Dehydration effects on imbibitional leakage from desiccation-sensitive seeds

Changes in electrolyte leakage and viability in response to dehydration stress were examined in two species of seeds that do not survive desiccation. Leakage from silver maple (Acer saccharinum L.) seeds increased markedly as seed moisture contents decreased from 45 to 35% (fresh weight basis) and germination decreased from 97 to 5%, coincidentally. Time course curves of imbibitional leakage from areca palm (Chrysalido-carpus lutescens [Bory] Wendl.) embryos showed an increase in both initial leakage and steady-state leakage rates in response to dehydration from an original moisture content of 84 to as low as 53%. Absorbance at 530 nanometers of extracts from triphenyl tetrazolium chloride stained embryos of areca palm was used as a measure of viability. Absorbance decreased significantly in response to dehydration as embryo moisture content decreased from 80 to 30%. Collectively, the data suggest that membranes in the desiccation-sensitive seed tissues studied are damaged by dehydration below a critical moisture content, 40% in silver maple seed and 55% in areca palm embryos, and that the membrane damage contributes to loss of viability.     

The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees

Proteins that have homology with dehydrins have been identified immunologically in the desiccationsensitive (recalcitrant) seeds of English oak (Quercus robur L.), European chestnut (Castanea sativa L.), horse chestnut (Aesculus hippocastanum L.), sycamore (Acer psuedoplatanus L.) and silver maple (Acer saccharinum L.), and in the desiccation-tolerant seeds of Norway maple (Acer platanoides L.). The mRNA for a late embryogenesis abundant (LEA) protein (dehydrin) was also detected by Northern blotting, using a cDNA clone (D11) from cotton embryos, in the recalcitrant and orthodox seeds. Medium-stringency washing was required to detect this hybridization. InQ. robur the amount of dehydrin protein increased during seed development, andLEA mRNA was induced by limited desiccation and by abscisic acid. Confirmation of the presence of dehydrin mRNA in matureQ. robur andC. sativa seeds was obtained by in-vitro translation of the extracted polyadenylated RNA followed by analysis of the immunoprecipitation products. Thus the presence of dehydrin proteins is not sufficient to confer desiccation tolerance on truly recalcitrant seeds, nor can their presence or absence be used as clear criteria for identification of recalcitrant seeds.     

Desiccation and storage studies on Dipterocarpm seeds

The relationship of seed moisture content (fresh weight basis) to germination, and the effect on viability of various storage conditions were examined for five species of the tropical forest tree genus Dipterocarpus. It was shown that seeds fall into two groups with regard to desiccation tolerance. Firstly, D. obtusifolius and D. turbinatus cannot be dried below about 45% moisture content without damage; a sigmoid curve was found to fit the relationship between germination and moisture content for the latter species. Secondly, D. intricatus, D. tuberculatus and D. alatus can be safely dried to 10%, 12% and 17% moisture contents respectively, but desiccation to near 7% moisture content reduced viability by at least a half. Storage studies showed that seed of D. intricatus and D. tuberculatus possessed increased longevity as moisture contents were reduced within the range 6–20%. It was concluded that seeds in the first group are ‘recalcitrant’ and that those in the second group are ‘orthodox’ in their storage physiology, according to the categories described by Roberts (1973). Wide differences between species in seed desiccation rates were observed. In 15% relative humidity D. intricatus dried to 7% moisture content within a week, whilst D. obtusifolius retained 30% moisture content even after 5 wk; other species had intermediate desiccation rates. Seed size and structure may partly account for the differences observed. Correlations were observed between seed storage physiology and other factors which were investigated. ‘Orthodox’ seeds had quicker desiccation rates, were derived from drier habitats, and had smaller embryos than those of ‘recalcitrant’ seeds. ‘Orthodox’ seeds, with the possible exception of D. alatus, should be kept at 0–3°C with about 12% moisture content in the short term and, provided less than 10% germination is lost on freezing, at-18°C with about 8% moisture content in the long term. ‘Recalcitrant’ seeds should be stored in ventilated containers at 21°C and with moisture contents above 45–50%.     

