仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

酒酒球菌液氮超低温保存

【目地】为安全、长期的保藏酒酒球菌,本文研究了菌体生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响,找到最优液氮超低温保存方法。【方法】采用平板计数法测定冷冻存活率。【结果】实验结果表明酒酒球菌的最佳保存方法为:首先在稳定期前期离心收集菌体;其次加入保护剂(20 g/L酵母浸提物,40V/V甘油,20 g/L蔗糖,30 g/L谷氨酸钠)稀释菌体,使菌密度为109CFU/mL;然后直接投入液氮冷冻;最后在37℃温水浴中迅速解冻。保存6个月后,其中21株酒酒球菌的冷冻存活率达到99%以上。【结论】初步研究表明酵母浸提物,甘油,蔗糖,谷氨酸钠复合保护剂对酒酒球菌的保护效果较好,液氮超低温保存可用于酒酒球菌的长期保存。     

金黄仓鼠卵子冷冻及在人精子受精能力检测中的应用

通过对比试验,筛选出含３．０％牛血清白蛋白的ＢＷＷ培养基和２·０ｍｏｌ／Ｌ最终浓度的二甲基 亚砜（ＤＭＳＯ）冷冻保护剂配制成冷冻液,将金黄仓鼠卵以０．３－１·０℃／ｍｉｎ的冷冻速率分别降温至 －４０℃和－８０℃,再浸入液氮中冷冻保存（３～３１天）。结果表明：冷冻降温速率以０·３℃－０·５℃／ｍｉｎ 为宜,降温到一８０℃浸入液氮的冻卵存活率（６３％）比－４０℃（４６％）的高。将解冻后存活卵去除 透明带,与人获能精子进行体外穿透试验,穿卵率达３４． ４％,与新鲜卵对照组（５０％）相比,无统 计学差异（Ｐ＞０·０５）;扫描电镜观察结果与压片光镜观察相吻合。     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

胚脑神经元冻存和培养的研究进展

影响胚脑神经元冻存效果的关键因素有:冷冻保护剂及其浓度、降温和复温速率、储存温度等。以10％DMSO作为冷冻保护剂,以接近1—2℃/分的速率降温,37℃水浴中快速复苏,储存在液氮中,能较好的保证冻存神经元的存活率。冻存的胚脑神经元,培养后继续存活的细胞数比未经冻存的明显减少,但生长、分化情况却无显著差别,仍保持了神经元的形态学特征和特异性酶的表达及递质合成的功能,并能在宿主脑内继续生长、发育、发生整合。     

乳酸乳球菌冷冻保藏条件的研究

乳酸乳球菌能有效定植在人体肠道内,抑制有害菌的增殖,保护肠道,增强人体健康。但乳酸乳球菌在实际应用中的一大难题是难于保存其活性,尤其是冷冻保藏过程中。本实验通过单因素实验和正交实验确定最优保护剂组合为30%海藻糖,20%蔗糖,30%脱脂乳和10%甘油,使乳酸乳球菌的冷冻存活率达到91.96%。进一步研究不同的冷冻降温速率和解冻温度对乳酸乳球菌存活率的影响,发现降温速率在-20℃/min,解冻温度在0℃时,乳酸乳球菌的存活率最高,达93.14%。     

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。     

3株抗水稻和荔枝病原菌的海洋真菌的分离鉴定

本研究采用平板对峙法和菌丝块法测定分离自广西北部湾的3株海洋真菌MF-3、MF-6和MF-13的抗荔枝和水稻病原菌抗菌活性,并通过形态学观察结合分子生物学方法鉴定其分类地位。结果表明,当采用平板对峙法时,3株海洋真菌对荔枝霜疫霉、炭疽病菌、水稻纹枯病菌和水稻稻瘟病菌等4种指示菌的菌丝生长均有强的抑制作用;菌丝块法试验结果发现3株真菌的发酵液均对荔枝霜疫霉菌有很好的抑制效果,菌丝相对生长抑制率分别达到94.29%、98.16%和86.37%,对水稻纹枯病病原菌抑制效果次之,其菌丝相对生长抑制率达到80%左右;其中,MF-3菌株对荔枝霜疫霉、炭疽菌、水稻纹枯病菌和水稻稻瘟病菌都有较好的抑制作用,MF-6对霜疫霉、水稻纹枯病菌和水稻稻瘟病菌也有较好的抑菌效果和广谱性。结合形态学观察和ITS rDNA序列比对分析,初步将MF-3鉴定为布雷正青霉(Eupenicillium brefeldianum),MF-6为微紫青霉(Penicillium janthinellum),MF-13为短棒曲霉(Aspergillus clavatonanicus)。耐盐度试验结果表明这3株真菌是适应海洋环境的兼性海洋真菌。该3株海洋真菌表现的抗真菌活性,对应用于荔枝或水稻病害的生物防治和新型农药的研发有潜在的应用价值。     

