Evidence that ethylene, light and abscisic acid interact to inhibit germination in the recalcitrant seeds of Quercus robur L.

The effects of ethylene, light and abscisic acid (ABA) on thegermination of prematurely-harvested and mature Q. robur seedswere studied. There was evidence of active biosynthesis of ethylene,but not of ABA in seeds from both harvests. Addition of ABA,the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid(ACC) or the ethylene-releasing compound Ethephon (E) to germinationmedia and exposure to light individually inhibited germinationand their effects were additive. The ethylene biosynthesis inhibitoraminoethoxyvinylglycine (AVG) tended to promote germinationof premature seeds only. Key words: Quercus robur, germination, ethylene, light, abscisic acid, recalcitrant seed     

Embryo Water Status and Survival in the Recalcitrant Species Quercus robur L: Evidence for a Critical Moisture Content

The responses of Q. robur L. fruits, seeds and embryonic axesto desiccation are characterized and discussed in relation tocurrent knowledge of recalcitrant seed behaviour. A relationshipbetween viability and seed moisture content is described. Thisrelationship was unaffected by rate of drying, year of harvestor presence of the pericarp. Desiccation sensitivity did notincrease with storage. Excised embryonic axes survived to lower moisture contents thanintact seeds. However, in the intact seed, loss of viabilityappeared to be determined by a critical moisture content inthe cotyledons. Consequently, the level of desiccation tolerancewithin the axis attached to cotyledons was not determined byaxis drying rate. A link is drawn between the difference in the desiccation toleranceof embryonic axes and of cotyledons, and estimates of theirdifferent levels of matrix-bound water. The results presentedare consistent with a critical moisture content for survivalwhich is determined by the loss of all free cellular water.This hypothesis takes account of the differential desiccationsensitivity of seed tissues and differences in desiccation tolerancebetween species.     

Responses to Drying of Recalcitrant Seeds of Quercus nigra L.

It has been suggested that rate of desiccation can influencethe expression of recalcitrant behaviour in seeds, thus complicatingthe task of determining which seeds are truly recalcitrant.The objective of this study was to see if variable rates ofdesiccation influenced such behaviour inQuercus nigra L., atree seed known to be recalcitrant. Acorn moisture content, leachate conductivity, and germinationwere determined at various times during desiccation at threerates at 27 and 40°C. Moisture contents and germinationdecreased as the severity of desiccation increased. Leachateconductivity increased slightly but was not a sensitive indicatorof loss of viability. The critical (lethal) moisture contentfor these acorns was 10–15%, although rehydration within48 h of reaching this level prevented death in about 25% ofthe acorns. At 27°C any desiccation treatment that producedlosses of 30–50mg of moisture per g of acorn dry weightper day should be suitable as a test for recalcitrance in thegenus. Apparent physiological or fungal damage at 40°C rulesout the higher temperature for such a test. Quercus nigra L.; desiccation rate; temperature; recalcitrant; leachate conductivity; germination; viability     

The recalcitrant plant species, Castanospermum australe and Trichilia dregeana, differ in their ability to produce dehydrin-related polypeptides during seed maturation and in response to ABA or water-deficit-related stresses

