植物组织培养物的超低温保存

引言十九世纪末,诞生了一门新的科学——低温生物学(Cryobiology)。气体液化技术使人类可以获得近乎生命的凝固状态。1949年发现了甘油可以防止冰冻对活细胞的伤害,对低温生物学有重要贡献。从而导致了在许多不同领域内广泛应用低温保护技术。超低温通常指低于-80℃的低温,常用的有干冰(-79℃)、深冷冰箱、液氮(-196℃)及液氮蒸汽相(-140℃)。这样低的温度下保存生物材料,可以大大减慢、甚至终止代谢和衰老过程,因而能     

顽拗性竹柏种子的贮藏特性

文章对竹柏(Podocarpus nagi)种子的脱水耐性和贮藏特性进行了研究,结果表明:竹柏种子成熟时初始含水量约为(35±0.7)%,种子对脱水敏感,其最低安全含水量约为(16.86±0.73)%,具有顽拗性种子的典型特征;湿藏和半干藏都可以作为短期保存竹柏种子的方法,且以4℃保存优于15℃保存,但不管种子含水量如何,零下低温保存对竹柏种子都是致命的;半干藏法保存实验中,未进行脱水处理的种子(对照)在4℃贮藏6个月,种子萌发率没有发生明显下降,但贮藏期延长到9个月时,临界含水量的种子萌发力保存最高;不管贮藏介质的含水量高低,也无论贮藏在4℃还是15℃,湿藏种子在9个月的贮藏期内萌发率均没有明显的降低,但当贮藏到12个月时,15℃湿藏种子的萌发率显著高于4℃贮藏的种子,但15℃湿藏的种子在贮藏到3个月时即发现种子在贮藏期间萌发,且随着贮藏介质含水量的升高和贮藏期的延长,萌发的种子增多;竹柏的离体胚经过2 h硅胶快速脱水至含水量7%后再冷冻即可获得90%以上的融后存活率,且超低温保存1年的离体胚解冻后,与只保存1周的存活率没有明显差异,表明超低温长期保存竹柏种子是可行的。本研究可以为进一步探究顽拗性种子的短期贮藏和长期保存提供理论基础和基础资料。     

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。     

顽拗性竹柏种子的贮藏特性?

文章对竹柏( Podocarpus nagi)种子的脱水耐性和贮藏特性进行了研究,结果表明：竹柏种子成熟时初始含水量约为(35±0?7)％,种子对脱水敏感,其最低安全含水量约为(16?86±0?73)％,具有顽拗性种子的典型特征;湿藏和半干藏都可以作为短期保存竹柏种子的方法,且以4℃保存优于15℃保存,但不管种子含水量如何,零下低温保存对竹柏种子都是致命的;半干藏法保存实验中,未进行脱水处理的种子(对照)在4℃贮藏6个月,种子萌发率没有发生明显下降,但贮藏期延长到9个月时,临界含水量的种子萌发力保存最高;不管贮藏介质的含水量高低,也无论贮藏在4℃还是15℃,湿藏种子在9个月的贮藏期内萌发率均没有明显的降低,但当贮藏到12个月时,15℃湿藏种子的萌发率显著高于4℃贮藏的种子,但15℃湿藏的种子在贮藏到3个月时即发现种子在贮藏期间萌发,且随着贮藏介质含水量的升高和贮藏期的延长,萌发的种子增多;竹柏的离体胚经过2h硅胶快速脱水至含水量7％后再冷冻即可获得90％以上的融后存活率,且超低温保存1年的离体胚解冻后,与只保存1周的存活率没有明显差异,表明超低温长期保存竹柏种子是可行的。本研究可以为进一步探究顽拗性种子的短期贮藏和长期保存提供理论基础和基础资料。     

螺旋藻的超低温保存法

【目的】建立螺旋藻藻种的超低温保存法,并探究该方法对不同种类螺旋藻藻种保存的适用性。【方法】采用碘量法筛选出耐低温螺旋藻藻株,通过单因素和正交试验设计对耐低温螺旋藻超低温保存法进行条件优化,并以优化后的超低温保存法对8株不同种类的螺旋藻进行保藏实验。【结果】FACHB-351为筛选出的耐低温螺旋藻藻株;优化后的超低温保存方案为:以10%蔗糖溶液做冷冻保护剂,将藻丝体密度为1.0×107 CFU/m L的藻悬液于4°C驯化72 h,再将藻液和保护剂分别在0°C预冷30 min后混匀,混匀后于0°C停留3 h,然后投入液氮保存。保藏实验结果表明,保藏6个月时除了耐低温性较差的FACHB-350、FACHB-1070、FACHB-902螺旋藻存活率为0,不能恢复生长繁殖,其它5种耐低温性较好的螺旋藻均能在一定时间内恢复正常的生长繁殖,其中FACHB-351的存活率最高,为39.33%。【结论】建立的超低温冷冻保存法可用于耐低温性较好的螺旋藻藻种的长期保存。     

Cryopreservation of domestic animal sperm cells

Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15–60°C/min) gives rise to best sperm survivals after freezing–thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 16–20, 2007, Vale de Santarém, Portugal.     

