Chilling Stress to Soybeans during Imhibition

Embryos, excised from seed coats of soybeans (Glycine max Merr. cv. `Wayne'), leak profusely during the first minutes of imbibition. A discontinuity of temperature/leakage patterns occurs between 10 and 15 C; as embryos imbibe at 10 C or lower, disproportionately more solutes leak out per unit of water imbibed. Short periods of imbibition at or below 12 to 14 C reduce embryo germination and axis elongation; injury results from imbibition at 2 C for as little as 5 minutes. Humidifying embryos to 35 to 50% moisture before imbibition reduced leakage during imbibition and imparted some resistance to imbibitional chilling injury.

The period of profuse leakage is interpreted as a time of membrane reorganization. Imposing a low temperature during this period prolongs the rapid leakage, suggesting delayed or faulty membrane reorganization. Reduced cold sensitivity of embryos with an initial 35 to 50% moisture content is presumed to be due to at least partial membrane reorganization in the embryo before imbibition. These data collectively are taken to indicate that low temperature interferes with normal membrane reorganization during imbibition, probably by modifying the physical state of membrane phospholipids, and that the consequent abnormal organization of membranes is a basic cause of low temperature injury.

     

A Physical Explanation for Solute Leakage from Dry Pea Embryos During Imbibition

The pattern of solute leakage from imbibing dead pea (Pisumsativum L.) embryos was the same as that from living embryos,with an initially high leakage declining to a low constant rateof leakage in the first 3 min of imbibition. The same patternof leakage occurred during each imbibition phase of repeatedimbibe/dry cycles of dead embryos. Living and dead seeds alsoshowed this pattern of leakage. These observations are usedto argue that leakage during imbibition of embryos and seedsis a physical diffusion phenomenon. Vital staining of livingembryos after imbibition revealed positive staining for dehydrogenaseenzymes in the cells on the outer surface of the cotyledonsonly when 0.5 mM sodium succinate solution was present duringimbibition and/or staining. This is discussed in relation tothe effect of rapid water uptake on these cells.     

Studies of the Surfaces of Desert Plant Seeds: I. Effect of Day Length upon Maturation of the Seedcoat of Ononis sicula Guss

Pods of Ononis sicula may contain three types of seeds, allwith embryos ready to germinate but differing in the time whichthey take to imbibe water: seeds which have matured during longdays take longer than those matured in short days. The scanningelectron microscope has provided evidence that the delay inimbibition by the ‘long day’ seeds is related tothe thickness, i.e. the degree of development, of the cuticlesof their seedcoats.     

Alcohol Stress on Soya Bean Seeds

When soya bean seeds were exposed to pure aliphatic alcohols,the shorter alcohols were the most damaging (methanol > ethanol> n-propanol > n-butanol); when the alcohols were appliedin equal volumes of water, the opposite was found (n-propanol> ethanol > methanol), leakage of solutes from the pre-treatedtissue during subsequent imbibition in water was associatedin each case with the loss of germination and a decline in axisgrowth. Damage by the pure alcohols was related to the extentof their penetration and the amount of phospholipid eluted,injury caused by alcohols in the presence of water did not exhibitthese functions. It is proposed that damage to seeds by alcoholsis due to the elution or displacement of cellular phospholipidsand possibly the partial denaturation of membrane proteins Membranedamage is considered to be a prime cause of injury to the seed. Glycine max (L.) Merr., soya bean, seed, denaturation of membranes, alcohols, phospholipids     

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     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development

We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4-cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue.     

Transient changes during soybean imbibition

Air dry cotyledons of soybean (Glycine max Merr. var. Wayne) imbibe water rapidly for about 10 minutes followed by a slower, linear rate of uptake. Leakage of solutes out of the coytledon likewise shows an initial rapid period, followed by a slower, nearly linear rate after 5 to 10 minutes; both the rapid and the steady rate leakage are greater for initially drier seeds. Respiratory activity of cotyledons as measured by manometric techniques becomes apparent after about 10 minutes of imbibition while polarographic studies of ground particles suggest that O₂ comsumption begins almost immediately upon wetting. Initial wetting of the seed causes the release of adsorbed gases, and a series of changes in volume of the seed-water mixture are charted. The data are interpreted as indicating that extensive physical changes occur in the first few minutes of water entry, including a rearrangement of membranes changing them from a relatively porous to a less permeable condition, and a release of adsorbed gases which cause an inflation or swelling of the seed.     

