藻类种质超低温保存研究概况

藻类种质的超低温保存技术已受到广泛的重视。目前已对数千种、株的淡水和海水藻类进行过超低温保存。其中,绝大多数藻类是采用两步冰冻法保存。影响藻类存活的主要因素是两步法冰冻保存程序和藻类自身的抗冻性。鉴定存活率是超低温保存技术中的重要环节。由于两步法保存技术的局限性,玻璃化和包埋脱水法等新技术在某些藻类的种质保存中可能有较大的应用潜力。     

冰冻保存小新月菱形藻的包埋-玻璃化法

采用包埋-玻璃化法对小新月菱形藻进行冰冻保存,探讨玻璃化溶液(PVS)配方、装载液浓度和装载时间、脱水时间以及洗涤方法对冰冻保存存活率的影响。结果表明:小新月菱形藻在0℃预冷后50%PVS2装载60min,100%PVS2脱水60min,1mol·L-1蔗糖梯度洗涤30min的条件下存活率最高,为74.1%。包埋-玻璃化法不需要特殊的冷冻设备,冰冻程序操作简单,在藻类种质的超低温保存中有较大的应用潜力。     

植物种质的玻璃化超低温保存

植物种质的玻璃化超低温保存技术已受到广泛重视。玻璃化法主要由装载、玻璃化保护液脱水、降温、复温、洗涤这5个环节构成。目前已对百余种植物进行过玻璃化冻存研究,但主要应用于高等植物,而用该法保存藻类获得成功的报道很少。将玻璃化法用于某些藻类种质的冻存将会有广阔的应用前景。     

猕猴桃茎段的超低温保存

近年来的研究结果表明,超低温(-196℃)冰冻保存是长期保存植物种质最理想的方法,并已在40多种植物上取得初步成功。我国猕猴桃的品种资源十分丰富,但其种质的超低温保存尚未见报道。我们以猕猴桃茎段为材料进行了试验,试材经120天的液氮贮存后,在再培养中表现出很高的存活率,并产生出大量的新植株。现将这一初步结果简报如下。     

果树种质资源超低温保存研究进展

综述了国内外在超低温保存果树种质资源方面的最新研究成果,从材料选择、材料预处理、冰冻保护剂、冰冻方法及超低温保存对组织超微结构的影响等几个方面总结了果树超低温保存的原理、影响因素及关键操作技术,展望了果树种质资源超低温保存技术的应用前景.     

甘蔗愈伤组织超低温保存中一些因素的研究

对甘蔗愈伤组织超低温保存中几个主要因素进行了多方面的对比试验,为甘蔗愈伤组织的超低温保存提供了一套较佳的技术。实验结果表明:愈伤组织的适宜培养时间是10—15天。较好的冰冻保护剂是10%二甲亚砜(DMSO)+0.5mol/l 山梨糖醇,在0℃预处理30—45分钟。较佳的冰冻程序是以1℃/分的降温速度从0℃降到-40℃,停留1—3小时,然后浸入液氮中贮存。用自来水冲洗化冻同40℃水浴中化冻的效果一样良好。化冻后的愈伤组织在黑暗培养中生长好,存活率高。经过半年超低温保存后的愈伤组织在再培养中生长良好,并产生大量的新植株。此项结果为甘蔗种质的长期保存提供了可能性。     

低温保存技术在顽拗性种子种质保存中的利用

由于顽拗性种子不耐脱水且对低温敏感,常规保存方法难以达到长期保存的目的。因此,(超)低温保存顽拗性种子种质是最理想的方法。顽拗性种子的低温保存,应用较多的是玻璃化法和两步法。诸多因素影响着低温保存的成败,如种子或胚的含水量水平、溶液低温保护剂效应、降温冰冻与解冻方式、水合过程以及后培养等,这些需深入探索与解决。除顽拗性种子脱水耐性和低温敏感性机理外,植物细胞的冻害和抗冻机理也亟需探明,以便找到最佳冷冻方法,制定长期保存种质基因的最佳方案。     

