A novel means for cryopreservation of germplasm of the recalcitrant-seeded species, Ekebergia capensis

Cryopreserved zygotic embryonic axes offer the best means of genetic diversity conservation of recalcitrant-seeded species, but frequently shoots fail to develop following processing for, and after, cryostorage. The present work offers a means to overcome this, by generating adventitious shoots from seedling roots produced after axis cryopreservation. Embryonic axes of Ekebergia capensis were exposed to cryoprotectants, flash dried, and rapidly cooled in nitrogen slush. Cryoprotection was an essential step, with both glycerol and DMSO permitting survival after cryogen exposure, but sucrose alone, or in combination with glycerol, was deleterious. Adventitious shoots were formed from seedling roots developed by axes germinated after cryogen exposure, after being subjected to intermittent flushing with a BAP-containing medium for 24 h in a temporary immersion system and subsequent culture on a semi-solid BAP-containing medium. After excision, a high proportion of the adventitious shoots produced roots in vitro, with most of these rooted plantlets being subsequently successfully acclimated.     

Improvement of cryopreservation results in garlic using low temperature preculture and high-quality in vitro plantlets

The efficiency of garlic cryopreservation is, amongst other factors, depending on the origin of the donor explants. So far, in vitro grown material has always been the least responding one with respect of the regrowth rates. On the other side, the possibility to produce virus-free material via meristem culture and to keep these clones then under isolated conditions in a clean culture induced studies to increase the efficiency of cryopreservation using this kind of material. Experiments have been performed to use various materials and cultivation temperatures for a vitrification protocol. Best results (up to 70 percent regrowth) were obtained with cultures grown for only 10 months under in vitro conditions including a cold preculture of two months either at alternating or at permanently low temperatures. The conclusion was drawn that the quality of the explants and temperature conditions play a major role for the efficiency of cryopreservation using in vitro plantlets.     

The impact of OECD best practice on the validation of cryopreservation techniques for microorganisms

The Organisation for Economic Co-operation and Development (OECD) Biological Resource Centre Initiative (BRC) was established after the 1998 Working Party on Biotechnology endorsed a proposal by Japan to examine support for Biological Resource Centres (BRCs) as a key element of the scientific and technological infrastructure for the life sciences and biotechnology. As part of this Best Practice Guidelines for the operation of Biological Resource Centres (BRCs) were published. Cryopreservation is widely used in BRC's and is seen as the method of choice for the preservation of most organism groups. This paper reviews the developments of BRC standards, how they are applied, current practices in cryopreservation and methods for validating the success of cryopreservation methodology.     

Thermal analysis of the plant encapsulation-dehydration cryopreservation protocol using silica gel as the desiccant

The encapsulation-dehydration cryopreservation protocol is critically dependent upon the evaporative desiccation step, which must optimise survival with the retention of glass stability on sample cooling and rewarming. Desiccation is usually achieved evaporatively by drying in a sterile airflow. However, chemical desiccation using silica gel has advantages for laboratories that do not have environmental control and/or which are exposed to high relative humidities and risks of microbial contamination. This study characterised thermal profiles of silica gel-desiccated encapsulated shoot-tips of two Ribes species using Differential Scanning Calorimetry. For both species silica gel-desiccation at 16 degrees C for 5 h decreased bead water content from ca. 75 to 28% fresh weight (3.8 to 0.4 g x g(-1) dry weight); further desiccation (for 6 and 7 h) reduced the bead water content to 21% (0.3 g x g(-1) dry weight). These changes in water status altered the thermal properties of beads for both species. After 7 h desiccation over silica gel stable glass transitions were observed on both cooling and rewarming of beads containing meristems. Tg mid-point temperatures ranged from -78 to -51 degrees C (cooling) and from -88 to -54 degrees C (warming) [at cooling and warming rates of 10 and 5 degrees C min(-1), respectively] after 5 to 7 h silica gel-desiccation. Post-cryopreservation viability of both species was ca. 63%. Thermal analysis studies revealed that an encapsulation/dehydration protocol using silica gel as a desiccant should comprise a minimum 5 h drying (at 16 degrees C). This reduces bead moisture content to a critical point (ca. 0.4 g x g(-1) dry weight) at which stable glasses are formed on cooling and rewarming. It is concluded that silica gel has advantages for use as a desiccant for alginate-encapsulated plant meristems by promoting stable vitrification and is useful in laboratories and/or geographical locations where environmental conditions are not under stringent control.     

Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification

This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 degree C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 degree C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 degree C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 percent.     

