Dehydration in cryopreservation of moist plant tissues and seed maturation

The feasibility of long-term cryopreservation of plant objects depends on their moisture content. In orthodox seed, dehydration takes place at the late stage of maturation and is accompanied by accumulation of protective sugars and proteins. Such seed easily withstand cryopreservation. Moist objects such as meristems and recalcitrant seed must be desiccated in the presence of sucrose before freezing. In both naturally dry and dehydrated tissues, differential scanning calorimetry and electron paramagnetic resonance reveal intracellular vitrification; this glassy state is supposed to determine the success of cryostorage. Methodical approaches to cryopreservation of moist plant tissues are reviewed.     

The ascorbate system in recalcitrant and orthodox seeds

Recalcitrant seeds of Ginkgo biloba L., Quercus cerris L., Aesculus hippocastanum L. and Cycas revoluta Thunb. are shed by the plant at a high moisture content, contain a large amount of ascorbic acid (ASA) and maintain high ascorbate (ASC) peroxidase (EC 1.11.1.11) activity. Three proteins showing ASC peroxidase activity are present in G. biloba seeds. Conversely, dry orthodox seeds ( Vicia faba L., Avena sativa L., Pinus pinea L.) are completely devoid of ASA and ASC peroxidase. Experimentally induced rapid variations of the water level in both recalcitrant and orthodox seeds do not affect the ASC peroxidase; slow dehydration affects the ASC peroxidase activity moderately in recalcitrant seeds, but provokes a complete loss of germinability. Another peculiar difference between orthodox and recalcitrant seeds concerns the ascorbate recycling enzymes, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and dehydroascorbate (DHA) reductase (EC 1.8.5.1). The DHA reduction capability is low in recalcitrant seeds, but is high in the orthodox ones. In contrast, AFR reductase activity is high in recalcitrant seeds and low in the orthodox ones. Data reported here concerning the ASC system appear to contribute to better understanding the recalcitrance. The presence of three different proteins showing ASC peroxidase activity in the archaic seed-bearing plant G. biloba and its involvement in the spermatophyte evolution is discussed.     

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.     

Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival

In most species, arrest of growth and a decrease in water content occur in seeds and pollen before they are dispersed. However, in a few cases, pollen and seeds may continue to develop (germinate). Examples are cleistogamy and vivipary. In all other cases, seeds and pollen are dispersed with a variable water content (2-70%), and consequently they respond differently to environmental relative humidity that affects dispersal and maintenance of viability in time. Seeds with low moisture content shed by the parent plant after maturation drying can generally desiccate further to moisture contents in the range of 1-5% without damage and have been termed 'orthodox'. Pollen that can withstand dehydration also was recently termed orthodox. Seeds and pollen that do not undergo maturation drying and are shed at relatively high moisture contents (30-70%) are termed 'recalcitrant'. Since recalcitrant seeds and pollen are highly susceptible to desiccation damage, they cannot be stored under conditions suitable for orthodox seeds and pollen. Hence, there are four types of plants with regard to tolerance of pollen and seeds to desiccation. Orthodoxy allows for dispersal over greater distances, longer survival, and greater resistance to low relative humidity. The advantage of recalcitrance is fast germination. Orthodoxy and recalcitrance are often related to environment rather than to systematics. It has been postulated that certain types of genes are involved during presentation and dispersal of pollen and seeds, since molecules (sucrose, polyalcohols, late embryogenic abundant proteins, antioxidants, etc.) that protect different cell compartments during biologically programmed drying have been detected in both.     

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...     

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.     

Global Changes in DNA Methylation in Seeds and Seedlings of Pyrus communis after Seed Desiccation and Storage

The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m⁵C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m⁵C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2–3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m⁵C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds.     

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。     

DNA methylation of Quercus robur L. plumules following cryo-pretreatment and cryopreservation

Pedunculate oak (Quercus robur) is an ecologically and economically important forest tree species which produces seeds that are classified as recalcitrant. Thus, cryopreservation of seed meristems is a method for long-term preservation of this germplasm in gene banks. During cryopreservation, many factors, such as desiccation, cryoprotection and cooling/rewarming, can induce stress in the frozen meristems. In this study, in vitro survival and the global DNA methylation level of plumules after cryoprotection, desiccation and cryostorage was evaluated. Results indicated that both desiccation and storage in liquid nitrogen have negligible influence on DNA methylation status of Q. robur plumules. These findings support the cryopreservation of plumules as an appropriate method for conservation of Q. robur germplasm.     

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。     

Orthodox Behaviour of Oil Palm Seed and Cryopreservation of the Excised Embryo for Genetic Conservation

Desiccation experiments have shown the oil palm seed to be orthodox,not recalcitrant, in character. There is a significant differencein water content between the whole seed and the embryo whichis maintained despite desiccation, and results in the failureof sub-zero storage of whole seeds. The successful recoveryof viable, excised embryos from liquid nitrogen, and their subsequentregrowth in vitro, suggests a practical technique for the longterm conservation of the genetic resources of the species. Elaesis guinensis L, oil palm embryo, cryopreservation, recalcitrant seed     

The effect of time of harvest on the viability and pre-emergence mortality in soil of pea (Pisutn sativum L.) seeds

A series of harvests from field plots of peas showed that the younger seeds were at the time of harvest the lower was their percentage viability and the greater their percentage mortality in soil. Young seed also showed a high degree of solute leaching into steep water and had a high moisture content at harvest compared with older seeds. Heavy rainfall just prior to harvesting reduced the percentage viability of the dried seed and increased the moisture content of the seed at harvest as well as the leaching of solutes from the dried seed into steep water. Comparisons of moisture content at harvest and seed condition after drying suggested that cotyledons and embryo axes were only able to withstand enhanced drying after removal from the plant if the seed moisture content had already begun to decline whilst the seed was still on the plant.     

Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.     

Seed development and maturation in Phaseolus vulgaris I. Ability to germinate and to tolerate desiccation

The onset and development of both the ability to germinate andto tolerate rapid enforced desiccation were investigated duringthe development and maturation of seeds of bean (Phaseolus vulgahsL.) at different temperatures and also after different slow-dryingtreatments. The onset of germinability occurred when seeds wereless than half-filled in the absence of both a post-ovule abscissionprogramme and water loss from the seeds. Maximum ability togerminate normally and maximum tolerance to rapid enforced desiccationto 14–16% moisture content did not occur until 2–23d and 6–23 d after mass maturity (end of the seed-fillingperiod), respectively. The slow-drying of immature seeds for7 d ex planta before rapid enforced desiccation increased theability to germinate and stimulated the onset of desicationtolerance. Holding seeds moist for 7 d (during which time moisturecontent declined by <5%) had similar effects, but seed germinationafter rapid enforced desiccation was consistently greater inseeds first dried slowly than held moist. Comparisons betweenseeds less than half-filled dried slowly ex planta and fullseeds undergoing maturation drying in planta showed that a similar(slow) rate of water loss over a 7 d period had a similar effecton the subsequent ability of seeds to tolerate rapid enforceddesiccation. Thus, neither a post-ovule abscission programmenor loss of water were required for the onset of the abilityto germinate in developing bean seeds, but both were requiredfor the development of the ability to germinate and resistanceto solute leakage, when rehydrated, after rapid enforced desiccation. Key words: Bean, Phaseolus vulgaris L., seed germination, seed development, desiccation tolerance     

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.     

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。     

The use of plant stress biomarkers in assessing the effects of desiccation in zygotic embryos from recalcitrant seeds: challenges and considerations

Zygotic embryos from recalcitrant seeds are sensitive to desiccation. In spite of their sensitivity, rapid partial dehydration is necessary for their successful cryopreservation. However, dehydration to water contents (WCs) that preclude lethal ice crystal formation during cooling and rewarming generally leads to desiccation damage. This study investigated the effects of rapid dehydration on selected stress biomarkers (electrolyte leakage, respiratory competence, rate of protein synthesis, superoxide production, lipid peroxidation, antioxidant activity and degree of cellular vacuolation) in zygotic embryos of four recalcitrant‐seeded species. Most biomarkers indicated differences in the levels of stress/damage incurred by embryos dried to WCs < and >0.4 g·g^?1, within species; however, these changes were often unrelated to viability and percentage water loss when data for the four species were pooled for regression analyses. Dehydration‐induced electrolyte leakage was, however, positively related with percentage water loss, while biomarkers of cellular vacuolation were positively related with both percentage water loss and viability. This suggests that electrolyte leakage and degree of cellular vacuolation can be used to quantify dehydration‐induced stress/damage. Biomarkers such as superoxide production, whilst useful in establishing the nature of the dehydration stress incurred may not be able to distinguish the effects of different WCs/drying times. Irrespective of which biomarker is used, the data suggest that understanding differences in desiccation sensitivity across recalcitrant‐seeded species will remain a challenge unless these biomarkers are related to a generic desiccation stress index that integrates the effects of percentage water loss and drying time.     

Water and Seed Survival

Between about –350 and –14 MPa the rate of lossof viability in orthodox seeds is a positive function of waterpotential. The relative effect of water potential has been analysedin an oily seed (lettuce) and a non-oily seed (barley) and foundto be more or less identical. The lower limit for the relationin various species coincides with a seed moisture content (wetbasis) between about 2 and 6%. Below this level there is littleor no improvement in longevity with reduction in moisture content.The upper limit coincides with moisture contents of between15 and 28%, depending on whether the seeds are oily or non-oily.A water potential of about –14 MPa is the threshold forrespiration which increases more-or-less linearly with increasein water potential above this level. Above this threshold, andproviding oxygen is available to sustain respiration, seed longevityincreases with increase in water potential except that, unlessthe seeds are dormant, germination may be initiated at a waterpotential of about –1·5 to –0·5 MPa.In the absence of oxygen there may be a slight further declinein longevity with increase in water potential above –14MPa before longevity reaches a minimum value Since they cannot be dried very much without immediate lossof viability, recalcitrant seeds survive longest in the presenceof oxygen at maximum water potential commensurate with preventinggermination. The threshold water potential for immediate lossof viability has not been determined for most species but itis probable that it is close to the water potential typicalof the permanent wilting point in these plants, say –2MPa Lactuca saliva L., lettuce, Hordeum oulgare L., barley, seed storage, moisture content, relative humidity, water potential, temperature, oxygen