水稻单倍体不定芽超低温保存和植株再生及其遗传稳定性研究

通过水稻单倍体幼穗离体培养,诱导形成了大量的不定芽。将３ｍｍ左右的不定芽转入半固体继代培养基继代培养７ｄ,而后转入１．８ｍＬ的安瓿瓶中,冰浴条件下加入预冻了的冰冻保护剂淹没材料,在冰上平衡４５￣６０ｍｉｎ后,转入程序降温仪,以１．０℃／ｍｉｎ的速率从４℃降至－４０℃,在－４０℃停留１ｈ后,投入液氮保存。将液氮保存３０ｄ左右的不定芽于３８￣４０℃的水浴中快速解冻,随即转入半固体的再生培养基,以待恢复     

液氮保存疫霉属菌种存活期检测

对Phytophthora和Peronophythora所属12个种29株菌在液氮中保存5年零8个月至6年零3个月后检测证明有68.9％的菌种存活下来。但有些种未能存活,这些种有Phytophthora colocasiae(XH30),P.drechsleri(ATCC15428),P.erythroseptica(ATCC36302)与Peronophythora litchii(ATCC 34595)。即使存活的菌种也不一定每个保存的菌株与菌块都存活。说明在液氮中也有存活力下降的现象。除菌种本身耐深冻贮藏特性不同外,贮藏前菌种培养的旺盛程度明显影响存活。对一些菌的致病性测定证明长期保存后致病力维持不变。液氮保藏不失为保持菌种长期不变的良好方法,只是要严格按要求掌握,并对菌的耐冻力先行了解。     

液氮冻结法保藏毛霉目菌种的效果

本文报告了用液氮冻结法保藏毛霉目菌种的效果。对14属26种32株进行了液氮保藏试验,其结果慢速冻结孢子成活率高于快速冻结。用10%的甘油、10%的二甲基亚砜和蒸馏水作保护剂制成的孢子悬液,经慢速冻结后储存在液氮罐气态中二年,以甘油作保护剂的孢子成活率为90%,二甲基亚砜的为85%,蒸馏水的为79%。并检测了某些菌株的反丁烯二酸、蛋白酶、α-半乳糖苷酶、脂肪酶、果胶酶、葡萄糖苷酶等生理活性,结果冻结后的活性与冻结前相比,无明显差异。     

Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation

Basidiomycetes present specific problems with regard to their preservation, because most of them do not form resistant propagules in culture but exist only as mycelium. Usually these fungi can only be preserved by serial transfer on agar (labour-intensive procedures that can increase the danger of variation or loss of physiological or morphological features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at −80 °C and lyophilisation could be good alternatives.In this work we set up and tested six protocols of cryopreservation at −80 °C, and 12 protocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The tested protocols were mainly characterized by the use of different growth media, protectants, time and number of perfusion with protectants and finally by the typology and origin of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise (mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisation outcomes were checked, at morphological (macro- and microscopic features), physiological (growth rate and laccase, Mn-independent and Mn-dependent peroxidases activities) and genetic level (Amplified Fragment Length Polymorphisms analysis - AFLP). Vitality of all fungi was successfully preserved by all cryopreservation protocols at −80 °C, and by two lyophilisation methods. Our results showed that cryopreservation at −80 °C did not produce morphological changes in any isolate, while two isolates were affected by lyophilisation. None of the physiological features were lost, even though growth rate and enzyme activities were somehow influenced by all preservation methods. AFLP analysis showed that only the two isolates that varied in their morphology after lyophilisation produced a different DNA fingerprint pattern in comparison with that obtained before lyophilisation. These findings provide evidence that cryopreservation at −80 °C and lyophilisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of some WRF strains.     