桃花粉低温和超低温保存方法比较研究

桃(Prunus persica(L.)Batsch)是我国重要的无性繁殖作物种质资源,目前主要保存于3个国家无性繁殖作物种质圃。随着以茎尖、花粉、休眠芽为保存载体的超低温保存技术的发展,超低温保存已成为无性繁殖作物重要备份保存方式。本研究以15份桃种质花粉为研究对象,开展含水量、回湿处理和保存温度(4℃低温保存和液氮超低温保存)对保存后花粉离体萌发率的影响研究。研究结果:明确了桃种质花粉超低温保存的含水量;揭示了回湿处理对部分桃种质花粉超低温保存产生显著影响;超低温保存后花粉离体萌发率最高可达83%;4℃低温保存和超低温保存比较研究结果表明,超低温保存4年后14份桃种质花粉离体萌发率仍可保持30%以上,11份桃种质花粉离体萌发率与保存前花粉离体萌发率相比无显著变化甚至显著提高,而4℃低温保存的花粉离体萌发率降至0。该研究为国家种质库建立花粉规模化超低温保存提供技术支撑。     

植物种质资源超低温保存概述

简要回顾了植物种质资源超低温保存的历史,说明了超低温保存植物材料的多样性,阐述了超低温耐性的生物学基础及超低温伤害产生的原因和类型,介绍了各种常用超低温保存方法的技术要点,并对生产顽拗性种子的植物种质资源的超低温保存作了专门的论述,分析了生产顽拗性种子的植物种质资源超低温保存的潜力、现状和困难,指出顽拗性种子的超低温保存是植物种质资源超低温保存的重点和难点,而真正实现用超低温保存技术贮藏顽拗性植物种质资源还有很长的路要走。     

枇杷茎尖二步玻璃化法超低温保存的研究

超低温保存是目前植物种质资源长期稳定保存最理想的方法,而近几年发展的玻璃化超低温保存法具有设备要求简单、材料处理步骤简便及效果和重演性好等特点,倍受人们的青睐。国内外用玻璃化法成功地保存许多果树的种质资源。在对枇杷（Eriobotrya japonica Lindl．）花粉超低温保存取得成功的基础上,作者进行了枇杷茎尖玻璃化超低温保存的研究,以期建立枇杷茎尖超低温保存体系,为长期稳定保存枇杷种质资源提供技术支持。     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.     

Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods published. The aim of this study was to evaluate some important factors in the freezing process in an effort to find an optimized approach to the cryopreservation of precision-cut liver slices. A comparative study of a slow and a fast freezing technique was carried out to establish any differences in tissue viability for a number of endpoints. Both freezing techniques aim at the prevention of intracellular ice formation, which is thought to be the main cause of cell death after cryopreservation. Subsequently, critical variables in the freezing process were studied more closely in order to explain the differences in viability found in the two methods in the first study. For this purpose, a full factorial experimental design was used with 16 experimental groups, allowing a number of variables to be studied at different levels in one single experiment. It is demonstrated that ATP and K(+) content and histomorphology are sensitive parameters for evaluating slice viability after cryopreservation. Subsequently, it is shown that freezing rate and the cryopreservation medium largely determine the residual viability of liver slices after cryopreservation and subsequent culturing. It is concluded that a cryopreservation protocol with a fast freezing step and using William's Medium E as cryopreservation medium was the most promising approach to successful freezing of rat liver slices of those tested in this study.     