Osmoregulation, growth and sucrose accumulation in germinated Trigonella foenum-graecum (fenugreek) seeds treated with polyethylene glycol

Germinated seeds of Trigonella foenum-graecum L. (fenugreek) were grown in water or in polyethylene glycol (PEG) solutions. After endosperm removal, the water relations, growth, dry weight, sucrose and reducing sugar content of the embryo were determined. Under water sstress conditions, water content and osmotic potential (π₀) at saturation, growth and dry weight were lower than in non-stressed controls. The reduction in dry weight indicated a lower uptake of solutes from the endosperm and the decrease in π₀ was not accompanied by an increase in the amount of the accumulated solutes. It is suggested that embryos of stressed fenugreek seeds control osmotic potential by reduction of water uptake and that this results in reduction of growth. Embryos isolated from germinated seeds ("naked" embryos) were grown in water or in PEG solutions, with or without galactose (as an external solute source substituting for the endosperm). The results indicate that a decrease in the external solute did not account for growth reduction under conditions of water stress, and that decreased solute transport to the embryo may be important. The sucrose contents of "naked" embryos and of embryos from whole seeds were higher after PEG treatment, while reducing sugar contents were lower compared to non-stressed controls. The increased sucrose accumulation may be due to decreased sucrose hydrolysis.     

Sensitivity to Abscisic Acid and Osmoticum Changes During Embryogenesis of Alfalfa (Medicago sativa)

Developing embryos of alfalfa can be placed into nine stagesof development according to their morphological characteristics.The intact seeds do not germinate when removed from the podand placed on water until at least stage VI, and complete germinabilityis not achieved until the seeds are at the stage when they startto undergo maturation desiccation. Isolated embryos are germinableas early as stage III, and will germinate within 24 h on Murashigeand Skoog medium containing 3% sucrose. Osmoticum can inhibitembryo germination, but only proper osmotic conditions can maintaindevelopment in vitro; development does not occur at germination-inhibitingconcentrations of abscisic acid. The sensitivity to abscisicacid and osmoticum changes with stage of development. Early-stageembryos have the highest abscisic acid sensitivity and thisdeclines to the extent that mature dry embryos require a highconcentration (1?0 mol m^–3) to prevent their germination.Sensitivity of the embryos to osmoticum is maximum at stageVII of development. The combined inhibitory effect of abscisicacid and osmoticum on germination during development is greaterthan their individual effects. This combined effect is stage-dependent.Thus studies on the effects of abscisic acid and osmoticum onembryogenesis and associated synthetic events should be expectedto vary according to the sensitivity to these agents at differentstages of development. Key words: Medicago sativa, embryogenesis, abscisic acid, osmoticum, seed development     

The Damaging Effect of Water on Dry Pea Embryos During Imbibition

When pea seeds were imbibed in water without their seed coats,vital staining revealed that cells on the abaxial surface ofthe cotyledons were dead. No damage occurred on the surfaceof cotyledons when the seeds were imbibed intact, or beneaththe testa when only half of the testa was removed. Cell deathoccurred as a result of rapid water uptake within the first2 min of imbibition, since reducing the rate of imbibition insolutions of Carbowax 4000 lessened the damage. Cell death wasrestricted to the outer layers of the cotyledons; inner tissuesremained alive. These observations supported the hypothesisthat rapid early leakage during imbibition of dry embryos resultedfrom the death of cells caused by the physical disruption ofmembranes. Imbibition damage resulted in reduced respirationand germination, a decline in the rate of food reserve transferfrom the cotyledons to the growing axis, and a lower growthrate in the seedlings produced. Greater sensitivity of embryosto imbibition damage at low temperature, and similarities betweenfeatures of imbibition damage and chilling injury led to thesuggestion that so-called chilling injury is the result of imbibitiondamage rather than the effects of low temperature.     

The Influence of Embryo Condition on the Leaching of Solutes from Pea Seeds

Differences in the leaching of solutes from different seed lotsof peas were found to be associated with the condition of theembryos and not the testae. Seeds with areas of dead tissueon the abaxial surfaces of their cotyledons exhibited the highestlevels of leaching but the large range of levels found amongseed lots resulted from differences in leaching from livingtissue. When dry embryos were placed in water a rapid declinein the leaching of solutes was seen over the first 30 min forall lots tested but differences between lots were seen fromthe first minute in water and were related to the ability ofthe embryos to retain solutes rather than their solute content.Differential retention of solutes by the cytoplasm and sensitivityto the damaging effects of rapid imbibition are both discussedas possible explanations for differences in leaching.     

