcus pluvialis Flotow /
- Cryopreservation /
- Survival rate /
- Akinete /
- Vegetative cells
-
-
[1] 胡鸿钧, 魏印心. 中国淡水藻类——系统、分类及生态[M]. 北京:科学出版社, 2006. [2] 耿金峰,张惠敏,杨建强,马欣欣,石蕾,滕杰,冯倩,张凯. 雨生红球藻中虾青素含量的快速测定方法[J].食品研究与开发, 2016, 37(12):125-128. Geng JF,Zhang HM,Yang JQ,Ma XX,Shi L,Teng J,Feng Q,Zhang K. A method for rapid determination of astaxanthin from Haematococcus pluvialis[J]. Food Res Dev, 2016, 37(12):125-128.
[3] 潘克厚, 朱葆华. 微藻的保种技术及其应用[J]. 青岛海洋大学学报:自然科学版, 2002, 32(3):403-408. Pan KH, Zhu BH. Storage techniques of microalgae and their applications[J]. Journal of Ocean University of Qingdao:Natural Science Edition, 2002, 32(3):403-408.
[4] 洪森荣,尹明华,王艾平. 江西铅山红芽芽胚性愈伤组织的包埋玻璃化超低温保存[J]. 植物科学学报,2014, 32(1):80-87. Hong SR, Yin MH, Wang AP. Cryopreservation of Jiangxi Yanshan red bud taro embryogenic calli by encapsulation-vitrification[J]. Plant Science Journal, 2014, 32(1):80-87.
[5] Holm-Hansen O. Viability of blue-green and green algae after freezing[J]. Physiol Plantarum, 1963, 16(3):530-540.
[6] Friedl T, Lorenz M. The culture collection of algae at Got-tingen University (SAG):a biological resource for biotechnological and biodiversity research[J]. Procedia Environ Sci, 2012, 15:110-117.
[7] Rhodes L, Smith KF, MacKenzie L, et al. The cawthron institute culture collection of micro-algae:a significant national collection[J]. New Zeal J Mar Fresh, 2016, 50(2):291-316.
[8] Choi YH, Nam TJ, Kuwano K. Cryopreservation of gametophytic thalli of Porphyra yezoensis (Rhodophyceae) by one-step fast cooling[J]. J Appl Phycol, 2013, 25(2):531-535.
[9] Day JG. Cryopreservation of microalgae and cyanobacteria[J]. Methods Mol Biol, 2007, 368:141-151.
[10] Macdonald SM, Lee RW. A survey of Polytomella (Chlorophyceae, Chlorophyta) strains in public culture collections[J]. J Phycol, 2016, 52(4):656-663.
[11] Lee YN, Nam KW. Cryopreservation of gametophytic thalli of Ulva prolifera (Ulvales, Chlorophyta) from Korea[J]. J Appl Phycol, 2016,28(2):1207-1213.
[12] Rhodes L, Smith J, Tervit R, et al. Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae[J]. Cryobiology, 2006, 52(1):152-156.
[13] Chong G, Tsai S, Wang LH, et al. Cryopreservation of the gorgonian endosymbiont Symbiodinium[J]. Sci Rep-UK, 2016, 6:18816.
[14] Robert A. Andersen Algal Culturing Techniques[M]. Bur-lington:Elsevier Academic Press, 2005:165-187.
[15] Mori F, Erata M, Watanabe MM. Cryopreservation of cyanobacteria and green algae in the NIES-collection[J]. Microbiol Cult Coll, 2002, 18(1):45-55.
[16] Canavate JP, Lubinn LM. Some aspects on the cryopreservation of microalgae used as food for marine species[J]. Aquaculture, 1995, 136(95):277-290.
[17] Boussiba S, Vonshak A. Astaxanthin acculation in the green alga Haematococcus pluvialis[J]. Plant Cell Physiol, 1991, 32(7):1077-1082.
[18] Morris GJ. The cryopreservation of Chlorella. 2. Effect of growth temperature on freezing tolerance[J]. Arch Microbiol, 1976b,107(3):309-312.
[19] Morris GJ. The cryopreservation of Chlorella. 1. Interactions of rate of cooling protective additive and warming rate[J]. Arch Microbiol, 1976a, 107(1):57-62.
[20] Joseph I. Panigrahi A, Chandra PK. Tolerance of three marine microalgae to cryoprotectants dimethyl sulfoxide, methanol and glycerol[J]. Indian J Geo-Mar Sci, 2000, 29(3):243-247.
[21] Hubálek Z. Protectants used in the cryopreservation of microorganisms[J]. Cryobiology, 2003, 46(3):205-229.
[22] Santarius KA. Freezing of Isolated thylakoid membranes in complex media:X. Interactions among various low mole-cular weight cryoprotectants[J]. Cryobiology, 1996, 33(1):118-126.
[23] Motham M, Peerapornpisal Y, Tongsriri S, Vacharapiyasophon P, Pumas C. High subzero temperature preservation of Spirulina platensis (Arthrospira fusiformis) and its ultrastucture[J]. Chiang Mai J Sci, 2012, 39(4):554-561.
计量
- 文章访问数: 1221
- HTML全文浏览量: 5
- PDF下载量: 851
下载:
