Dehydration in the cryopreservation of moist plant tissues and in seed maturation

The possibility of long-term cryopreservation of plant objects depends on their water content. In orthodox seeds, it decreases at the late stage of maturation and is accompanied by the synthesis of protectors--sugars and proteins. These seeds easily withstand cryopreservation. Organs with a high water content, meristems, and recalcitrant seeds are dried in presence of sucrose before plunging in liquid nitrogen. In orthodox seeds, artificially dried moist seeds, and meristems, the cellular content forms glass structures that are estimated in frozen materials by differential scanning calorimetry and electron paramagnetic resonance methods. It is proposed that the glass cellular content is connected with the duration of cryopreservation. Methodical approaches to successive cryopreservation of moist plant tissues are described.     

Maize seedlings produced from dry seeds exposed to liquid nitrogen display altered levels of shikimate pathway compounds

In light of climate change and risks of food insecurity, it is becoming increasingly important to preserve plant germplasm in genebanks. Storage of seeds, particularly via cryopreservation, is one of the most proficient methods for ex situ plant germplasm conservation. Whilst seed cryo-banking can have little, to no, or even beneficial effects on subsequent seedling vigor in some species, it can lead to a number of plant abnormalities (morphological and physiological). This study investigated the effects of maize seed cryopreservation on seedling growth (until 14 d) and levels of selected amino acids produced in the shikimate pathway, a major link between primary and secondary metabolism. Seed cryopreservation reduced FW in recovered seedlings, reduced caffeic acid (2.5-fold decrease), and increased levels of all other shikimate pathway–related compounds assessed: phenylalanine (2.9-fold increase), tyrosine (2.6-fold increase), and shikimic (2.1-fold increase) and protocathecuic (3.1-fold increase) acids in cotyledons. Our results suggest that maize seed cryopreservation results in seedlings that exhibit signs of an ‘overly’ efficient and caffeic acid–deficient shikimate pathway, possibly related to their reduced growth during a highly vulnerable growth stage. However, these metabolic abnormalities manifested most severely in the maternal (cotyledonary), as opposed to vegetative (roots, stems, and leaves), tissues and hence are likely to disappear when the seedlings shed the cotyledons and become completely autotrophic.

     

Use of biotechnologies for the conservation of plant biodiversity

In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.     

Plant cryopreservation: a continuing requirement for food and ecosystem security

This issue of In Vitro Cellular and Developmental Biology—Plant is dedicated to current developments in liquid-nitrogen cryopreservation methods and their use in plant biology and germplasm preservation. The development of cryopreservation for storage of plant cells, tissues, and organs began in the 1960s and continues to this day. Long-term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains an important requirement for many scientists, organizations, and companies. The continued development of cryopreservation techniques and their application to new plants is the subject of this issue.     

Effects of cryopreservation of Phaseolus vulgaris L. seeds on early stages of germination

In this work, we studied the effects of cryopreservation on various parameters of early stages of germination of Phaseolus vulgaris seeds (0, 7 and 14?days). Percentages of germination, fresh mass of different plant parts, levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and protein were determined. No phenotypic changes were observed visually in seedlings recovered from cryopreserved seeds. However, several significant effects of seed liquid nitrogen exposure were recorded at the biochemical level. There was a significant negative effect of cryopreservation on shoot protein content, which decreased from 3.11?mg?g^?1 fresh weight for non-cryopreserved controls to 0.44?mg?g^?1 fresh shoot weight for cryopreserved seeds. On the other hand, cryopreservation significantly increased levels of other aldehydes than malondialdehyde in shoots at day 7, from 56.47?μmol?g^?1 for non-cryopreserved controls to 253.19?μmol?g^?1 fresh shoot weight for cryopreserved samples. Liquid nitrogen exposure significantly reduced phenolics contents (free, cell-wall linked, total) in roots at day 7 after onset of germination. In general, roots were more affected by cryostorage compared with other plant parts, while leaves were the least affected. The effects of seed cryopreservation seem to decline progressively along with seedling growth.     

种质资源保存的战略问题和面临的挑战

植物种质资源保存,特别是种子库保存是各种迁地保护措施中最为经济有效的方法。通过对成千上万个物种的有效保存,种子库为减缓物种的灭绝和气候变化对生物多样性的影响发挥了特别关键的作用。本文较为详细地介绍了“中国西南野生生物种质资源库”的立项背景和最新进展,同时介绍了世界上其它几个主要的植物迁地保存设施,特别是英国皇家植物园的“千年种子库”。结合“全球植物保护策略”讨论了中国植物濒危状况,估计我国受威胁的物种比例达20—25％,甚至更高。本文还简要讨论了种子保存中的一些科学问题,包括超低温保存,并强调了植物分类学和种子生物学的学科建设在植物种质资源保护中的重要意义。     

Plant cryopreservation: Progress and prospects

Summary Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cells suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.     

