Increased efficiency using the encapsulation-dehydration cryopreservation technique for Arabidopsis thaliana

Arabidopsis thaliana shoot tips were successfully cryopreserved using encapsulation-dehydration cryopreservation methods. Between one and seven shoot tips were encapsulated within 4 mm calcium-alginate beads. Beads were formed in the presence of 2 M glycerol + 0.4 M sucrose. The time required to make 10 beads, each containing five shoot tips (4 min), was less than the time required to make 50 beads containing one shoot tip (12 min). Shoot tip regrowth after cryoexposure was between 60 and 68%, with one to seven shoot tips per bead. Using five Arabidopsis shoot tips per bead, alginate beads were formed either in the presence of 2 M glycerol + 0.4 M sucrose or 0.5 M sucrose. Beads formed in the presence of glycerol were immediately air-dried to moisture contents between 0.21 to 0.26 g H2O/g FW (0.27 to 0.38 g H2O/g DW). Alginate beads formed in 0.5 M sucrose were incubated in solutions of 0.5, 0.75, and 1 M sucrose for one day each prior to air-dehydration, achieving moisture contents of 0.19 to 0.21 g H2O/g FW (0.23 to 0.27 g H2O/g DW). Shoot tip regrowth after cryoexposure was between 42 and 65%, with no significant differences among treatments. We successfully reduced the amount of time needed for shoot tip processing for Arabidopsis by encapsulating five shoot tips per alginate bead and by using a glycerol-encapsulation method, without lowering shoot tip regrowth levels after cryopreservation.     

硅胶除湿转轮性能优化实验研究

为了提高除湿转轮的除湿性能,降低吸附热对除湿性能的影响,将导热硅脂作为传热基质负载在不同扇形区域的硅胶转轮转芯基体表面,搭建硅胶除湿转轮性能优化实验台,研究了除湿转轮动态除湿性能.实验结果表明:硅胶除湿转轮的除湿性能随导热硅脂负载区域的增大先增大后减小,导热硅脂负载区域为2/8时,具有最佳的除湿性能,除湿量达到了0.3...     

Apex cryopreservation of several strawberry genotypes by two encapsulation-dehydration methods

This paper presents results from a study to develop cryopreservation procedures for apices of several strawberry genotypes. Five Fragaria x ananassa Duch. cultivars and two wild species (F. chiloensis and F. virginiana) have been screened using the encapsulation-dehydration method and/or a protocol which compromises vitrification and encapsulation-dehydration. Apices were encapsulated in an alginate gel, precultured on media containing high levels of sucrose (0.8 M, conventional protocol), or a combination of 0.4 M sucrose and 2 M glycerol. Recovery rates varied among genotypes (23-63%). The latter method reduced considerably the time needed for the cryogenic procedure by eliminating the pre-treatment with 0.8 M sucrose for 19 h prior to dehydration, as required by the conventional procedure.     

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.     

An improved cryopreservation protocol for pineapple apices using encapsulation-vitrification

Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0 degree C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 percent) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in 0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0 degree C for 60 min before immersion into liquid nitrogen. Following this procedure 54 percent and 83 percent of apices from MD-2 and Puerto Rico varieties respectively survived.     

分级再生式转轮除湿空调系统的性能分析

提出了分级再生式转轮除湿和蒸发冷却相结合的空调系统,依据除湿和再生过程的质量守恒和能量守恒方程,结合边界条件和初始条件建立除湿转轮和空调系统的数学模型,对比分析了分级再生空调系统的性能。研究结果表明:采用分级再生的转轮可达到与一般再生转轮相同的除湿效果。与一般再生的空调系统相比,分级再生式空调系统的再生能耗降低了约50%,热力性能系数提高了约90%。     

V-cryo-plate procedure as an effective protocol for cryobanks: case study of mint cryopreservation

A vitrification procedure using aluminium cryo-plates (V-Cryo-plate procedure) was successfully developed and adjusted for in vitro-grown mint (Mentha spp.) shoot tips. Shoots were cultured at 25°C on MS medium containing 0.088 M sucrose for 7 to 14 days after the last subculture. Shoot tips with a basal part (1-1.5 mm × 1 mm) were dissected from the shoots and precultured at 25°C for 1 day on the same medium. Precultured shoot tips were placed on aluminium cryo-plates with 10 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in 25 ml pipetting reservoirs filled with PVS2 for 20 min at 25 degree C. Then the cryo-plates were transferred in uncapped 2 ml cryotubes and directly plunged into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in cryotubes containing 2 ml 1 M sucrose solution at room temperature. Using this procedure, regrowth of cryopreserved shoot tips of line 'Fukuyamajisei' reached over 90 percent. This protocol was successfully applied to 16 additional Mentha lines, with regrowth ranging from 73 percent to 100 percent. This V-Cryo-plate method will facilitate the cryostorage of mint germplasm in our genebank.     