Preservation of basidiomycete strains on perlite using different protocols

For preservation of 31 basidiomycete strains on perlite in cryovials we used five different perlite protocols to compare their applicability in laboratories with different equipment, namely a viability of the controlled freezing device or the electric deep-freezer and liquid nitrogen supply. The viability of the strains, macromorphological characteristics and the production of laccase were tested after 48 h, six months and one year of storage in the respective device. Our results indicated that the different response to the freezing/thawing process is an intrinsic feature of the respective strain. Nevertheless, the highest viability and preservation of laccase production in our tested strains was found when we used pre-freezing to −80 °C at a freezing rate of 1 °C/min in a programmable IceCube 1800 freezer or in freezing container Mr. Frosty before storage in liquid nitrogen or at ultra-low temperature freezer at −80 °C, respectively. The two abovementioned protocols enable all tested strains to survive three successive freezing/thawing cycles without substantial reduction of growth rate. The majority of the strains also do not lose laccase production. Our results showed that direct immersion of the strains into liquid nitrogen or placing them into −80 °C without pre-freezing is not suitable for basidiomycete cryopreservation.     

Proceedings: Preservation of rust fungi in liquid nitrogen

Spores of rust fungi can be expected to retain viability without loss of infectivity for at least several years when stored in liquid nitrogen (?196 °C). Addition of liquid suspending media is harmful and not necessary. Some rust fungi experience cold-induced dormancy when exposed to less than 0 °C for a minute or longer but germinability is dependably restored on applying a heat shock by heating the spores to 40 °C for at least 15 sec during or after thawing. Most rust fungi are not sensitive to moisture content at the time of freezing but Puccinia striiformis must be vacuum dried before freezimg. The need for heat shock may not show up until several days after thawing. All of the rust strains tested to date have retained their properties to the extent tested and for the duration of storage. Data are available for up to 11 years. Preliminary experiments to preserve saprophytic mycelial cultures of P. graminis have so far failed, with and without use of 10% glycerol and 5% DMSO. The successful preservation of rust spores has made feasible the development of a collection of living rust fungi at ATCC beginning in 1965 and which now has over 80 strains in 20 species in 7 genera.     

霜疫霉菌侵染对荔枝氧化作用的影响

淮枝、桂味和糯米糍 3种荔枝经接种霜疫霉菌后 ,果皮超氧化物歧化酶 (SOD)和过氧化氢酶 (CAT)活性下降 ;而过氧化氢 (H2 O2 )含量升高 ,超氧阴离子自由基 (O2 .)产生速率增加 ,丙二醛 (MDA)积累增多 ,与果实感病指数增加相一致 ,表明霜疫霉菌侵染加速荔枝氧化作用的进程。实验还表明不同荔枝品种对霜疫霉菌抵抗力与其自由基清除能力有关     

Freezing damage to isolated tomato fruit mitochondria as modified by cryoprotective agents and storage temperature

Isolated tomato (Lycopersicon esculentum var. Kc 146) fruit mitochondria could be stored successfully in the frozen state without a cryoprotective agent if the mitochondria were frozen quickly by immersion in liquid nitrogen and later thawed quickly at 30 C. Criteria of freezing damage were rate of respiration, adenosine diphosphate to oxygen ratio, and respiratory control ratio. Marked reduction in respiration and loss of respiratory control occurred when mitochondria were transferred from liquid nitrogen to −5, −10, or −18 C for 15 minutes prior to thawing at 30 C. Dimethylsulfoxide (5%) prevented freezing damage when mitochondria were incubated at −5 C but did not prevent freezing damage at −10 or −18 C. Isolated tomato mitochondria show promise as a model system for studying the nature of freezing damage and the mode of action of cryo-protective agents.     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

荔枝霜疫霉的研究

对华南的荔枝霜疫霉(Peronophythora litchii Chen ex Ko et al)的形态和营养特性进行了研究,并和新模式种进行了比较。发现此菌孢囊梗的生长是一种有限-无限生长类型,或称之为多级有限生长。即孢囊梗上的小分枝大多数是有限生长的,在其顶端同时形成孢子囊。但有时在同一孢囊梗上有的小分枝会继续生长,形成二级、三级甚至四级孢囊梗。在营养要求上与疫霉无大差别,能在天然和合成培养基上旺盛生长,需要硫胺素,ca~(++)和有机二元酸,能利用NH_4~+ 或No_3~-为其氮源,并能利用淀粉为其碳源,菌体匀浆中测出淀粉酶活性。根据孢囊梗的独特生长方式,我们认为完全有理由承认这菌是一个新属,并可成为新科,霜疫霉科。本文中已将霜疫霉科作了修改描述。孢囊梗已被修改为:孢囊梗多级有限生长。无疑这菌是腐霉科和霜霉科的中间类型。在营养类型与有性器官上和疫霉相近,而其孢子囊的形成和霜霉相似。但其孢囊梗的多级有限生长方式则和这两科都不相同。     

Quick freezing of rat embryos

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).