深燃LNG工艺低温绝热技术应用

分析了深燃LNG工艺低温绝热技术的应用情况,并就LNG工厂中使用的几种低温绝热材料及结构型式作了介绍,对今后LNG工艺设计具有一定参考作用。     

Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections

A simple method, based on the binomial distribution, is proposed to calculate the probability of recovering at least one (or any other fixed number of) plant(s) from a cryobank sample using four given parameters: the percentage of plant recovery observed from a control sample, pobs, the number of propagules used for this control, n1, the number of propagules in the cryobank sample, n2, a chosen risk for the calculation of a confidence interval for the observed plant recovery, alpha. Using this method, it is possible to assess the number of propagules which should be rewarmed immediately after freezing in order to estimate the plant recovery percentage as a function of the total number of propagules available. It also allows the calculation of the minimum plant recovery percentage to ensure that the probability to recover at least one (or A, with A>1) plant(s) is higher than a fixed probability level, as a function of the control and the cryobank sample sizes. Reciprocally, once the plant recovery percentage has been estimated, it is possible to assess the minimum size of the cryobank sample to obtain a probability to recover at least one (or A, with A>1) plant(s) higher than some fixed level.     

Cryopreservation of garlic (allium sativum L.) using plant vitrification solution 2

Most cryopreservation procedures are optimized using a small number of germplasm accessions. We classified the garlic (Allium sativum L.) accessions that were selected for our studies based on genotype as identified using amplified fragment length polymorphism markers. Although recovery was variable, shoots regenerated from a broad range of the accessions after cryo-exposure. Garlic shoot tips were excised from cloves, surface sterilized, and placed on media at 5 degree C for 2 days prior to cryopreservation. Shoot tips were then treated with sucrose-glycerol for 20 minutes, plant vitrification solution 2 (PVS2; 15 percent w/v ethylene glycol, 15 percent w/v DMSO, 30 percent w/v glycerol, 13.7 percent w/v sucrose) at 0 degree C, and then plunged on foils into liquid nitrogen slush. Explants were recovered in 1.2 M sucrose for 20 minutes and then plated onto Gamborgs B5 medium containing alpha-naphthaleneacetic acid (NAA) and 6-(gammagamma-dimethylallylamino purine) (2-iP). Our results demonstrate that genotypically diverse accessions of garlic can be successfully cryopreserved.     

Evaluation of critical points in technology transfer of cryopreservation protocols to international plant conservation laboratories

Cryopreservation of plant tissues in liquid nitrogen is now used for long-term conservation of vegetatively-propagated crops. Development of standard techniques for cryopreservation is important to the international plant-conservation community for successful implementation of storage protocols in diverse and internationally dispersed laboratories. Evaluation of the critical points of each preservation technique will greatly assist in developing and validating internationally-used cryopreservation protocols. The goals of this project were to assess critical points of two major cryopreservation techniques (PVS2 vitrification and encapsulation dehydration) during their transfer to international laboratories; analyze post-storage viability for each technique and location; and develop recommendations based on the assessments and data from the participating laboratories. Investigators from Germany, Kazakhstan, Poland and UK participated in a 2-week training workshop in cryopreservation methods after which the techniques were tested in the home laboratories of the participants. After one-year site visits by the technology trainers identified critical points in the protocols. Critical points were identified as 1) Cryogenic (cryoprotection, LN exposure, rewarming); 2) Non-cryogenic (plant health status, pre- and post-storage culture); 3) Operational (skills transfer, training, interpretation of procedures); 4) Facility (growth room, ambient conditions, media preparation, equipment). The most critical factors in all laboratories were culture health, operator skills and experience, and clarification of the technical details of the procedures. Final results showed that correction of critical factors improved the post-storage recovery in all the laboratories.     

Cryopreservation of plant germplasm using the encapsulation-dehydration technique: review and case study on sugarcane

Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allows the subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be highly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices.     

Comparison of three techniques for cryopreservation and reestablishment of long-term Gentiana tibetica suspension culture

Cryogenic storage of cell suspensions allows long-term maintenance of cultures. The main purpose of the study was to develop a successful cryogenic protocol for 10-year-old embryogenic cell suspensions of G. tibetica. We examined three techniques of freezing: (I) controlled-rate cooling with various cryoprotectants (0.1-0.5 M DMSO, 0.5-1.0 M sucrose, 0.5-1.0 M glycerol, 0.25-1.0 M proline) or preculture with 0.4 M sorbitol and cryoprotectants (0.065-0.1 M DMSO, 0.2-0.8 M proline), (II) vitrification (PVS2) and (III) encapsulation. Cell viability was assessed by the TTC test and biomass increase. After controlled-rate cooling the majority of cells were lethally damaged, with only 3% viability observed. Vitrification and encapsulation approaches were more effective, assuring high levels of post-thaw viability ca. 85% and 7%, respectively. The encapsulation procedure gave faster recovery of the culture suspension than did vitrification, and ensured culture homogeneity and embryogenic competence.     