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique

Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.     

Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii

The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved.     

A cryopreservation protocol for embryos of the endangered species Zizania texana

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.     

Deployment of the encapsulation-dehydration protocol to cryopreserve microalgae held at the Sammlung von Algenkulturen, Universitat Gottingen, Germany

Encapsulation-dehydration was applied to cryopreserve 14 diverse algal strains, representing eukaryotic terrestrial microalgae; of these 12 survived to form cell colonies after recovery from cryostorage. Surviving algae had varying degrees of tolerance to osmotic dehydration and desiccation in this vitrification-based cryoprotective strategy. The extent of algal regrowth was affected by the mode of desiccation (silica gel or air-flow), the duration of evaporative desiccation and exposure to light during early recovery phase. This paper: (i) demonstrates the versatility of the encapsulation/dehydration method to cryopreserve diverse microalgae; (ii) confirms the successful transfer of this cryostorage technology to the Culture Collection of Algae at Gottingen University (SAG); and (iii) recommends encapsulation/dehydration as a feasible alternative to controlled rate cooling for preserving algae held in international culture collections.     

Thermal conductivity of neutron-irradiated silica

Low temperature thermal conductivity Λ(T) of vitreous silica has been measured after exposure to various neutron fluences. It has been observed that the increase in Λ(T) saturates for approximately the same fluence for which the change in mass density ? saturates. A correlation between Λ(T) and the fictive temperature has been found.     

Deployment of the encapsulation-dehydration protocol to cryopreserve polar microalgae held at the Czech Republic Academy of Sciences Institute of Botany

Polar isolates of four chlorococcal microalgae originating from the Arctic and Antarctica withstand cryopreservation using encapsulation-dehydration. Viability assessments, which initially used chloroplhyll fluorescence (Kautsky) induction kinetics, revealed that all strains suffered photosynthetic impairment during early post-cryopreservation recovery. This cryoinjury was reversible, as indicated by cell regrowth in three of the four strains. Lack of growth in the fourth isolate was due to contaminating bacteria rather than cryogenic factors.     

转轮除湿空调系统能耗分析及节能措施研究

针对常用的转轮除湿空调系统能耗高的问题,通过理论分析,提出了转轮除湿机与预冷器或热回收装置不同组合下的三种节能型转轮除湿空调系统,并建立相应的能耗数学模型。实例能耗分析表明:有预冷的转轮除湿空调系统比无预冷的转轮除湿空调系统总能耗低40.9%~43.8%;有热回收转轮的除湿空调系统比无热回收转轮的除湿空调系统总能耗低16.1%~20.2%;预冷热回收型转轮除湿空调系统能耗最低,比传统冷却除湿空调系统节能12.8%。处理空气先预冷后除湿和增加热回收装置的措施可大大降低转轮除湿空调系统能耗。     

Ultrasound-mediated gene delivery into suspended plant cells using polyethyleneimine-coated mesoporous silica nanoparticles

Sonoporation, ultrasound-mediated membrane perforation can potentially puncture plasma membrane and rigid cell wall on presumably reversible basis which benefit gene transfection and plant biotechnology. Herein, positively charged poly-ethyleneimine (PEI)-coated mesoporous silica nanoparticles (MSNs) with an average diameter of 100 ± 8.7 nm was synthesized for GUS-encoding plasmid delivery into the suspended tobacco cells using the ultrasound treatment. The overall potential of PEI-MSN for DNA adsorption was measured at 43.43 μg DNA mg⁻¹ PEI-MSNs. It was shown that high level of sonoporation may adversely upset the cell viability. Optimal conditions of ultrasonic treatment are obtained as 8 min at 3 various intensities of 160, 320 and 640 W. Histochemical staining assay was used to follow the protein expression. It was shown that PEI-coated MSNs efficiently transfer the GUS-encoding plasmid DNA into the tobacco cells. The results of this study showed that ultrasonic treatment provides an economical and straightforward approach for gene transferring into the plant cells without any need to complicated devices and concerns about safety issues.     

Implementation of garlic cryopreservation techniques in the national plant germplasm system

The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.