In constrast to seeds of orthodox species, those of recalcitrantspecies do not acquire desiccation tolerance during their developmentand are shed from the parent plant at high water contents. Dehydrinproduction in seeds of recalcitrant species was examined duringdevelopment and germination, in response to abscisic acid (ABA),and following the imposition of various water-deficit-relatedstresses, including desiccation, water stress, high salt, highosmolarity, and low temperature. Two tropical species exhibiteda differential capacity to produce dehydrin-related proteinsduring seed maturation. Dehydrins were present in axes and cotyledonsof Castanospermum australe seeds during mid-maturation and atmaturity. In Trichilia dregeana, no dehydrin-related polypeptideswere detected in the mature seed. During the development ofC. australe seeds, the nature of the dehydrin related polypeptidesaccumulated in the cotyledons and axis changed and new polypeptideswere detected in the mature seeds that were not present duringmid-maturation. The dehydrins present in cotyledons of matureseeds (31, 37 and 40 kDa) were still detectable after germination(i.e. in untreated seedlings). These dehydrins became less abundantin the cotyledons of C. australe seedlings following ABA andall stress treatments except cold, although most of the dehydrinswere still detectable. An exception was the desiccation-treatedseedlings, in which no dehydrins were detected. In the rootsof C. australe seedlings, no dehydrins were found after germinationnor were they induced in the root by ABA or any of the stresstreatments imposed on seedlings. Seedlings of Trichilia dregeanadid not produce dehydrins in the roots or cotyledons when exposedto ABA or water-deficit-related stresses. Key words: Dehydrin, ABA, desiccation, recalcitrant, seed     

ROS production and antioxidative system activity in embryonic axes of <Emphasis Type="Italic">Quercus robur</Emphasis> seeds under different desiccation rate conditions

The seeds of pedunculate oak (Quercus robur L.) were subjected to slow (S) and rapid (R) desiccation at desiccation rates of 0.16 and 0.39% H₂O per hour, respectively. Till ca. 40% water content (WC) the germination capacity of seeds in the S and R variants was high (ca. 100%). Between 40 and 28% WC, germination capacity declined to 20 and 50% in S and R variants, respectively. The decrease in seed viability was accompanied by a significant increase of electrolyte leakage from embryonic axes (28% for S and 15% for R variants). In the embryonic axes of seeds subjected to slow desiccation, malondialdehyde (MDA) and free fatty acid (FFA) contents were significantly higher than those in R variants, indicating greater membrane damage due to lipid peroxidation. The production of ROS (H₂O₂ and O₂^·−) was significantly higher in S than in R variants. The low molecular weight antioxidants α-tocopherol, ascorbic acid (ASA), and phenolic compounds indicated different reactions in response to desiccation stress. ASA levels decreased during desiccation to a similar degree in both the S and R variants. A significant decrease of total phenols was observed in R variant, which coincided with a significant increase of guaiacol peroxidase (POX) activity. α-Tocopherol content was significantly higher in the embryonic axes of seeds subjected to rapid drying. The activities of the enzymatic scavengers APX and GR had similar runs and were slightly higher in R variant. The activities of POX and SOD were significantly higher in the embryonic axes of seeds subjected to rapid drying. These results show that rapid dehydration of Q. robur seeds leads to the greater mobilization of antioxidant system in embryonic axes, particularly increased levels of α-tocopherol and POX and SOD activities, in the first stages of water loss. This mobilization has a greater impact on maintenance of higher viability of seeds after drying to lower level of WC.     

Seed Development in the Recalcitrant Species Quercus robur L: Water Status and Endogenous Abscisic Acid Levels

Water status and endogenous ABA levels were measured duringthe development of the embryonic axis and cotyledons in fruitsof Quercus robur L. As dry matter accumulated in the embryoduring development, both moisture content and osmotic potential(     

Water Status Changes During Development in Relation to the Germination and Desiccation Tolerance of Aesculus hippocastanum L. Seeds

Seed growth characteristics of Aesculus hippocastanum were examinedin detail during development from about 70 to 140 d after anthesis(DAA), mainly in 1988 and 1989. Mean fresh and dry weights increasedfor both the axis and the whole seed up to the time of peakseed fall at 135 DAA with no cessation before fruit abscission.Water per seed increased up to 100 DAA, after which no furtherincrease occurred; moisture content declined for the embryonicaxis and whole seed respectively from above 75 and 65% at 95DAA to 65 and 50% at 130 DAA. At fruit shedding in 1990 waterpotential values of -1·2, -2·6 and -1·1MPa were observed for the testa, cotyledon and axis tissuesrespectively; relevant sorption isotherms are presented. Decreases in seed moisture content during development were accompaniedby increases in desiccation tolerance and in germinability,both reaching their maximum at the time of peak seed fall. Atmaturity, only about 10% viability was retained on drying seedto 20% moisture content; it is confirmed that the seeds are'recalcitrant'. The exact relationship between moisture contentand germination during development was dependent on the deptof dormancy, as judged by the period of chilling required; eachduration of chilling at 2°C within the range 3-12 weeksyields a curve of sigmoid shape. No germination occurred at26°C without chilling, but nearly full germination can beobserved for samples collected at 6 weeks before maximum seedfall with 12 weeks chilling. The rate of moisture loss duringdesiccation at 15°C and 15% rh becomes reduced during development.The ontogeny of these 'recalcitrant' seeds is compared withthat of 'orthodox' seeded species and the implication of sigmoid-shapedcurves for the relationship between seed moisture content andgermination are considered.Copyright 1993, 1999 Academic Press Aesculus hippocastanum L., horse chestnut, seed development, water status, germination, desiccation intolerance, desiccation rate     

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.     

Quantal Response of Fruit and Seed Germination Rate in Quercus robur L. and Castanea sativa Mill, to Constant Temperatures and Photon Dose

A linear relationship between constant temperatures in the sub-optimaltemperature range and germination rate is shown in both Quercusrobur L and Castanea sativa Mill germinated under nominal darkconditions. The mean base temperature was interpolated for Qrobur as 0 8 ? or 2-4 ?, depending on seed lot provenance, andfor C. sativa as 1 -4? The optimum temperature for germinationin Q. robur was about 20? compared with around 28 ? in C. sativaOver the sub-optimal temperature range the distribution of thermaltimes was log-normal for each population studied their spreadvarying both between Q robur seed lots and between species However,in C. sativa germinated close to the mean base temperature,the distribution in thermal times was reduced Thermal timesto germination were decreased in Q. robur and C sativa by approximately0 3 and 0-5 log-units, respectively, when the pericarp was removed,i.e in the seeds, but the sensitivity of the response remainedrelatively unaltered In both species the germination rate was the same when nominaldark or safe green light conditions were employed during thegermination test. However, at 21 ? Q robur exhibited the highirradiance reaction (HIR) at photon doses above 30mmol m^–2d^–1. HIR first affected the germination rate by an inhibitionof radicle extension The sensitivity of the response to thermaltime was reduced as photon dose increased. This photo-inhibitionwas exacerbated at supra-optimal temperatures. In contrast,C. sativa germination rate at 26 ? was little influenced bylight at a photon dose of 752 mmol m^–2 d^–1 Key words: Seed germination rate, temperature, thermal time, light, photon dose     

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     

Dynamics of Water and Abscisic Acid During Embryogeny and Embryo Drying in the Recalcitrant Seeds of Vateria indica L.

Vateria indica L. is a critically endangered tree species in South-Western Ghats of India, commercially exploited for its valuable resins. Seed recalcitrance is a major problem hindering the natural regeneration of this species and it poses a great challenge in seed storage and conservation. There was a continuous import of water from the maternal tissues to seed tissues till maturity and the seeds were released in a fully hydrated state. Differential accumulation of water has been noticed in the cotyledons and embryonal axis. There was a positive correlation between seed moisture content and rate of germination which is a character of recalcitrant seeds. The critical moisture content was found to be 40% in the axis and 23.5% in the cotyledons, below which the embryo will not germinate. Loss of germination ability as a result of desiccation was attributed to the cell membrane damage, expressed as the electrolyte leakage exceeding 0.79 μS/cm. ABA peaked in the mid embryogenesis, then dropped drastically and maintained a lower level till seed maturity. On desiccation, ABA started to increase but gradually dropped down. Both cotyledons and embryonal axis had differential ABA content but exhibited a general pattern of ABA level during embryogeny. Due to the thin seed coat/embryo ratio and low investment in the seed coat, this recalcitrant seed could not hold water as efficient as orthodox seeds. Thus, it germinated as soon as it was shed from the mother plant. On desiccation, ABA shot up and moisture content decreased along with electrolyte leakage and cell membrane damage. All these hindered germination of the seed. Thus, we can see a clear interplay between moisture content and ABA levels during embryogeny and desiccation. Since the seed biology of this species has not been well documented, the present work is mainly intended to study the dynamics of water and ABA during embryogeny and embryo drying. This study can surely contribute to the long-term storage and conservation of recalcitrant seeds which is a less explored area.

     

Role of Abscisic Acid in the Induction of Desiccation Tolerance in Developing Seeds of Arabidopsis thaliana

In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation-specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance.     

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     

Global 5-methylcytosine alterations in DNA during ageing of Quercus robur seeds

Background and Aims Epigenetic regulation plays an important role in the management of plant growth, development and response to stress factors, and several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (m⁵C) levels in the DNA of seeds during ageing, a process that has important implications for plant conservation and agriculture.Methods Changes in the global level of m⁵C were measured in mature seeds of oak, Quercus robur. The extent of DNA methylation was measured using a protocol based on two-dimensional thin-layer chromatography. Viability of seeds was determined by germination and seedling emergence tests.Key Results An ageing-related decrease in total m⁵C during storage of recalcitrant seeds was highly and significantly correlated with a decrease in seed viability, as reflected by a reduction in germination (r = 0·8880) and seedling emergence (r = 0·8269).Conclusions The decrease in viability during ageing of Q. robur seeds is highly correlated with a global decline in the amount of m⁵C in genomic DNA, and it is possible that this may represent a typical response to ageing and senescence in recalcitrant seeds. Potential mechanisms that drive changes in genomic DNA methylation during ageing are discussed, together with their implications for seed viability.     

Different Types of Major Storage Seed Proteins in Fagaceae Species

The seed proteins of Fagus sylvatica, Quercus Hex and Q. robur,of the Fagaceae family, were extracted by the sequential methodof Koie and Nielsen (1977). The storage proteins of Fagns werefound to be globulins as has previously been shown in Castanea,a member of the same family. Quercus was found to store glutdins.The amino acid composition and the preliminary characterizationby electro-phoresis of Fagus globulins and Quercus glutelins,suggest that both contain proteins similar to the 1 IS globulinsof leguminous plants. Both genera showed considerable differencesin their albumins. Prolamins were found only in Fagus. Key words: Fagaceae, seeds, storage protein     

The Effects of Priming and Ageing on Resistance to Deterioration of Tomato Seeds

The influence of seed priming and ageing treatments on viabilityand rate of germination of tomato (Lycopersicon esculentum Mill.)seeds was examined under both long-term and controlled-deteriorationstorage conditions. Seeds of a single lot of tomato were eitherprimed or aged to increase or decrease the rate of germination(Argerich and Bradford, 1989). They were then stored at 6% moisturecontent (dry weight basis) at either 4 ?C or 30 ?C for 1 year.Both viability and germination rate were unaffected by eitherstorage temperature in control seeds, or by 4 ?C storage inprimed or aged seeds. At 30 ?C, however, viability and germinationrate of primed and aged seeds was markedly reduced after 6 monthsof storage. The temperature dependence of the germination rateand the spread of germination times within the population wasalso adversely affected by high temperature storage, particularlyfor primed seeds. Under controlled deterioration conditions(13.5% moisture content and 50 ?C), the rate of loss of viabilitywas greater for primed seed than for control or aged seeds.The relationship between seed viability and the mean germinationrate, however, was not influenced by the seed treatments. Thesedata are analysed in relation to current models of seed deteriorationduring storage and seed repair during priming. The results indicatethat enhancement of seed germination rates by priming treatmentssimultaneously lowers the resistance of seeds to deterioration.Primed tomato seeds must, therefore, be considered to be vigorousseeds with a reduced storage life. Key words: Tomato, controlled deterioration, seed germination rate, seed viability     

