Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Cryopreservation of sugarbeet germplasm

Meristems aseptically isolated from shoots developed on sugarbeet (Beta vulgaris L.) inflorescences were precultured on modified MS agar medium containing 19.4 M 6-benzylaminopurine, 6 M triiodobenzoic acid, and supplemented with 5% DMSO. After two days the meristems were transferred to liquid modified MS medium and the cryoprotectants sorbitol and DMSO added in varying concentrations. The meristems were frozen to –40°C and stored in liquid nitrogen. Growth resumed when the meristems were quick-thawed at 39°C.     

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。     

卵叶泥炭藓茎尖包埋玻璃化法超低温保存研究

该研究通过对脱水时间和化冻温度的探索,检验了包埋玻璃化法在超低温保存湿润生境中苔藓的可能性。结果表明:卵叶泥炭藓无菌苗在4℃条件下预培养3d后,在0℃用60% PVS_2装载30min,PVS_2脱水60min后迅速投入液氮保存,24h后用40℃水浴快速化冻2min再培养,成活率可达42.41%,且再生植株与常温状态下的植株形态指标没有显著性差异。研究认为,包埋玻璃化法超低温保存湿润环境中生长的苔藓植物是可行的。     

Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.     

Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L.

Uniformly applicable techniques for germplasm preservation are important to the international genetic resources community and validation of techniques among working genebanks will enable the integration of new technologies into plant genetic resources programs. Apical meristems from micropropagated plants of Ribes nigrum L. cv. Ojebyn and R. aureum cv. Red Lake were used to test three cryopreservation protocols (controlled freezing, plant vitrification solution no. 2 (PVS2) vitrification and encapsulation–dehydration) at the USDA-ARS National Clonal Germplasm Repository (NCGR), Corvallis, OR, USA and the University of Abertay Dundee (UAD), Scotland. Similar results were obtained with PVS2 vitrification at both locations but meristem regrowth varied greatly for the other techniques. Variable results between the locations were noted for controlled freezing and were largely attributed to differences in ice crystal initiation by the controlled rate freezers. Low survival of `Red Lake' at UAD with all three techniques was attributed to poorly performing shoot cultures. Attention to protocol details is important for limiting variation between locations and step by step instructions for procedures and solution preparation aided protocol standardization. These studies suggest that source plant status, cryogenic facilities, and culture conditions may be the most likely causes of variation when validating cryopreservation methodologies in different locations. However, in-house optimization of standard procedures offers considerable potential in ensuring that cryopreservation methodologies can be transferred between international laboratories.     

Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.     

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

防风愈伤组织的玻璃化法超低温保存及再生

为避免连续继代造成的愈伤组织变异,探索新的种质资源保存方法,对防风愈伤组织进行了超低温冷冻保存及植株再生研究。以关防风3周龄的愈伤组织为材料,单一变量法研究适宜的玻璃化法超低温保存程序。结果显示:(1)防风愈伤组织超低温保存的最佳方案为:4℃条件下于MS+1.0mg/L 6-BA+1.0mg/L NAA+5%DMSO的继代培养基中预培养3d,60%PVS2常温装载20min,100%PVS2于2℃脱水45min后直接投入液氮。(2)防风愈伤组织经超低温保存后的相对存活率最高为79.24%,其中预培养和脱水是实现超低温冻存的关键环节,且1.0mol/L蔗糖的MS溶液洗涤、暗培养14d以上有助于冻后愈伤组织恢复生长。研究表明,玻璃化超低温冻存可以作为防风愈伤组织的保存方法,冻后愈伤可以恢复生长并再生成完整植株。     

Ex situ conservation of plant germplasm using biotechnology

Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human generations. However, the conservation and subsequent use of such resources are complicated by cultural, economical, technical and political issues. Over the last 30 years, there have been significant increases in the number of plant collections and in accessions in ex situ storage centres throughout the World. The present review is of these ex situ collections and the contribution biotechnology has made and can make to conservation of plant germplasm. The applications and limitations of the new, molecular approaches to germplasm characterization are discussed. In vitro slow growth is used routinely for conserving germplasm of plants such as banana, plantain, cassava and potato. More recently, cryopreservation procedures have become more accessible for long-term storage. New cryopreservation techniques, such as encapsulation-dehydration, vitrification and desiccation, lengthen the list of plant species that can not only tolerate low temperatures but also give normal growth on recovery. Extensive research is still needed if these techniques are to be fully exploited.V.M. Villalobos is with the Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalia, 00100 Rome, Italy. F. Engelmann is with the International Plant Genetic Resources Institute (IPGRI), Via delle Sette Chiese 142, 00145 Rome, Italy.     

Rationalising germplasm collections: a case study for wheat

In total 70 genebank accessions comprising 50 hexaploid, 12 tetraploid and 8 diploid wheats of the Gatersleben collection were selected based on the screening of the passport data for identical cultivar names or accession numbers of the donor genebanks. Twelve potential duplicate groups consisting of three to nine accessions with identical names/numbers were selected and analysed with DNA markers (microsatellites). A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity. Although several homogeneous groups were identified it became clear that cultivar name identity alone did not allow the determination of duplicates. A combination of SSR-analysis followed by the bootstrap method and database survey considering the botanical classification and other data (origin, growth habit and donor) available is recommended in order to determine duplicates. A procedure for the identification of duplicates and their further handling in ex situ genebanks is discussed.     

