植物种质资源超低温保存概述

简要回顾了植物种质资源超低温保存的历史,说明了超低温保存植物材料的多样性,阐述了超低温耐性的生物学基础及超低温伤害产生的原因和类型,介绍了各种常用超低温保存方法的技术要点,并对生产顽拗性种子的植物种质资源的超低温保存作了专门的论述,分析了生产顽拗性种子的植物种质资源超低温保存的潜力、现状和困难,指出顽拗性种子的超低温保存是植物种质资源超低温保存的重点和难点,而真正实现用超低温保存技术贮藏顽拗性植物种质资源还有很长的路要走。     

种子顽拗性的形成机理及其保存技术

根据种子的脱水行为将种子分为正常性种子、顽拗性种子和中间性种子。顽拗性种子在发育的末期不经历成熟脱水．脱落时有相对高的含水量,并且对低温和脱水干燥非常敏感。在自然界,顽拗性种子存在一个连续群,即低度、中度和高度顽拗性种子,其差异在于对脱水伤害的敏感程度。影响种子顽拗性的因素,既有种子本身的生理生化物质基础,也有种子在母株上发育过程中所经受的外界环境的影响。目前,对种子脱水耐性的分子机制及其保存技术研究得较多。本文综述了有关顽拗性种子研究的近期进展。     

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。     

种子顽拗性:最新评价

根据种子的脱水行为,可以把种子分为正常性、顽拗性和中间性种子三种类型.种子脱水耐性和脱水敏感性的鉴定是制定种子的贮藏策略和物种基因资源长期保存的依据.除了物种的内在特性外,种子的发育状态、脱水速率、脱水时和重新水合时的环境是影响种子脱水耐性的重要因子.种子的存活率、电解质渗漏速率和存活种子的萌发生长速率是衡量种子脱水耐性的良好的综合参数.种子的脱水耐性是一种数量性状,用"临界含水量"的概念来判断是不正确的,且在评价种子顽拗性中引起了一些混乱.本文还提出了一种全新的评价种子顽拗性的工作模式.     

种子顽拗性：最新评价

根据种子的脱水行为,可以把种子分为正常性、顽拗性和中间性种子三种类型。种子脱水耐性和脱水敏感性的鉴定是制定种子的贮藏策略和物种基因资源长期保存的依据。除了物种的内在特性外,种子的发育状态、脱水速率、脱水时和重新水合时的环境是影响种子脱水耐性的重要因子。种子的存活率、电解质渗漏速率和存活种子的萌发生长速率是衡量种子脱水耐性的良好的综合参数。种子的脱水耐性是一种数量性状,用“临界含水量”的概念来判断是不正确的,且在评价种子顽拗性中引起了一些混乱。本文还提出了一种全新的评价种子顽拗性的工作模式。     

试论种子顽拗性的复合数量性状特征

文章简要概述了从顽拗型种子到顽拗性种子这样一个对顽拗性种子认识提高的过程,在综述了前人关于顽拗性种子数量性状特征论述的基础上,把种子顽拗性的数量性状特征划分为种间、种内和个体发育三个层面,提出种子顽拗性是一种复合性状,其核心至少包括脱水耐性、低温耐性和贮藏耐性三方面的内容,其伴随性状包括种子尺度、千粒重、初始含水量、休眠特性、分类地位、地理分布和保护性物质含量等多方面的内容,而且这些核心内容和伴随性状的每一个方面都具有渐变的、过渡的、数量型的特征,从而阐明种子顽拗性是一种复合数量性状.种子顽拗性的复合数量性状特征的提出及其三个层面的划分,有助于加深对种子顽拗性的认识,并用以指导我们在实践中解决种子顽拗性方面的具体问题.     

顽拗性竹柏种子的贮藏特性?

文章对竹柏( Podocarpus nagi)种子的脱水耐性和贮藏特性进行了研究,结果表明：竹柏种子成熟时初始含水量约为(35±0?7)％,种子对脱水敏感,其最低安全含水量约为(16?86±0?73)％,具有顽拗性种子的典型特征;湿藏和半干藏都可以作为短期保存竹柏种子的方法,且以4℃保存优于15℃保存,但不管种子含水量如何,零下低温保存对竹柏种子都是致命的;半干藏法保存实验中,未进行脱水处理的种子(对照)在4℃贮藏6个月,种子萌发率没有发生明显下降,但贮藏期延长到9个月时,临界含水量的种子萌发力保存最高;不管贮藏介质的含水量高低,也无论贮藏在4℃还是15℃,湿藏种子在9个月的贮藏期内萌发率均没有明显的降低,但当贮藏到12个月时,15℃湿藏种子的萌发率显著高于4℃贮藏的种子,但15℃湿藏的种子在贮藏到3个月时即发现种子在贮藏期间萌发,且随着贮藏介质含水量的升高和贮藏期的延长,萌发的种子增多;竹柏的离体胚经过2h硅胶快速脱水至含水量7％后再冷冻即可获得90％以上的融后存活率,且超低温保存1年的离体胚解冻后,与只保存1周的存活率没有明显差异,表明超低温长期保存竹柏种子是可行的。本研究可以为进一步探究顽拗性种子的短期贮藏和长期保存提供理论基础和基础资料。     