Mitochondria and membrane cryoinjury in micropatterned cells: Effects of cell-cell interactions

The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell “viability”. Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell-cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell-cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell-cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 °C/min to −40 °C after extracellular ice nucleation at −5 °C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell-cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell “viability” does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.     

Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella. S. K. Singh and M. K. Rai contributed equally to this work.     

Propagation of three orchid genera using encapsulated protocorm-like bodies

Summary Protocorm-like bodies (plbs) of the orchid Dendrobium ‘Sonia’ were obtained from fractionated plb explants on Murashiga and Skoog (1962) medium (MS) supplemented with 4.44 μM 6-benzylaminopurine (BA). The developmental stage of plbs to be encapsulated, concentrations of sodium alginate (2–5%) and calcium chloride (25–100 mM), and concentration of MS salts in the matrix were assessed. Fractionated plbs 13–15 d after culture were at the leaf primordia stage and found suitable for encapsulation studies. The best encapsulation response was observed with 3% sodium alginate upon complexation with 75 mM CaCl₂.2H₂O. An encapsulation matrix prepared with MS medium (three-quarter strength) supplemented with 0.44 μM BA and 0.54 μM α-naphthaleneacetic acid gave 100% conversion of encapsulated plbs to plants after storage. A viability of >85% of encapsulated plbs of Dendrobium ‘Sonia’ was observed for 75 d at 4°C. Encapsulated plbs of orchids Dendrobium, Oncidium, and Cattleya were stored at 4°C for 75, 60 and 30 d, respectively, with >88% germination. All plants survived potting in media having either wood charcoal pieces alone or in combination with brick pieces.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes

With the development of genomic research technologies, comparative genome studies among vertebrate species are becoming commonplace for human biomedical research. Fish offer unlimited versatility for biomedical research. Extensive studies are done using these fish models, yielding tens of thousands of specific strains and lines, and the number is increasing every day. Thus, high-throughput sperm cryopreservation is urgently needed to preserve these genetic resources. Although high-throughput processing has been widely applied for sperm cryopreservation in livestock for decades, application in biomedical model fishes is still in the concept-development stage because of the limited sample volumes and the biological characteristics of fish sperm. High-throughput processing in livestock was developed based on advances made in the laboratory and was scaled up for increased processing speed, capability for mass production, and uniformity and quality assurance. Cryopreserved germplasm combined with high-throughput processing constitutes an independent industry encompassing animal breeding, preservation of genetic diversity, and medical research. Currently, there is no specifically engineered system available for high-throughput of cryopreserved germplasm for aquatic species. This review is to discuss the concepts and needs for high-throughput technology for model fishes, propose approaches for technical development, and overview future directions of this approach.     

Cryopreservation of Human Embryos

Cryopreservation of human embryos from the 2-cell stage up to the morula stage is a safe procedure which has been carried out for the last 25 years. Experience with blastocyst cryopreservation is still limited and pregnancy rates after the use of frozen, thawed blastocysts vary extremely. Vitrification has improved the success of embryo cryopreservation. However, this technique cannot yet be considered as a routine procedure. Despite all of the advantages for infertile couples, cryopreservation of human embryos creates severe ethical problems, because of surplus frozen embryos which either have to be destroyed or perhaps used for research. Embryo adoption may provide a solution to solve imminent medical, ethical and social problems.     

动物种质细胞的超低温冷冻保存

目前不少种类的动物种质细胞都可在超低温条件下保存,随着低温生物学和生殖生物学的不断发展,超低温保存技术也在不断发展,可以保存的动物种质细胞范围也在不断扩大。但目前超低温保存技术面临的困难和需要解决的问题是如何提高种质细胞冷冻保存的效果,寻求最佳冷冻方案,降低种质细胞的冷冻损伤,进而运用超低温冷冻技术保护物种的多样性。从目前的研究情况来看,动物种质细胞超低温保存的常用方法有程序降温法和玻璃化法。防冻     

香石竹种质离体保存研究进展

香石竹是世界四大切花之一,具有重要的观赏价值。其种质资源主要依靠田间种质圃和离体库进行保存。离体保存包括试管苗保存和超低温保存,这两种方法作为田间种质圃保存的补充可以分别对种质资源进行短中期和长期保存。本文对香石竹离体保存的相关研究进行了概括总结,旨在为香石竹种质资源的保存提供参考。