Cryopreservation of seed of Western Australian native species

The ability of seed of native Western Australian species to be stored using cryopreservation methods was investigated by subjecting seed of 90 native species representing 84 genera and 33 families to storage in liquid nitrogen. Seed of 68 native Western Australian species were germinated after storage in liquid nitrogen for two weeks following treatments which involved direct plunging into liquid nitrogen or slow cooling at 0.4°C min^–1 in 15% dimethyl sulphoxide (DMSO) or slow cooling at 0.4°C min^–1 in 35% DMSO. The largest number of species (37) responded positively to direct plunging without pretreatments, with only 10 species responding to slow cooling in 15% DMSO. Thirty one species had enhanced germination and 10 species depressed germination after any of the liquid nitrogen treatments. There were no trends in a species ability to survive liquid nitrogen storage and freezing regime, moisture content, seed size or taxonomic relatedness. However, hard seeded species belonging to the families Caesalpinaceae and Papilionaceae showed a consistently high degree of tolerance to liquid nitrogen storage. Significant physical damage to seed and cotyledons only occurred in Templetonia retusa (Papilionaecae) and this was alleviated by nicking the seed coat. This study indicates that seed of a large proportion of native Western Australian species may be amenable to storage in liquid nitrogen and that at least 40% of the listed rare and endangered species of Western Australia could be maintained in this way.     

Cryopreservation of Dunaliella salina and Platymonas helgolandica var. tsingtaoensis

Dunaliella salina (Dunal) Teod. and Platymonas helgolandica Kylin var. tsingtaoensis were cryopreserved in liquid nitrogen with a two-step cryopreservation method. With 5% and 20% dimethylsulphoxyde (DMSO) concentration respectively through 30 min equilibrium time, and under -40℃, 60 min and -30℃, 30 min respectively as prefreezing temperature and sustained time, and by using slow dilution method to remove DMSO from the sample at 0℃ and room temperature respectively after sample thawing, D. salina and P. helgolandica var. tsingtaoensis gained the highest survival rates that were 68.5% and 78.3% respectively.     

盐生杜氏藻和青岛大扁藻的超低温保存

采用两步法冷冻技术超低浊保存盐生杜氏藻（Ｄｕｎａｌｉｅｌｌａ ｓａｌｉｎｕ（Ｄｕｎａｌ）Ｔｅｏｄ．）和青岛大扁藻（Ｐｌａｔｙｍｏｎａｓ ｈｅｌｇｏｌａｎｄｉｃａ Ｋｙｌｉｎ ｖａｒ．ｔｓｉｇｔａｏｅｎｓｉｓ）。当二甲基亚砚（ＤＭＳＯ）浓度分别为５％和２０％,平衡时间均为ｍｉｎ,预冻温度及保持时间分别为－４０℃,６０ｍｉｎ和－３０℃,３０ｍｉｎ;化冻后分别在０℃和到温下采用慢速稀释法去除ＤＭＳＯ时,     

An in vitro method for the conservation and storage of garlic (Allium sativum) germplasm

Shoot cultures of three garlic (Allium sativum) cultivars were kept in various temperatures and media in order to maintain their viability without subculture. A high level of viability was recorded after 16 months of culture at 4°C with 100 gl^-1 sucrose in B-5 medium.     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

In dry pear (Pyrus communis L.) pollen,membranes assume a tightly packed multilamellate aspect that disappears rapidly upon hydration

Summary Ultrastructural details of dry (7% moisture content) and hydratedPyrus communis L. pollen are revealed following freezesubstitution preparation for electron microscopy. Dry pollen is characterized by tightly packed, multilamellate membranous profiles found in association with plasma membrane, vesicles, ER, dictyosomes and some double-membrane bound organelles. Dry pollen also shows unit-membrane bound, densely osmiophilic bodies often with tightly packed multilamellations contained within and, at times, in their bounding membranes. These features are not evident in hydrated pollen. Results suggest that multilamellate membranes form as the plasma membrane, vesicles, ER, and double-membrane bound organelles undergo dehydration, and that upon hydration they rapidly resume normal unilamellate structure.Abbreviations DOB densely osmiophilic body - IMP intramembrane particles - MO multilamellate organelle     

Large-scale compatible methods for the preservation of human embryonic stem cells: current perspectives

Human embryonic stem cells (hESCs) and hESC-derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality-controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time-consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high-throughput drug screen and how many cells can be preserved using these techniques.Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here,the potential to lyophilize hESCs for long-term storage is considered as one such alternative.     

Cryopreservation of the unicellular marine alga, Nannochloropsis oculata

In microalgal culture collections, as in many biological resource centres, cryoconservation is the most attractive method for the long-term, secure storage of living material. Nannochloropsis oculata, a marine unicellular alga, is of interest in the field of biotechnology due to its high lipid content. Of various cryoprotectants tested for their toxicity and for their ability to prevent cryoinjury, glycerol (final concentration 1.1 M) was the most efficient. When glycerol-treated cultures were submitted to a strictly regulated cooling rate (-3 degrees C min(-1)), they attained the control culture density within 13 d after thawing.