顽拗性竹柏种子的贮藏特性

文章对竹柏(Podocarpus nagi)种子的脱水耐性和贮藏特性进行了研究,结果表明:竹柏种子成熟时初始含水量约为(35±0.7)%,种子对脱水敏感,其最低安全含水量约为(16.86±0.73)%,具有顽拗性种子的典型特征;湿藏和半干藏都可以作为短期保存竹柏种子的方法,且以4℃保存优于15℃保存,但不管种子含水量如何,零下低温保存对竹柏种子都是致命的;半干藏法保存实验中,未进行脱水处理的种子(对照)在4℃贮藏6个月,种子萌发率没有发生明显下降,但贮藏期延长到9个月时,临界含水量的种子萌发力保存最高;不管贮藏介质的含水量高低,也无论贮藏在4℃还是15℃,湿藏种子在9个月的贮藏期内萌发率均没有明显的降低,但当贮藏到12个月时,15℃湿藏种子的萌发率显著高于4℃贮藏的种子,但15℃湿藏的种子在贮藏到3个月时即发现种子在贮藏期间萌发,且随着贮藏介质含水量的升高和贮藏期的延长,萌发的种子增多;竹柏的离体胚经过2 h硅胶快速脱水至含水量7%后再冷冻即可获得90%以上的融后存活率,且超低温保存1年的离体胚解冻后,与只保存1周的存活率没有明显差异,表明超低温长期保存竹柏种子是可行的。本研究可以为进一步探究顽拗性种子的短期贮藏和长期保存提供理论基础和基础资料。     

植物顽拗性种子研究进展

对植物顽拗性种子的概念、物种分类、形态、分布及一些生理生态特征进行了综述,分析了顽拗性种子脱水敏感性的原因和对环境的生态适应性,探讨其可能的进化地位和贮存技术。同时,对顽拗性种子研究的发展趋势和应用前景进行了展望,即加强生境调查,更新顽拗性种子植物数据库,从形态学、生理生态学和分子生物学分析种子顽拗性本质。     

顽拗性种子

本文讨论了顽拗性种子的基本特性,在劣变中的生理生化变化,脱水敏感性的细胞生物学基础,以及贮藏顽拗性种子的必要性和可能性。     

Desiccation and Freezing Sensitivity in Recalcitrant Seeds of Tea, Cocoa and Jackfruit

Investigations were undertaken on the desiccation and freezingsensitivity of recalcitrant seeds of three species: tea [Camelliasinensis (L.) O. Kuntze], cocoa (Theobroma cacao L.) and jackfruit(Artocarpus heterophyllus Lamk.). All species showed changesin the physiological characteristics, desiccation and freezingsensitivity of both the seed and the embryonic axes with increasingseed maturity. Fully mature seeds of tea, cocoa and jackfruitsurvived desiccation to 24, 35 and 31% moisture content, respectively,but at these moisture levels seeds were not able to toleratefreezing in liquid nitrogen (-196 °C). Some survival ofcryopreservation was, however, achieved for excised embryonicaxes of partially and fully-mature tea and jackfruit seeds afterdrying to 14% moisture content; cocoa axes were totally freezingsensitive at all three stages of physiological maturity studied.Biochemical investigations on fully mature axes after desiccationand freezing showed that the decline in viability with moisturelevel was associated with increased leachate conductivity, lipidperoxidation products and/or soluble carbohydrates. Evidencefor disruption of cell membranes during desiccation and freezingwas supported by ultrastructural studies.Copyright 1995, 1999Academic Press Camellia sinensis (L.) O. Kuntze, Theobroma cacao L., Artocarpus heterophyllus Lamk., Phaseolus vulgaris L., tea, cocoa, jackfruit, french bean, seeds, embryonic axes, desiccation, freezing, cryopreservation     

Desiccation sensitivity and successful cryopreservation of oil seeds of European hazelnut (Corylus avellana)

The sensitivity of a Polish provenance of dormant Corylus avellana seeds to extreme desiccation and cryopreservation in liquid nitrogen (LN, ?196°C) was investigated. No sensitivity to desiccation was observed as seeds readily germinated and exhibited seedling emergence, even when the critical water content (WC) of seeds (nuts devoid of pericarp) was reduced to 0.027 g H₂O/g dry mass, g g^?1 by drying. Results of germination and seedling emergence tests indicated that seeds tolerated cooling in LN when desiccated to a WC in the range of 0.05–0.10 and 0.08–0.10 g g^?1, respectively. The results of this study demonstrate the feasibility of long‐term cryopreservation of European hazelnut seeds. As the seeds of this species have been classified in several different categories (orthodox, suborthodox and recalcitrant), based on their response to desiccation and low temperature, the assignment of the seeds of hazelnut to a specific category is provided and discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> conservation of Malaysian biodiversity—achievements,challenges and future directions