Storage Behaviour of Salix alba and Salix matsudana Seeds

Effects of dehydration, storage temperature and humidificationon germination of Salix alba andS. matsudana seeds were studied.Newly released seeds showed 100% germination before and afterdehydration to 11–12% moisture content. Germination ofthe high vigour lot (100% initial normal germination) was notaffected by dehydration to 6.7% moisture content but germinationdecreased with further dehydration to 4.3%. The lower vigourlot (75% initial normal germination) was more susceptible todehydration and germination decreased following dehydrationto 6.7% moisture content. Dry seeds of both species survivedimmersion in liquid nitrogen without loss of viability. Thegermination of seeds stored with 9% moisture content decreasedto 35–40% in 5 months at -20°C or in 2 months at 5°C.However, at 25°C seeds entirely lost viability within 2weeks. Seeds showed improved performance when stored at -70°C> - 20°C > 5°C > 25°C and tolerated dehydrationto a moisture content in equilibrium with 15% relative humidity.Results suggest that they are orthodox in storage behaviouralthough they are short-lived. Humidification treatment of lowvigour seed lots resulted in a remarkable increase in germinationpercentage. Copyright 2000 Annals of Botany Company Salix alba, Salix matsudana, willow, seed storage behaviour, dehydration, humidification, cryopreservation     

Response Breadths on Environmental Gradients of Germination and Seedling Growth in Two Dominant Forest Tree Species of Central Himalaya

The responses of Pimis roxburghii (chir pine), an early successionalspecies, andQuercus leucotrichophora(banj oak), a late successionalspecies were compared in terms of seed germination and seedlinggrowth. Seed germination was observed on single-factor gradientsof temperature, water stress, seed desiccation and light, andseedling growth on gradients of shade and soil moisture. Thesuppression of germination caused by continuous dark and far-redwas greater in Pinus than in Quercus. Pinus seed germinationshowed wider response breadth on gradients of water stress andseed desiccation, and narrower response breadth on the gradientof light quality, compared withQuercus. In terms of seedlinggrowth, the response breadth of Pinus was wider on the gradientof soil moisture and narrower on the gradient of shade comparedwith Quercus. Under higher soil moisture stress, the seedlingsof both species attained similar heights, butQuercus seedlingsachieved higher dry weights, a higher root: shoot ratio andlower leaf weight ratio than Pinus.     

Seed Dormancy in Acer: The Role of Testa-imposed and Embryo Dormancy in Acer velutinum

The embryo dormancy shown in freshly harvested samples of Acervelutinum seeds is weakly established and very short-lived.Loss of this embryo dormancy occurred during post-harvest fruitstorage at either 5 or 17 C. In contrast, the dormancy of intactfruits and seeds was overcome only during storage at the lowertemperature. Removal of the cotyledons from embryos of freshlyharvested fruits allowed more rapid germination of the embryonicaxes, indicating that the cotyledons exert an inhibitory effect,although the axes still retained a measure of innate dormancy.The inhibitory effect of the cotyledons became less marked withincreasing duration of fruit storage, this loss of inhibitoryeffect occurring at both storage temperatures. Applied ABA stronglysuppressed germinative capacity in intact embryos and isolatedembryonic axes from freshly harvested fruits, but when ABA wasapplied to embryos of fruits that had been stored for variousperiods at 5 or 17 C, the inhibitory effect was first weakenedand then lost with increased storage. Although dormancy in the seeds of A. velutinum may be describedas intermediate between testa-imposed dormancy and true dormancy,it is perhaps more properly included in the former category. Acer velutinum Boiss. var. vanvolxemii, abscisic acid, embryo dormancy, germination, seed storage, testa-imposed dormancy, tissue sensitivity     

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