Proteolytic Activities in x Haynaldoticum sardoum Seeds of Different Ages

Carboxypeptidase, aminopeptidase and proteinase activities weremeasured in endosperms from dry and germinating x Haynaldoticumsardoum naturally aged seeds. Carboxypeptidase activity, presentin dry seeds, decreased slightly during germination and remainednearly unchanged during the storage period. Aminopeptidase activityincreased during germination in younger seeds, but decreasedin non-viable seeds. Proteinase activity was absent in dry seeds,increased during germination in younger seeds and disappearedin the older ones. Proteinase activity was not recovered in old endosperms followingtransplantation of young embryos, and was recovered only toa very small extent in young endosperms following transplantationof old embryos. Young endosperms onto which young embryos hadbeen transplanted gave maximum recovery of enzyme activity,although this was lower than in young intact seeds. These results suggest that the reduced or delayed availabilityof nutrients to the embryo axis is not the only factor causingthe failure of seeds to germinate, the ageing process beinga progressive phenomenon affecting both embryo and endosperm. x Haynaldoticum sardoum, Denti de cani, seed ageing, proteolytic activities, embryo-transplantation     

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     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.): II. DEVELOPMENT OF GERMINABILITY, VIGOUR, AND DESICCATION TOLERANCE

The germinability, vigour, and desiccation tolerance of muskmelon(Cucumis melo L. cv. Top Mark) seeds was studied in relationto changes in seed water content during development within thefleshy fruit. Seed water content (fresh weight basis) declinedfrom 91% to 42% between 10 d and 35 d after anthesis (DAA) (whenmaximum dry weight was attained), then declined more slowlyto a minimum of 35% at 50 DAA before increasing again to 43%at 65 DAA. Fresh intact seeds were first germinable at 25 to30 DAA and attained maximum germination percentages at 45 DAA.Between 15 and 35 DAA, cotyledons, hypocotyls, radicles andepicotyls of isolated embryos (testa and perisperm enveloperemoved) sequentially developed the ability to grow when incubatedon water. Dehydration to water contents less that those attainedwithin the fleshy fruit is not a requirement for developmentof germination capacity of muskmelon seeds. Seeds became tolerantof rapid desiccation after 25 DAA, and drying of immature seeds(25 to 40 DAA) increased their germination percentages uponsubsequent imbibition. Washing, drying, or washing followedby drying increased seedling vigour (root length) as comparedto fresh seeds, which had very poor vigour. Water absorptionisotherms were constructed to test whether changes in water-bindingcomponents were correlated with the development of desiccationtolerance. Isotherms for seeds older than 25 DAA fit well tothe D'Arcy/Watt model, which postulates the existence of high-affinity,low-affinity and multi-molecular water-binding sites. Desiccation-intolerantseeds younger than 25 DAA lacked the component of the absorptionisotherm characteristic of the high-affinity water-binding siteswhich have been hypothesized to confer desiccation tolerance.However, we were unable to determine whether the absence ofhigh-affinity binding characteristics was specifically relatedto desiccation intolerance or was artifactual due to the lossof volatiles when immature seed samples were dried at high temperatures. Key words: Muskmelon, embryo, germination, development, vigour, desiccation     

The Role of the Lens in Seed Imbibition and Seedling Vigour of Sesbania punicea (Cav.) Benth. (Leguminosae: Papilionoideae)

Seeds of Sesbania punicea (Cav.) Benth. are largely impermeable,and the natural, low, percentage which germinate are permeableat the lens. Scarifying seed in acid stimulates germinationand causes various types of damage to the testa, including thelens. Placing seeds in boiling water stimulates germinationby rendering the seeds permeable at the lens. Seeds were mechanicallyscarified at precise sites on the testa to assess influenceof the site and degree of damage on germination. Results indicatethat both the site of initial water entry and the rate of waterentry are important in determining seed and seedling vigour.Damaging the lens is more effective in stimulating germinationand reducing seedling abnormalities than complete excision ofthe lens. This effect can be reversed if seeds from which thelens was excised are allowed to imbibe gradually in inert osmotica.The leguminous lens thus acts as the site of initial water entryand regulates the rate of water uptake, thereby increasing seedand seedling vigour. Sesbania punicea (Cav.) Benth., hardseededness, lens, seed germination, imbibition damage, seedling vigour     