Malaysia is fortunate and proud to contain some of the world’s richest biodiversity. In Malaysia, there are an estimated 185,000 species of fauna and 12,500 species of flowering plants, many of which are endemic to tropical forests in this region. Indeed, such diversity is an important and invaluable national asset to safeguard both present and future generations. In vitro conservation offers possible techniques for the preservation of plant germplasm that at present is difficult to maintain or is maintained with limited success. Research at the Universiti Kebangsaan Malaysia (The National University of Malaysia) focuses on the cryopreservation of woody fruit species with seeds that cannot tolerate cryopreservation (recalcitrant or intermediate). Among the plants with recalcitrant seeds are such traditionally important edible tropical fruits as mangosteen, langsat, and rambai (Garcinia mangostana, Lansium domesticum, and Baccaurea motleyana). Citrus aurantifolia, Citrus suhuiensis, Citrus madurensis, Citrus hystrix, and Fortunella polyandra are among the Citrus and Citrus-related species studied. Cryopreservation studies include the Nepenthes species (pitcher plants) of Malaysia. Fundamental research on desiccation and low-temperature tolerance and on the physiology of desiccation are used to understand seed behavior, a prerequisite for the development of successful conservation techniques. At the same time, cryopreservation protocols for several Citrus and forestry species were developed for embryonic axes and adventitious shoots, mainly using rapid dehydration and PVS2 vitrification techniques. There are no successful standard techniques or protocols for species with highly recalcitrant seeds such as Garcinia species. Modification of existing protocols or development of new methods is required, but this can be accomplished only when a detailed understanding of the recalcitrant nature of the seeds or explants is achieved. While we have considerable knowledge concerning the basics of biochemical processes and some molecular data from work on desiccation-tolerant seeds, a great need remains for understanding the cause of the recalcitrance or desiccation sensitivity of these seeds. It may be necessary to use a systems biology approach that exploits the “omics” technologies to generate global molecular data. In combination with bioinformatics for data integration and analyses, this approach would move toward improved modeling of the biological pathways associated with the development of recalcitrant seeds.     

Homoiohydrous (recalcitrant) seeds: Developmental status,desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer

The desiccation sensitivity in relation to the stage of development was investigated in embryonic axes from the homoiohydrous (recalcitrant) seeds of Landolphia kirkii. Electrolyte leakage, used to assess membrane damage after flash (very rapid) drying, indicated that axes from immature (non-germinable) seeds were the most desiccation-tolerant, followed by those from mature seeds, while axes from seeds germinated for increasing times were progressively more desiccation-sensitive. Differential scanning calorimetry was used to study the relationship between desiccation sensitivity and the properties of water in the tissues. Axes from immature seeds had a lower content of non-freezable water than that of any other developmental stage and a higher enthalpy of melting of freezable water. For mature and immature axes electrolyte leakage increased at the point of loss of freezable water. At other developmental stages the water content at which electrolyte leakage increased markedly correlated with the other properties of the water, such as the change in the shape of the melting endotherm and the onset temperature. Ultrastructural studies of axes at the various developmental stages showed changes in the degree and pattern of vacuolation, the presence and quantities of lipid and starch, and the degree of endomembrane development. The results are discussed in relation to current hypotheses on the basis of desiccation tolerance.Abbreviation DSC differential scanning calorimetry     

Water relations in germinating seeds

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.     

木波罗种子脱水敏感性与膜脂过氧化的研究

刚采收的木波罗种子含水量为５８．６％。随着含水量下降,种子的发芽率和发芽指数迅速下降,种子对脱水非常敏感,是典型的顽拗性种。自然脱水时,种子胚轴和子叶中超氧物歧化酶的活性先上升,然后下降,丙二醛和脂质氢过氧化物的含量显著增加。其脱水敏感性的原因可能是当种子脱水时,植物酶ＳＯＤ的活性下降,膜脂过氧化作用加强,从而使膜的结构和功能受到破坏,种子生活力丧失。     

Differential responses of Mimusops elengi and Manilkara zapota seeds and embryos to cryopreservation

Several dehydration protocols were evaluated for their ability to cryopreserve intact seeds and excised embryonic axes of Mimusops elengi and Manilkara zapota (Sapotaceae). Both interspecific and intraspecific variations in cryotolerance were found. M. zapota embryonic axes were more tolerant of cryopreservation than those of M. elengi, and showed higher desiccation tolerance, higher post-thawing survival and development, and a much wider range of moisture contents for cryopreservation. Maximum development rates were 94% and 27% for M. zapota and M. elengi, respectively. Intact seeds of both species tolerated desiccation to low moisture levels, but were sensitive to liquid nitrogen exposure, and cryopreserved seeds failed to germinate. Assessment of developing embryos excised from cryopreserved seeds associated nonviability of cotyledons and plumules with germination failure. Other structures survived at variable rates; most hypocotyls and radicles (up to 76% and 98% for M. elengi and M. zapota, respectively) were viable. The different cryotolerance between hypocotyls and cotyledons is a critical cause for failure in cryopreservation, contributing to the difficulty in developing protocols for such intermediate oily seeds.     

An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut ( Castanea sativa ). Axis excision was accompanied by a burst of superoxide (O₂^·−), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial 'burst' in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O₂^·− production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.     

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.     

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.