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。     

Effects of Moisture Content on Germination of Seeds of Hancornia speciosa Gom. (Apocynaceae)

Seeds of Hancornia speciosa germinated best at a temperatureof 20–30 °C. The viability of the seeds during storagewas short and the best storage conditions for viability entailedkeeping the seeds in polyethylene bags. Seed viability was maintainedonly when the seeds were stored at a moisture content above30%; storage conditions which allowed dehydration resulted ina rapid loss of viability (the seeds showed recalcitrant behaviour). Low temperature during storage did not improve longevity. Arelationship between germination and moisture content was established,but when the moisture content fell below 25% there was a drasticreduction of germination. After 9 weeks of storage, even athigh moisture content, seeds lost viability. Loss of seed viability during seed dehydration was associatedwith increased leakage of electrolytes and organic solutes,and reduced tetrazolium staining during subsequent imbibition. Hancornia speciosa, germination, recalcitrant seeds, storage, moisture     

Desiccation Intolerance in Seeds of Zizania palustris is Not Related to Developmental Age or the Duration of Post-harvest Storage

Freshly harvested seeds of Zizania palustris L. representingfour distinct stages of development were intolerant of desiccation.Rapid drying did not improve survival. The relationship betweenprobit viability and embryo moisture content for freshly harvestedmature seeds was identical to that described in an earlier publicationfor seeds previously stored fully imbibed at low temperaturesto remove dormancy. A small reduction in embryo moisture contentduring the first 3 d of drying at 15% r.h. and 15°C ledto a highly sigificant (P < 0.01) increase in the proportionof fresh seeds capable of germination in response to a rangeof dormancy breaking factors including giberellins. Resultsare discussed in relation to previous work on recalcitrant seedsand to the widely reported developmental changes in orthodoxseeds which are triggered by desiccation. Zizania palustris, wild rice, seeds, drying, desiccation intolerance, development     

Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desiccation sensitivity

Oxidative damage originating from uncontrolled metabolism is thought to be responsible for the sensitivity to drying in recalcitrant seeds. This study compares the responses of respiration to drying and the loss of membrane integrity in isolated axes and cotyledons of the recalcitrant seeds of Castanea sativa Mill. Electron spin resonance spectroscopy of two nitroxide spin probes introduced into the seed tissues was used to assess the cytoplasmic viscosity and the membrane permeability during fast and slow drying. Drying rates had not effect on the rise in viscosity in axes and cotyledons. In both tissues, the cytoplasmic viscosity during drying remained constant at 0.2 Poise until 1.6 g water/g DW (g/g), thereafter it increased exponentially. Axes were found to be more tolerant to drying than cotyledons: membranes showed minor changes in their permeability during drying and 50% viability was retained in dried axes containing 0.12 g/g. In contrast, plasma membranes in cotyledons lost their integrity below 0.6 g/g, regardless of the drying rate. Drying axes and cotyledons exhibited contrasting responses of their metabolism to drying. At the onset of drying, the rates of O2 uptake declined rapidly in drying axes. However, respiration in drying cotyledons sequentially increased to c. 1.4-fold at 1.2 g/g then decreased concomitantly with the loss of membrane integrity. The respiratory quotients (CO2 output/O2 input) remained constant around 0.9 until the loss of membrane integrity, then rose to 2.8. As a symptom of mitochondrial injury, the levels of reduction of cytochromes were assessed in situ in fresh and dried cotyledons using light spectroscopy. The levels of reduced cytochrome c and aa3 were lower in dried C. sativa cotyledons than in dried orthodox cotyledons of cowpea, indicating that a disruption in the electron transport chains may have occurred during drying. Desiccation sensitivity in recalcitrant seeds may be due to the inability to actively depress their metabolism during drying, thereby increasing the chances of initiating peroxidative damage during drying.Key words: Castanea satia, membrane permeability, recalcitrant seed, respiration, viscosity.      

Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds

Ascorbate–glutathione systems were studied during desiccation of recalcitrant seeds of the silver maple (Acer saccharinum L.). The desiccated seeds gradually lost their germination capacity and this was strongly correlated with an increase in electrolyte leakage from seeds. Simultaneously the increase of reactive oxygen species (ROS) (superoxide radical – O₂⁻ and hydrogen peroxide – H₂O₂) production was observed. The results indicate that remarkable changes in the concentrations and redox status of ascorbate and glutathione occur in embryo axes and cotyledons. After shedding, concentrations of ascorbic acid (ASA) and the reduced form of glutathione (GSH) are higher in embryo axes than in cotyledons and their redox status is high in both embryo parts. Cotyledons in freshly shed seeds are devoid of GSH. At the first stages of desiccation, up to a level of 43% of moisture content, ASA content in embryo axes and GSH content in cotyledons increased. Below this level of moisture content, the antioxidant contents as well as their redox status rapidly decreased. The enzymes of the ascorbate–glutathione pathway: ascorbate peroxidase (APX) (EC 1.11.1.11), monodehydroascorbate reductase (MR) (EC 1.6.5.4), dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) increased their activity during desiccation, but mainly in embryonic axes. The changes are probably required for counteracting the production of ROS during desiccation. The relationship between ascorbate and glutathione metabolism and their relevance during desiccation of recalcitrant Acer saccharinum seeds is discussed.     

Homeohydrous (Recalcitrant) Seeds: Dehydration, the State of Water and Viability Characteristics in Landolphia kirkii

Differential scanning calorimetry was used to study the relationships among drying rate, desiccation sensitivity, and the properties of water in homeohydrous (recalcitrant) seeds of Landolphia kirkii. Slow drying of intact seeds to axis moisture contents of approximately 0.9 to 0.7 gram/gram caused lethal damage, whereas very rapid (flash) drying of excised embryonic axes permitted removal of water to approximately 0.3 gram/gram. The amount of nonfreezable water in embryonic axes (0.28 gram H₂O/gram dry mass) did not change with drying rate and was similar to that of desiccation-tolerant seeds. These results suggest that the amount of nonfreezable water per se is not an important factor in desiccation sensitivity. However, flash drying that removed all freezable water damaged embryonic axes. Differences between desiccation-sensitive and -tolerant seeds occur at two levels: (a) tolerant seeds naturally lose freezable water, and sensitive seeds can lose this water without obvious damage only if it is removed very rapidly; (b) tolerant seeds can withstand the loss of a substantial proportion of nonfreezable water, whereas sensitive seeds are damaged if nonfreezable water is removed.     

The protective role of selenium in recalcitrant Acer saccharium L. seeds subjected to desiccation

Freshly harvested silver maple (Acer saccharinum L.) seeds were soaked in either sodium selenite (10 mg/L) or water for 6 h. After washing and air drying, seeds were desiccated at 22 °C at a RH of 45-50% to comparable water levels from 50 to 12%. Germination capacity was significantly higher in seeds treated with selenium and desiccated [from 50 to 40, 35 and 30% of water content (WC)] than in water-soaked seeds. At 20% WC, the seeds from both treatments had low viability (approximately 20%). The electrolyte leakage and the MDA content were significantly lower in the embryonic axes of seeds soaked in selenite than in seeds soaked in water. We also found that the activity of glutathione peroxidase (GPX) of embryonic axes from selenium-treated seeds that were not desiccated, or from seeds that were desiccated to 40 and 35% WC, was significantly higher than that of non-treated axes. No difference in GPX activity was detected in cotyledons. This was confirmed by activity staining of GPX after native PAGE of proteins extracted from embryonic axes and cotyledons. An increase in glutathione reductase (GR) activity was also observed in embryonic axes of seeds treated with selenium and dried to 35 and 30% WC compared to non-treated samples. Selenium appeared to have no such effect on cotyledons.     

Germination-associated events and the desiccation sensitivity of recalcitrant seeds — a study on three unrelated species

The storage behaviour of recalcitrant seeds was assessed using three diverse species: a gymnosperm, Araucaria angustifolia (Bert.) O. Kuntze; a herbaceous monocotyledon, Scadoxus membranaceus (Bak.) Friis Nordal; and a woody dicotyledon, Landolphia kirkii Dyer. Seeds were stored under conditions of high relative humidities that maintained seed moisture content and under low relative humidities that caused drying. At regular intervals moisture content was determined, germinability assessed and the ultrastructure of radicle meristem cells examined. Under storage at high relative humidity, seed moisture content was maintained at the original level and subcellular germination events were initiated in the short-term. Such seeds showed enhanced rates of germination when removed from storage and planted. Long-term storage under these conditions resulted in the initiation of subcellular damage which intensified with time and ultimately resulted in the loss of viability. The rate at which germination events proceeded varied among the three species, and could be directly correlated with the period of viability retention under humid storage conditions. Storage under desiccating conditions resulted in subcellular damage and rapid loss of viability. The rate at which the seeds dried varied among the three species. The proportion of water loss tolerated by the different species before loss of viability, correlated with the rate of drying. The storage behaviour of the seeds of these three species is discussed in terms of a previously described model.     

