Opposite effects of temperature on breaking of dormancy and on induction of frost resistance in apple embryos

Apples ( Pyrus mains L., ev. Golden Delicious) were placed at 0, 12 and 35°C. At different intervals, seeds were taken from fruits, and the ability of isolated embryos to germinate was determined. In parallel, the survival of similar embryos frozen for 30 min at -7°C was estimated. Storage of apples at 0°C led to complete removal of embryo dormancy within ca 10 weeks. At 12°C, dormancy was only partly eliminated, and at 35°C breaking of dormancy did not occur. The onset of secondary embryo dormancy was thus evident as the result of 35 and 12°C treatments. On the other hand, the storage of apples at 35°C was the most effective treatment for induction of frost resistance in the embryos. At 12°C the same level of frost resistance was reached much slower. Even a prolonged storage of apples at 0°C did not allow frost survival of all embryos. The above relations were confirmed in experiments with transfer of fruits from one temperature of storage to another. The temperature of fruit storage affected the dormancy removal in embryos and induction of resistance to rapid freezing in opposite directions.     

Morphological dormancy in seeds of the autumn-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae)

To better understand the germination ecophysiology of the genus Lonicera , the dormancy class, temperature requirements for embryo growth and radicle emergence and phenology of seedling emergence were determined for Lonicera caerulea var. emphyllocalyx . At maturity, seeds have an underdeveloped embryo (approximately 28% of the length of full-grown embryos). Embryos in fresh seeds grew to full length at 15, 20, 20/10 and 25/15°C within 3 weeks, but failed to grow at ≤ 10°C and at 30°C. Radicles emerged from 86–100% of freshly matured seeds in light at 15, 20, 20/10 and 25/15°C within 28 days, but failed to emerge at 10°C. Radicles emerged equally well in a 12 h photoperiod and in continuous darkness at 25/15°C. Rapid embryo growth and germination over a range of conditions indicate that seeds of this taxon have morphological dormancy (MD); this is the first report of MD in a species of Lonicera . Seeds are dispersed in summer, at which time high temperatures promote embryo growth. Embryos grow to the critical length for germination in approximately 1 month; the peak of seedling emergence occurs in early autumn. Radicles emerged within 2 months from 98% of seeds buried at soil depths of 2 cm and 10 cm in the field in August in Sapporo, Japan; thus, seeds have no potential to form a persistent soil seed bank. However, seeds sown too late in autumn for embryos to grow remained viable and germinated the following summer when temperatures were high enough to promote embryo growth.     

Levels of endogenous abscisic acid in achenes of Rosa rugosa during dormancy release and germination

Germination experiments on isolated embryos and intact achenes from Rosa rugosa L. var. rubra revealed the existence of both coat-induced and embryo dormancy. Studies were made on the quantitative changes in abscisic acid (ABA) of achenes during both a stratification period at 4°C and a subsequent germination period at 20°C. Controls were run in parallel at 17°C. Dormant, unimbibed achenes contained large amounts of ABA. The level fell rapidly during the early stages of stratification at 4°C, mainly due to leakage, and then the decline levelled off. The reduction of ABA content during germination was less and followed a tri-phasic pattern. Under the control conditions, the ABA level also fell although still faster, the amounts present being consistently below those detected in the corresponding cold-stratified samples. The content of alkali-hydrolysable ABA was low and not affected either by the duration or by the temperature of stratification. The investigation shows that termination of dormancy is not simply a consequence of a fall in the content of endogenous ABA and supports the current debate questioning the central role of ABA in regulation of dormancy breakage and of germination.     

Changes in dormancy levels ofFraxinus excelsior L. embryos at different stages of morphological and physiological maturity

The dormancy status ofFraxinus excelsior embryos at different developmental stages under environmental conditions was examined over a period of 2 years. For each sampling date the length of the fruit, of the seed, and of the embryo were measured, and the embryological stage determined. The depth of dormancy was assayed by the germination behaviour of isolated embryos under aseptic conditions on an agar medium without nutrients. As an approach towards a quantitative estimate of the dormancy status, the degree of inhibiton of germinative growth in the embryonic organs was evaluated on the basis of four categories from none to full germinative growth. From these ratings a dormancy index was calculated, expressing the mean dormancy status of the embryos at a given date. Embryo dormancy already became apparent during embryogenesis and reached its highest level during the later phase of reserve deposition in the seed. A marked loss of embryo dormancy occurred during the phase of maturation drying in autumn, followed by a moderate increase in winter. In hydrated seeds in spring the embryo was gradually released from dormancy and enlarged further. In maintaining the embryo ofF. excelsior in a developmental but not germinative mode, dormancy mechanisms within the embryo and the endosperm, combined with environmental factors, may be involved.     

Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels

The main aims of the present work were to investigate whether a chilling treatment which breaks dormancy of Douglas fir ( Pseudotsuga menziesii (Mirb.) Franco) seeds induces changes in the sensitivity of seeds to exogenous ABA or in ABA levels in the embryo and the megagametophyte, and whether these changes are related to the breaking of dormancy. Dormant seeds germinated very slowly within a narrow range of temperatures (20–30°C), the thermal optimum being approximately 25°C. The seeds were also very sensitive to oxygen deprivation. Treatment of dormant seeds at 5°C improved further germination, and resulted in a widening of the temperature range within which germination occurred and in better germination in low oxygen concentrations. In dry dormant seeds the embryo contained about one-third of the ABA in the megagametophyte. ABA content of both organs increased during the first 4 weeks of chilling. It then decreased sharply in the megagametophyte to the level in the embryo after 7–15 weeks of chilling. At 15°C, a temperature at which dormancy was expressed, the ABA level increased in the embryo and the megagametophyte of dormant unchilled seeds whereas it decreased in the organs of chilled seeds. The longer the chilling treatment, the faster the decrease in ABA after the transfer of seeds from 5°C to higher temperatures, and the decrease was faster at 25 than at 15°C. These results suggest that the breaking of dormancy by cold was associated with a lower capacity of ABA biosynthesis and/or a higher ABA catabolism in the seeds subsequently placed at 15 or 25°C. Moreover, the chilling treatment resulted in a progressive decrease in the sensitivity of seeds to exogenous ABA. However, seeds remained more sensitive to ABA at 15 than at 25°C. The possible involvement of ABA synthesis and of responsiveness of seeds to ABA in the breaking of dormancy by cold treatment is discussed.     

Acide abscissique lié et dormance embryonnaire chez Pyrus malus

Bound abscisic acid and embryo dormancy in Pyrus malus. The first part of this work was devoted to the study of the behaviour of Pyrus malus L. cv. Golden Delicious embryos cultivated in vitro. At the beginning of the experiment, either the root (RM) or the distal part of the cotyledons (CM) was immersed in the medium. For embryos directly isolated from the fruits at harvest time, as well as for embryos submitted to a 3-month post-maturation treatment at 4°C, the dormancy was deeper in CM cultures than in RM. The use of gibberellins (GA₄ or GA₇) emphasized these differences. The second part of this work was devoted to the study of free and bound forms of ABA (cis and trans isomers) in embryos isolated from the fruits at harvest time and cultivated by the RM or CM procedure during 3 weeks. The biochemical data obtained indicated in both cases the existence of the following three processes: (A) Mobilization of the bound ABA with the consequent release of free ABA; this was particularly important in CM. (B) Metabolism of free ABA: the isomerization into trans-ABA only partly accounted for the decrease in the content of free ABA which was much greater in RM than in CM. (C) Transport of ABA towards the root, the result being an accumulation of free ABA in the root, much greater in CM than in RM; this would account for the deeper dormancy in CM than in RM.     

Study of the inheritance of seed primary dormancy and the ability to enter secondary dormancy in Petunia: influence of temperature, light and gibberellic acid on dormancy

Abstract. The germination behaviour of two Petunia hybrida lines. M₃₀ and Th₇, and their reciprocal hybrids was studied. Two sets of experimental conditions appeared helped to distinguish between dormant and non-dormant parental lines: (1) 25 and 35 °C in the dark, in the latter case after 2 months of dry storage at 20 °C; (2) 35 and 40 °C in the light. Photosensitivity was tested in the first case and sensitivity to GA₃ in the second case. The predominance of paternal control over dormancy was evident. A maternal or tegumentary control of photosensitivity and of sensitivity to GA₃ was also shown. Transferring the seeds, originally imbibed in conditions expressing primary dormancy, to conditions which previously supported their germination, allowed us to show that secondary dormancy could be easily induced when a deeper primary dormancy had already developed in the seeds.     

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     

Seed Dormancy in Acer pseudoplatanus L.: the Role of the Covering Structures

The present studies with Acer pseudoplatanus L. suggest thatthe covering structures play an important and multiple rolein the dormancy of the fruit. Whole fruits and seeds with thetesta intact required a period of chilling at 5 °C beforedormancy was broken whereas bare embryos germinated immediatelyat 20 °C without pretreatment. This suggested that dormancywas coat-imposed and that the testa was responsible for thiseffect. Germination of dormant seeds was inhibited by lightwhereas the non-dormant bare embryos showed little response.Studies on the manner in which the testa imposed dormancy onthe embryo indicated that restriction on oxygen uptake, wateruptake, mechanical restriction to embryo enlargement, and thepresence of germination inhibitors in the testa were not limitingfactors at this stage of dormancy. Results from leaching experimentssuggest that dormancy was the result of the restriction by thetesta of the outward diffusion of a germination inhibitor(s)present in the embryo. In seeds that had nearly completed theirstratification requirements, the covering structures seemedto act in a manner other than by preventing the leaching ofan inhibitor from the embryo. At this point the physical propertiesof the covering structures seem to determine any further delaysin germination by the mechanical restriction of embryo enlargementby the testa and by restriction of oxygen uptake by the pericarp.     

