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。     

植物种质资源的保存

传统的保存植物种质资源的方法是建立种子库。采用低温保存可大大延缓正常性种子的寿命,但采用此法仍难以保存顽拗性种子。和种子库相比,采用植物细胞、组织或器官,能最大限度地抑制生理代谢强度,降低劣变发生频率,达到长期保存种质的目的。     

植物种质资源超低温保存现状及其研究进展

本文根据联合国粮农组织2010年发布的世界各国的《第二份世界粮食和农业植物遗传资源现状报告以及有关国际会议和相关文献资料,从超低温保存材料类型、基本程序、方法技术、理论基础、影响因素、保存费用、保存策略和保存现状、实际应用等方面综述了全球植物种质资源超低温保存现状及其研究进展,展望了植物种质资源超低温保存技术的应用前景,旨在加强非正常型种子植物种质资源的安全长期保存。文章最后分析了我国存在的差距,提出了今后努力方向和发展的建议。     

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     

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。     

枇杷茎尖二步玻璃化法超低温保存的研究

超低温保存是目前植物种质资源长期稳定保存最理想的方法,而近几年发展的玻璃化超低温保存法具有设备要求简单、材料处理步骤简便及效果和重演性好等特点,倍受人们的青睐。国内外用玻璃化法成功地保存许多果树的种质资源。在对枇杷（Eriobotrya japonica Lindl．）花粉超低温保存取得成功的基础上,作者进行了枇杷茎尖玻璃化超低温保存的研究,以期建立枇杷茎尖超低温保存体系,为长期稳定保存枇杷种质资源提供技术支持。     

猕猴桃茎段的超低温保存

近年来的研究结果表明,超低温(-196℃)冰冻保存是长期保存植物种质最理想的方法,并已在40多种植物上取得初步成功。我国猕猴桃的品种资源十分丰富,但其种质的超低温保存尚未见报道。我们以猕猴桃茎段为材料进行了试验,试材经120天的液氮贮存后,在再培养中表现出很高的存活率,并产生出大量的新植株。现将这一初步结果简报如下。     

香蕉茎尖超低温保存过程中的细胞超微结构观察(简报)

超低温保存(Cryopreservation)通常称为液氮保存或LN(-196℃)保存,是目前植物种质资源长期稳定保存的理想方法,已经成功应用于多种植物种质资源保存。玻璃化法(Vitrification)超低温保存植物种质资源始于20世纪80年代末,Uagami等首次     

桃花粉低温和超低温保存方法比较研究

桃(Prunus persica(L.)Batsch)是我国重要的无性繁殖作物种质资源,目前主要保存于3个国家无性繁殖作物种质圃。随着以茎尖、花粉、休眠芽为保存载体的超低温保存技术的发展,超低温保存已成为无性繁殖作物重要备份保存方式。本研究以15份桃种质花粉为研究对象,开展含水量、回湿处理和保存温度(4℃低温保存和液氮超低温保存)对保存后花粉离体萌发率的影响研究。研究结果:明确了桃种质花粉超低温保存的含水量;揭示了回湿处理对部分桃种质花粉超低温保存产生显著影响;超低温保存后花粉离体萌发率最高可达83%;4℃低温保存和超低温保存比较研究结果表明,超低温保存4年后14份桃种质花粉离体萌发率仍可保持30%以上,11份桃种质花粉离体萌发率与保存前花粉离体萌发率相比无显著变化甚至显著提高,而4℃低温保存的花粉离体萌发率降至0。该研究为国家种质库建立花粉规模化超低温保存提供技术支撑。     

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。     

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.     

超低温保存植物种质资源的新途径——小滴玻璃化法

超低温保存是一种安全、有效的种质资源保存途径,可长期保存种质资源。小滴玻璃化法是在滴冻法和玻璃化法上基础上发展起来的用于植物种质资源保存的新技术。本文综述了该方法的技术概念、主要优点、基本程序、应用前景及国内外研究现状。     

Cryopreservation of shoot tips of recalcitrant and tropical species: Advances and strategies

There is a pressing need for practical and successful conservation efforts to establish long-term germplasm collections of recalcitrant and tropical species, given the challenge and threat that these plants are facing. Cryopreservation is the only way of conserving some of these species, especially those with temperature or desiccation sensitive (recalcitrant) seeds. This review covers reports on cryopreservation studies of shoot tips (apical and axillary) of tropical and subtropical plants. Since many of these species have recalcitrant seeds, the cryopreservation successes, failures and problems involved with these seeds are also discussed. The methodologies, important factors and steps involved in successful cryopreservation protocols are analyzed. Finally strategies are suggested to develop a successful cryopreservation protocol for new plant species, in particular those with tropical recalcitrant seeds.     

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.     

果树种质资源超低温保存研究进展

综述了国内外在超低温保存果树种质资源方面的最新研究成果,从材料选择、材料预处理、冰冻保护剂、冰冻方法及超低温保存对组织超微结构的影响等几个方面总结了果树超低温保存的原理、影响因素及关键操作技术,展望了果树种质资源超低温保存技术的应用前景.     

植物种质的玻璃化超低温保存

植物种质的玻璃化超低温保存技术已受到广泛重视。玻璃化法主要由装载、玻璃化保护液脱水、降温、复温、洗涤这5个环节构成。目前已对百余种植物进行过玻璃化冻存研究,但主要应用于高等植物,而用该法保存藻类获得成功的报道很少。将玻璃化法用于某些藻类种质的冻存将会有广阔的应用前景。     

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.

     

包埋玻璃化法超低温保存植物种质的研究进展

包埋玻璃化法是在玻璃化法和包埋脱水法基础上发展起来的超低温保存植物种质的新技术.它具有能同时处理大量材料,处理后恢复生长快,对材料的毒害作用较小及成芽率高等优点,已成功地用于辣根、山嵛菜等20余种植物,在植物种质资源的保存上显示出了巨大的应用潜力.本文介绍了包埋玻璃化法产生的背景及其优点,阐述了包埋玻璃化法的基本方法和预培养、包埋、脱水、化冻及恢复培养等过程,比较了该法冻存后的效果和冻存后所形成植株的遗传稳定性,同时指出了进一步研究的重点.     

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.