Viability of rape (Brassica napus L.) seeds following selection on the basis of newly-emerged radicles then subsequent drying and storage

Germinating rape seeds selected on the basis of newly-emerged radicles (1 ± 0.5 mm) were dried to an equilibrium moisture content (c. 11%) in air at 20°C and 80% relative humidity without loss of viability. Storage life of these low-moisture-content germinating (LMCG) seeds at 15°C was limited to 7 days before viability was significantly reduced. However, viability of LMCG seeds was maintained for 84 days in storage at -20°C. Longer periods in store reduced viability, but 96% of seeds still remained viable after 336 days at - 20°C. Increasing periods of storage at -20°C reduced the subsequent seed longevity at 15°C, indicating a reduction in vigour during storage. Storage under reduced pressure or in a nitrogen atmosphere had little significant effect on seed longevity. Reduction of moisture content below 11% using vacuum drying at a range of temperatures reduced seed vigour.     

Changes in abscisic acid levels in embryo axes of Norway maple and sycamore seeds during maturation and dehydration

Changes in the abscisic acid (ABA) levels in embryo axes of seeds, belonging to the orthodox (Norway maple — Acer platanoides L.) and recalcitrant (sycamore — Acer pseudoplatanus L.) categories, were investigated throughout maturation using an ELISA (enzyme-linked immunosorbent assay) test. Concentration of ABA in embryo axes substantially differed depending on species and sampling date. ABA was always higher in Norway maple except at the end of seed maturation when ABA content was similar in both species. During maturation ABA decreased in both species but the decline was more marked in Norway maple than in sycamore (11 vs. 3 fold). These species also differed in the pattern of ABA changes, which in sycamore embryo axes was very regular, while in Norway maple a sharp decrease was recorded after acquisition by the seeds of tolerance to desiccation. Dehydration of embryo axes of Norway maple caused a further significant decrease of ABA level. In contrast, in dehydrated sycamore embryo axes ABA content did not decrease, but slightly increased. The role of ABA in desiccation tolerance and dormancy of Norway maple and sycamore seeds is discussed.     

Ascorbic acid metabolism in ageing recalcitrant sal (Shorea robusta Gaertn. f.) seeds

Changes in ascorbate content and its enzymatic utilization pattern were studied in embryonic axes and cotyledons of sal seeds undergoing rapid loss of viability, at ambient conditions. Ascorbate levels were significantly higher initially in the embryonic axes (0.32 mg/g fresh weight) and cotyledons (0.21 mg/g fresh weight) of freshly mature, relatively hydrated (42.2% moisture content) and 100% viable sal seeds. It declined sharply as the tissues; embryonic axes and cotyledons, desiccated with absolutely no detectable amount in non-viable seeds (21% moisture content). Significantly strong correlation was obtained between desiccation of embryonic axes (r = 0.96) and cotyledon (r = 0.97) with loss of ascorbate levels and loss of germinability. Higher rates of ascorbic acid utilization (AAU) recorded in the embryonic axes of 100% viable seed declined sharply as the seed viability reduced due to desiccation below 36.8% moisture content. AAU was not detected in the cotyledons.     