Sinapine Leakage from Non-viable Cabbage Seeds

Seeds leak many compounds during the early phases of germinationand seed viability may be associated with differential leakageof specific compounds. Leakage of a fluorescent compound fromnon-viable cabbage (Brassica oleracca var. capitata L.) wasdocumented and studies were performed to identify the fluorescentcompound. Imbibed samples of both heat-killed (HK) and viablecabbage seeds were submerged in a viscous colloidal gel. After2 h to 4 h, a fluorescent halo was observed under U.V. lightaround the heat-killed seed but not around the viable seed.Viable and HK seeds were imbibed in water for 8 h and the pHof the leachate was adjusted to either 7 or 10. The absorptionspectra of leakage from HK seeds revealed peak values at 322nm and 388 nm at pH 7 and 10, respectively. This pattern wasnot observed from viable seed leakage. Two-dimensional paperchromatography was conducted on the HK seed leachate. Four fluorescentspots were observed after development first with BAW (n-butanol:acetic acid: water, 4: 1: 5 by vol.) followed by 6% acetic acid.One of the fluorescent spots (spot 3) was studied further dueto its observed intensity. Sinapine thiocyanate was preparedfrom rapeseed oilmeal and used as a reference sample. Absorptionspectra of spot 3 and sinapine thiocyanate were similar at bothpH 7 and 10. Spot 3 had identical R_F values using three solventsystems and identical colour reactions in five tests comparedto sinapine thiocyanate. It was concluded that sinapine wasthe major compound responsible for the fluorescent leakage fromHK cabbage seeds. Key words: Leachate, viability, Brassica     

Imbibitional leakage from anhydrobiotes revisited

Dry desiccation-tolerant organ(ism)s leak cellular solutes when placed in water. Elevated temperatures at imbibition and elevated initial moisture contents reduce the leakage and promote growth. We have re-examined the effects of imbibitional stress imposed on cattail (Typha latifolia L.) pollen as a model anhydrobiotic system. A nitroxide spin probe technique and electron microscopy were used, allowing study of the permeability of the plasma membrane together with its visual intactness. Imbibitional leakage can be transient, or prolonged when associated with membrane damage. During the first 15 s of rehydration in medium, plasma membranes of pre-humidified pollen were highly permeable but became less permeable thereafter. The resulting transient leakage may affect vigour as measured by the rate of fresh weight increase, but did not reduce germination. A permanent, high permeability was observed when dry pollen was plunged into medium at low temperatures. This led to cell death and is associated with a phase change of the membranes from gel to liquid crystalline during imbibition. Freeze-fracture images indicate that the damage to plasma membranes is mechanically imposed by the pressure of the penetrating water rather than occurring structurally by a phase separation of membrane components. We suggest that a high rigidity of the plasma membranes in the gel phase at imbibition underlies imbibitional damage.     

Drought tolerance of Periploca sepium during seed germination: antioxidant defense and compatible solutes accumulation

Periploca sepium Bunge is a native and widespread shrub on the Loess Plateau, an arid and semi-arid region in China. To understand the adaptability of its seed germination to dry environments, we investigated the germination rate, water relations, lipid peroxidation, antioxidant capacity and accumulation of major organic solutes during seed germination under water deficit conditions. Results showed that seeds pre-treated by hydration–dehydration or ?0.9 MPa PEG germinated faster than control seeds, indicating strong resistance of P. sepium to drought condition. The re-dried seeds showed higher proline, total free amino acids (TFAA) and soluble proteins (SP) contents than control dry seeds, indicating the maintenance of physiological advancement when dehydrated. Osmotic stress made seed germination stay on the plateau phase (phase II). However, germinating seeds moved into phase III immediately once transferred into distilled water. Large increases in SP and soluble sugars (SS) of both re-dried and osmotic stressed seeds help themselves to resist drought stress. The re-hydrated seeds showed significantly higher levels of proline, TFAA, SP and SS than control seeds. The largely accumulated SS during osmotic stress declined sharply when transferred into distilled water. Our data demonstrate that P. sepium’s tolerance to drought stresses during germination is associated with enhanced activity of antioxidant enzymes and accumulation of some compatible solutes. Seed physiological advancement progressed slowly under low water conditions and it was maintained when seeds were air dried. This strategy ensures high and more rapid seed germination of P. sepium under drying and wetting conditions in drought-prone regions.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.