八棱海棠种子超低温保存中含水量对糖代谢的影响

含水量是影响种子超低温保存效果的关键因素，而其作用机制尚不完全清楚。为探讨含水量对种子超低温保存生活力的影响途径，该研究以八棱海棠种子为材料，通过硅胶干燥法获得不同含水量的种子，测定超低温保存后种子生活力、糖含量及相关酶指标的变化并分析相关性。结果表明：(1)超低温保存15 d后，不同含水量种子生活力不同，随着种子含水量的降低，种子生活力呈现先升高后降低的趋势，含水量为9.02%的八棱海棠种子生活力最高，为53.33%;超低温保存120 d后，种子生活力随着含水量下降一直升高，含水量为6.40%生活力最高，为27.78%。这表明八棱海棠种子含水量对超低温保存后的生活力有明显影响，但受液氮保存时间影响，随着液氮保存时间的延长，最适含水量降低。(2)相关分析显示，超低温保存后种子含水量与生活力呈极显著负相关(r=-0.82);与果糖和蔗糖含量、酸性转化酶、果糖激酶呈显著负相关，而种子萌发率与这些指标呈显著正相关。这表明种子含水量通过影响酸性转化酶活性而影响蔗糖和果糖含量，进而影响蔗糖代谢，响应低温和脱水胁迫，最终导致生活力差异。种子生活力还受到介导果糖激酶的果糖代谢影响。此外，海藻糖也是种...     

Transfusion and transplantation of cryopreserved cells and tissues

The modern era of cryomedicine began in 1949 in London and developed world-wide in the second half of the 20th century based on the first report of a novel method of cryopreservation of sperm and erythrocytes using glycerol that was reported in 1949 and 1950 by Polge and Smith. In 1951 at Hradec Kralove, Czech. Klen initiated a "tissue bank" using his unique freeze-drying system. In 1964, the initial meeting of the Society for Cryobiology was organized by its first president. B. J. Luyet in Washington, DC. Cryobiology including cryopreservation and cryosurgery, contributed immense advances for clinical medicine. Cryomedicine will realize the goals of the New Millennium medicine: regeneration, plasticity, and minimally invasive therapy. I explained the first one, regeneration in this paper in detail.Cryomedicine involved subzero-temperatures to freeze the biological objects either for preservation or for destruction. Cryopreservation involves the cooling of the target biological materials to below the temperature of solidification by consumption of energy, through continuously supplying inert cryogens to attain the necessary cryo-temperatures by Joule-Thompson's effect. Therefore biological materials for cryopreservation should be carefully selected and once frozen purposefully kept in the frozen state to be used later to regenerate human cells, tissues and organs, and also to relaize "plasticity". Recently, lyophilization of human cells and tissues came back to the main street of cryopreservation to provide low cost economical and ecological banking of cells and tissues as a hope of the New Millennium. The first attempt of that was made by Prof. Dr. Rudolf Klen and his colleagues.Finally, physicians and related scientists who are going to be interested in cryomedicine should not worry about "freezing and thawing" as being time consuming and labor intensive, otherwise they will not share in the crucial benefits of cryomedicine.     

五种豆科药用植物种子超低温保存技术研究

以豆科药用植物降香檀、决明、含羞草、灰毛豆和猪屎豆的成熟种子为材料,探讨含水量对其发芽率的影响,以及超低温冷冻方式对种子超低温保存的影响。结果表明,降香檀、决明、猪屎豆和灰毛豆种子发芽率均随含水量的下降而从80%左右降至20%以下,而含羞草种子含水量低于10%时,其发芽率仍在75%以上。经超低温冷冻后,五种豆科药用植物种子发芽率较对照组均有显著差异;适宜的含水量下,种子经过超低温冷冻后其发芽率与对照组差异不显著,甚至高于对照组。三种冷冻方法中,玻璃化冷冻法更适合降香檀种子的超低温保存,缓慢冷冻法更适合猪屎豆种子的超低温保存,快速冷冻法适合于决明种子、灰毛豆种子和含羞草种子的超低温保存。由此可知,液氮超低温冷冻法保存降香檀等五种豆科药用植物种子是可行的。     

Consequences of plant tissue cryopreservation (phenotype and genome)

The question about the correspondence of regenerants after cryopreservation to the initial organisms has long been in the focus of attention of cryobiologists. The correspondence has been established for morphological and physiological characteristics and the DNA composition in flower plants and algae. However, at least as many works point to some phenotypic changes and appearance of DNA polymorphism in representatives of both plant kingdoms. Changes in DNA methylation at cytosine after cryopreservation of flower plant tissues have attracted considerable interest. Thus, after freezing and thawing, changes in the phenotype and genome may occur, and the latter involve two mechanisms: mutations and modification of DNA methylation.     