134 Cryopreservation of Chlamydomonas Reinhardtii (Chlorophyceae): A Cause of Low Viability at High Cell Density

Cryopreservation provides a convenient method for long term storage of living organisms. Current protocols allow the successful cryopreservation of a wide range of algae, although many strains remain recalcitrant to cryopreservation. Chlamydomonas reinhardtii , a species utilized in many molecular and biochemical studies, survives cryopreservation best at low cell density. We show that reduced viability at higher cell densities is caused by the accumulation of a substance released from C. reinhardtii into the culture medium during cryopreservation. A mutant strain of C. reinhardtii (cw10) with a greatly reduced cell wall did not release a substance inhibitory to wild type or cw10 C. reinhardtii during cryopreservation, and could be cryopreserved with the same viability regardless of cell density. The inhibitory substance is small (mw<1300), polar, heat-stable and organic. Chlamydomonas moewusii Gerloff and Chlamydomonas zebra Korschikov ex Pascher both produce substances that reduce the viability of cryopreserved C. reinhardtii . However, neither is affected by the inhibitory substance produced by themselves or C. rienhardtii. Pandorina morum (Müller) Bory and Volvox carteri f. nagariensis Iyengar are colonial Volvocalean algae related to C. reinhardtii that cannot be successfully cryopreserved. They both generate substances that inhibit C. reinhardtii during cryopreservation. The identification of the substance inhibitory to C. reinhardtii during cryopreservation should explain why this alga cryopreserves best at a low cell density, and may lead to protocols that facilitate the more successful cryopreservation of C. reinhardtii and related algae.     

Effects of very rapid versus vapor phase freezing on human sperm parameters

The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.     

Proceedings: Preservation of marine and fresh water algae by means of freezing and freeze-drying

Several species of marine and fresh water algae have been isolated from various habitats. Recently they were examined for their viability after freezing and freeze-drying procedures.The addition of suspending agents to algal cultures has resulted in greater viability for most of the green algae, but has shown little effect on the blue-green algae.It is considered that the preservation of algae by means of freezing and freeze-drying procedures are of great benefit as they offer the possibility of long-term preservation of viable collections, especially for patent depository for industrial applications.     

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium)

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D₂O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H₂O:D₂O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.     

Cryopreservation of eukaryotic algae – a review of methodologies

Since pioneering work in the early 1960s, there has been growing interest and numerous experimental investigations into the cryopreservation of algal material. Mostly, these studies relate to the requirement for long term preservation and storage of algal material contained in culture collections or used in the seaweed mariculture industry. The present review deals with techniques used in the cryopreservation of biological samples and their application to both micro- and macroalgae. Methods for the prevention of cell damage and freezing injury during the cooling and low-temperature storage of algal material are discussed with reference to the effect on viability of such variables as cooling rates, final temperatures attained, the use of various types and concentrations of cryoprotectants, thawing rates, and storage times and temperatures. Some consideration is also given to the various methods used for increasing cell viability, including the induction of freezing tolerance. Cryopreservation protocols employed by numerous workers in this field are detailed, and concluding remarks are made on those techniques and conditions providing optimum viability of cryopreserved algae. This revised version was published online in June 2006 with corrections to the Cover Date.     

The cryopreservation of fish thymocytes and its potential application for immunological reconstitution experiments

Fish thymocytes were cryopreserved by two methods, a programmed freezing method by a direct control system and a mechanical freezing method. There was no loss of viability or ability to respond to Concanavalin A compared to control thymocytes, up to 5 months after cryopreservation, and no difference between the two methods.     

Simplified cryopreservation of the microalga Chlorella vulgaris integrating a novel concept for cell viability estimation

Microalgae currently receive growing attention as promising candidates for future bio‐economy concepts. However, the reliable maintenance of production strains remains challenging. The well‐established serial subculturing techniques suffer from low long‐time stability and high effort and are therefore stepwise being replaced by cryopreservation. Currently, available protocols are often deduced from cell culture technology and are rather complex. This study aimed to investigate if less complex approaches can be applied. We introduce an easy‐to‐use cryopreservation protocol based on the model organism Chlorella vulgaris. To overcome error‐prone viability estimation by plating techniques, an alternative method using growth pattern analysis was developed. As revealed by growth pattern analysis, the preservation of stationary phase cells proved superior to the commonly applied concept of freezing cells from the growing phase. Controlled‐rate cooling using simple devices resulted in reproducibly high post‐thawing viabilities in the range of 63 ± 2%. Moreover, the presented protocol highlights the potential of simplifying microalgal cryo‐preservation procedures, thereby reducing the required labor and material need to a minimum. Apart from the viability analysis of the cryopreserved microalga C. vulgaris, this approach seems to have the potential to be applied for other algae species and microorganisms, as well.