Differential drying rates of recalcitrant Trichilia dregeana embryonic axes: a study of survival and oxidative stress metabolism

Studies to elucidate the biochemical basis of survival of excised embryonic axes (EAs) of recalcitrant seeds of Trichilia dregeana at different drying rates revealed significant differences between slow and rapid drying. Rapid drying allowed these EAs to survive dehydration to much lower water contents (WCs; ca. 0.31 g g^?1 dry mass basis with 73% germination) compared with slow drying, where 90% of the EAs lost viability at a WC of ca. 0.79 g g^?1. In EAs slowly dried within seeds, the levels of hydroxyl radical (three‐ to fivefold at WCs >0.5 g g^?1) and lipid peroxidation (50% at similar WC) were significantly higher compared with those dried rapidly to comparable WCs. When EAs were dried slowly, enzymic antioxidant levels were not sustained and declined significantly with prolonged storage. In contrast, sustained activity of enzymic antioxidants was detected in rapidly dried EAs even at relatively low WCs. Furthermore, the greater decline in glutathione (GSH)/GSH disulphide ratio in EAs slowly dried within seeds compared with rapidly dried EAs and a shift in GSH redox potential to relatively more positive values in the EAs slowly dried within seeds was correlated with considerable viability loss. It is apparent from this study that greater retention of viability to lower WCs in rapidly dried EAs from recalcitrant seeds may at least be partly explained by the retention of functional antioxidant status. It is also suggested that the reduction of viability in rapidly dried EAs at very low WCs appears to be a non‐oxidative process.     

Seed germination during floatation and seedling growth of Carapa guianensis, a tree from flood-prone forests of the Amazon

Carapa guianensis Aubl. (Meliaceae), a hard wood tree from the Brazilian Amazon, has large recalcitrant seeds that can germinate and establish in both flood-free (terra-firme) and flood-prone (várzea) forests. These seeds, although large, can float. This study was designed to experimentally examine seed longevity under floating conditions ex-situ and its effects on subsequent germination and seedling growth. Many seeds germinated while floating, and radicle protrusion occurred from 3 to 42 d after the start of the floating treatment (tap water, room temperature 20–30 °C). Shoots of newly germinated floating seedlings may elongate up to 37.0 cm in 20 d without loss of viability. Epicotyl and first leaf emergence were delayed by floating. Seeds that did not germinate while floating were then placed on vermiculite and watered daily, where many seeds resumed germination. Germination during and after floating was affected by the length of the floating treatment: 88% germinated after 1 mo, 82% germinated after 2 mo and 70% germinated after 2.5 mo. These results indicate that Carapa guianensis has physiological variation regarding dormancy in response to seed floatation. The fact that floatation induces dormancy in recalcitrant seeds of this economically important species can be relevant to initiatives of ex situ storage of seeds.     

A study of the optimum drying conditions for cabbage seed following selection on the basis of a newly-emerged radicle

A series of experiments were carried out to study the effects of different drying regimes on the survival of newly-germinated (radicle emerged 0·5-1·5 mm through the seed coat) cabbage seeds and to establish a treatment for reducing the moisture content of these germinated seeds. A preliminary surface-drying step proved necessary to avoid seed agglutination during the main drying stage. Of the drying regimes compared both viability and vigour were most effectively maintained in air, conditioned to between 20 and 30°C and with 80% r.h., flowing through the seeds at 0·25 m s^-1. Under these conditions, the viability and vigour of newly-germinated seed was maintained during drying to an equilibrium moisture content of c. 14%. At this moisture content further radicle growth was prevented and seeds remained free-flowing and separate for sowing or for further treatment.     

Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.)

. Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2-5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Longevity of pearl millet (Pennisetum glaucum) seeds harvested at different stages of maturity

The storage potential of seeds harvested at weekly intervals after controlled pollination was studied in three diverse cytoplasmic male sterile pearl millet (Pennisetum glaucum) lines. In the first experiment in 1989, a comparison of p₅₀ (time for viability to decline to 50% during storage) among seeds of the line DSA 105A harvested 14, 21, 28, 35 and 42 days after pollination (DAP), and then stored at 35°C with 15% moisture content or 40°C with 13% moisture content, showed that those harvested 35 DAP had the greatest longevity. In the second experiment in 1990, a comparison of p₅₀ within the lines 5141A and L 67A harvested 28, 35 and 42 DAP, and then stored at 40°C with 13% moisture content, showed that seeds of both lines harvested 42 DAP had the greatest longevity. In both the seasons, and in all three lines, maximum seed longevity (p₅₀) was attained one week after physiological maturity (defined as the end of the grain filling period), which is therefore the optimum time of harvest to obtain good quality seeds for conservation.