植物种质超低温保存遗传稳定性的研究进展

超低温保存被认为是种质长期保存最有效的方法,其中生物材料低温保存的遗传稳定性是植物种质资源保存中最受关注的问题之一。本文对近年来超低温保存后植物材料的遗传稳定性及变异的研究情况进行了介绍,涉及表型性状分析、基因组遗传稳定性、表观遗传变化及超低温保存的筛选效应等,为进一步研究超低温保存的应用提供参考。     

Conservation between higher plants and the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>in response to the phytohormone abscisic acid: a proteomics analysis

Background  

The plant hormone abscisic acid (ABA) is ubiquitous among land plants where it plays an important role in plant growth and development. In seeds, ABA induces embryogenesis and seed maturation as well as seed dormancy and germination. In vegetative tissues, ABA is a necessary mediator in the triggering of many of the physiological and molecular adaptive responses of the plant to adverse environmental conditions, such as desiccation, salt and cold.     

Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures

Production of vaccines in plant cells provides an alternative system that has several advantages when compared to current vaccine production methods. Establishment of stable seed stocks for a continuous supply of a vaccine is a critical part of production systems. Therefore, a vitrification method for cryopreservation was applied to non-transgenic and three different antigen-expressing transgenic Nicotiana tabacum (NT-1) lines. Preculture of the suspension cultures 1 d prior to vitrification was sufficient for cell survival through the cryopreservation process. Inclusion of 0.3 M mannitol in the preculture medium was necessary for maintenance of cell viability. Cultures were also treated with and without heat shock prior to vitrification, and it was found that heat shock was unnecessary for growth recovery post cryopreservation. All cultures survived storage in liquid nitrogen at intervals ranging from 1 h to 1 yr. Antigen expression was measured by enzyme-linked immunosorbent assay for cultures that grew post cryopreservation and those that had never been cryopreserved. Expression levels in cultures derived from cryopreserved material were comparable to cultures that had not been cryopreserved. Transmission electron microscopy showed that the integrity of the cell structure was maintained post cryopreservation.     

拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

State and Phase Transition Behaviors ofQuercus rubraSeed Axes and Cotyledonary Tissues: Relevance to the Desiccation Sensitivity and Cryopreservation of Recalcitrant Seeds

Freezing and melting transitions of cellular water in embryonic axes and cotyledonary tissues of recalcitrantQuercus rubra(red oak) seeds were compared under slow and rapid cooling conditions. The relevance of desiccation sensitivity (critical water content) and state/phase transition behaviors to cryopreservation was examined. Under a slow to intermediate cooling condition (≤10°C min⁻¹), unfrozen water content in the tissues decreased to less than the critical water content, resulting in a dehydration damage. Under a rapid cooling condition (>100°C min⁻¹) using liquid nitrogen (LN₂), freeze-induced dehydration damage could be avoided if the initial water content was >0.50 g g⁻¹dry wt. However, at water content >0.50 g g⁻¹dry wt, the vitrified cellular matrix was highly unstable upon warming at 10°C min⁻¹. These results offered a theoretical explanation on the difficulty for successful cryopreservation of recalcitrant red oak embryonic axes. A complete state/phase transition diagram for red oak axes was constructed, and a vitrification-based cryopreservation protocol that employed predehydration and rapid cooling was examined. State/phase transition behaviors of cellular water are important parameters for cryopreservation; however, vitrification alone was not sufficient for seed tissues to survive the cryopreservation condition.     

束花石斛种子超低温保存的研究

以濒危兰科物种野生束花石斛（Dendrobium chrysanthum）的成熟种子为材料,研究了PVS2植物玻璃化液对其萌发的作用,以及快速冷冻法和玻璃化法对种子超低温保存的影响。结果表明,种子的萌发率随着PVS2处理时间的延长而下降。PVS2预处理能明显地增加种子的超低温耐性。当预处理时间为15～45min时,种子的超低温耐性随预处理时间的延长而增加;当预处理时间长于60min后,种子的超低温耐性随预处理时间的延长而下降。经液氮保存后,存活的种子能萌发成为正常的幼苗。结论是经PVS2预处理45min后,成熟的束花石斛种子能成功地进行超低温保存。     

Conservation of endemic species from the Russian Far East using seed cryopreservation

A seed response to cryopreservation has been studied in 11 endemic plant species from the Russian Far East to determine the possibility of their long-term storage. It has been shown that the cryogenic treatment does not kill seeds. The viability of 10 species did not decrease (or even increased) after their cryogenic storage. We have not revealed any deviations in the development of plants germinated from seeds stored at an extremely low temperature (−196°C). To monitor germination after long-term storage, procedures of seed germination under laboratory conditions have been developed. The results of this study contribute to the creation of low-temperature seed banks, able to conserve and restore a floral biological diversity.