Comparison of cryopreservation techniques for long-term storage of ash (Fraxinus excelsior L.)

The main purpose of this study was to develop a cryopreservation protocol for ash and to highlight the importance of testing different clones and plant material of different ontogenetic states. In vitro-grown ash (Fraxinus excelsior L.) shoot tips were successfully cryopreserved following optimization of the PVS2-vitrification protocol. Pretreatment conditions were optimized and three cryopreservation techniques (encapsulation/dehydration, PVS2-vitrification and encapsulation-vitrification) were tested one after another. PVS2-vitrification proved to be the most suitable technique. In vitro-grown shoot tips of ash were successfully cryopreserved with a mean regrowth of 73% for juvenile clones and 67% for selected mature trees. The optimum preculture conditions and the initial protocol were: 10 days cold hardening, preculture for 2 days on medium with 0.8 M glycerol, incubation in 2 M glycerol solution for 20 min at 22 degrees C followed by PVS2 for 25 min at 0 degrees C on ice and direct immersion in liquid nitrogen. Warming was carried out in 43 degree C water for 1 min followed by 22 degree C water for 10 sec. The encapsulation/dehydration method was not successful for shoot tips of F. excelsior because the shoots were sensitive to osmotic dehydration. The encapsulation/vitrification method resulted in a mean regrowth of only 16%. PVS2 vitrification can now be used to store important ash germplasm of either juvenile or mature trees.     

Implementation of the regularization and cumulant techniques for interpreting and modeling analytical signals

A new approach to the modeling of analytical signals based on the combined Tikhonov regularization and cumulant analysis of random non-Gaussian processes, allowing us to subtract the background, determine and refine the parameters, and to separate overlapping peaks is presented. The efficiency of the developed procedure in analyzing the stripping voltammetric measurements of analytical signals for Cd(II), Cu(II), and Pb(II) pollutants of natural water at mercury-graphite electrodes is demonstrated.     

动脉血管低温保存研究现状及展望

血管低温保存是满足临床移植的重要保证手段。从影响动脉血管低温保存的因素,低温保存后的活性恢复、细胞水平上的血管低温损伤机理、冻结/复温过程中的热物理性质和力学性质变化,以及低温断裂现象等方面综述了血管低温保存研究进展。     

太阳能热发电聚光系统的研究进展

概述了太阳能热发电技术的发展状况,介绍了用于太阳能热发电的5种聚光系统,包括槽式、碟式、塔式、线性菲涅耳式以及地面接收式。详细阐述了这些聚光系统的光学结构、聚光原理以及聚光器件的设计方法和制作工艺,指出了不同聚光系统在聚光过程中的优缺点。文中的讨论可为太阳能热发电聚光系统的设计提供参考。     

太阳能热发电聚光系统的研究进展

概述了太阳能热发电技术的发展状况,介绍了用于太阳能热发电的5种聚光系统,包括槽式、碟式、塔式、线性菲涅耳式以及地面接收式。详细阐述了这些聚光系统的光学结构、聚光原理以及聚光器件的设计方法和制作工艺,指出了不同聚光系统在聚光过程中的优缺点。文中的讨论可为太阳能热发电聚光系统的设计提供参考。     

微扫描技术对光电图像混淆噪声的影响

在分析采样理论和微扫描理论的基础上,引入了一个评价光电成像系统混淆噪声的品质因数,定量地分析了不同模式的微扫描对系统混淆噪声的改善程度。     

图像跟踪系统中机动目标预测的实现

针对装甲车上的图像跟踪系统，提出一种机动目标预测方法。将目标的运动分解为2部分，即全局运动和局部运动，并根据各自的动态特性分别进行预测。文章以局部运动预测为重点，通过对机动目标跟踪技术的研究，建立起性能优良的跟踪滤波器，并将其扩展到预测滤波器中对目标的未来运动状态进行预测，从而提出“交互式多模型预测算法”，并对该算法进行仿真，给出速度均方根误差的仿真结果。仿真结果表明:该算法预测精度高、自适应能力强。最后还给出了预测门的计算方法。     

Signal-processing techniques in a computerized hearing aid test system

General background is given to describe the factors leading up to the implementation of a computerized hearing aid test system in a production environment. The digital measurement methods for determination of the required acoustal components such as fundamental, rms, distortion, etc., in the presence of noise are discussed. The use of the concept of the average cycle for repetitive signals is described with its advantages in a Fourier type system. An algorithm for measuring rms for nonrepetitive signals that trades off resolution for memory size is described. Features and advantages of computerized testing of hearing aids are listed.