The dynamics of indole-3-acetic acid in Acer platanoides seeds during stratification and germination

During stratification at 5°C indole-3-acetic acid (IAA) levels in embryos of Acer platanoides decreased during the early stages but subsequently increased again throughout the remainder of a 144 day period. The reduction in IAA levels in embryos of fruits stored at 17°C was even more pronounced, and in addition, no increase was observed after longer storage periods at this temperature, the levels of IAA remaining very low. Germination in seeds maintained at 5°C was not observed until after 120 days or longer, but germination potential increased at an earlier stage, as shown by the fact that seeds transferred to 20°C gave appreciable increases in germination after much shorter chilling periods. Endogenous IAA levels in embryos from seeds transferred to 20°C after a chilling period, long enough to break dormancy, increased within 24 h, i.e. before visible germination, to levels similar to those observed in embryos from seeds chilled continuously for 144 days. Embryos from seeds chilled for 120 days, i.e. when the samples already showed visible germination and when the endogenous IAA content was already high, showed no further increase in endogenous IAA during a three day incubation at 20°C. None of the treatments employed was effective in inducing germination of seeds or embryos from fruits stored at 17°C.     

Imbibition conditions and seed dormancy of Arabidopsis thaliana

The optimal combinations of temperature in the range of 0 to 20°C and duration (1 to 14 days) of imbibition for the induction of germination of Arabidopsis thaliana (L) Heynh., ecotype "Landsberg-erecta", by red light were investigated. At 2°C, 10 days of imbibition are needed tor loss of dormancy, whereas at higher temperatures, e.g 15°C, it is already lost after 1 or 2 days. It is proposed that the development of light-inducible germination is governed by two temperature-dependent processes-the loss of primary or innate dormancy and the simultaneous induction of secondary dormancy. Data are discussed in terms of the availability of phytochrome, the availability of an unknown factor X and changes in sensitivity of the process of germination induction by the far-red absorbing form of phytochrome (Pfr).     

Inhibiteurs non acides et dormance embryonnaire chez Taxus baccata

Non-acidic inhibitors and embryo dormancy in Taxus baccata L. Embryo dormancy of Taxus baccata is eliminated when the embryos are continuously kept in sterile nutritive liquid medium. After 3 weeks of culture, an important non-acidic inhibitory complex can be extracted from this liquid medium. At least three substances are involved: two pigments and a compound with some properties that suggest xanthoxin. These substances are neither found in embryos taken directly from the seeds, nor in liquid medium after 8 days of culture, which is the time necessary and sufficient to allow germination after transfer on agar medium. Such behaviour is quite different from that of ABA previously studied and indicates that these non-acidic inhibitors appear late during the culture and are not directly involved in embryo dormancy.     

In Situ Abscisic Acid Synthesis : A Requirement for Induction of Embryo Dormancy in Helianthus annuus

When applied to young nondormant embryos of sunflower (Hellanthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy.     

Endosperm dormancy breakage in olive seeds

Seeds of Olea europaea L. ssp. oleaster Hoffm. and Link freed from the sclerous endoearp and incubated in water at 15 or 25°C in darkness or in 12:12 h white light:dark conditions, did not germinate, due to dormancy imposed by the endosperm. Seeds also did not germinate when incubated in abscisic acid, gibberellic acid, kinetin or zeatin in darkness and at cither 15 or 25°C. SAN 9789 |4-chloro-5-(methylamine)-2-(a,a,a-trifluoro-m-tolyl)-3-(2H)-pyridazmone] did not promote germination at 15°C but it did to a 75% level at 25°C. This promoting effect of SAN was counteracted by abscisic acid. Cultures of naked embryos grew equally well in the presence or absence of SAN 9789. 6-Benzylaminopurine promoted whole seed germination to a 15% level.     

Dormancy and germination of olive embryos as affected by temperature

Olive seeds do not germinate promptly when placed under favourable conditions, which is a problem in raising young plants for breeding or experimental purposes. In a series of experiments an investigation of the role of temperature in the germination of olive embryos was conducted. Naked, unchilled olive embryos ( Olea europaea L. cv. Chalkidikis), cultured in vitro at 20°C, had a germination capacity of 73%, whereas that of embryos which had previously been chilled at 10°C for 2 or more weeks reached 96%. Intact seeds did not germinate at 20°C unless they had previously been subjected to 10°C for 3 or 4 weeks. Embryos chilled while in the intact seed and excised just before transfer to 20°C, reacted in a similar way to naked embryos, but reached their maximum germination capacity after 4 weeks at 10°C. Under constant temperature conditions the highest germination percentage of embryos was observed at 10 and 15°C and the highest germination rate at 15°C, while a moderate capacity and rate of germination occurred at 20°C, and a very low percentage and rate at 25 and 30°C. Prechilling at 10°C did not affect germination at 15°C, but improved the percentage and the rate of germination at 20, 25 and 30°C. The germination percentages of embryos chilled for 1 or 2 weeks at 10°C and then transferred to 25°C were lower than those of similarly chilled embryos transferred to 20°C. The chilling effect could not be reversed at 25°C when the embryos had been chilled for 3 or more weeks. The results show that olive seeds exhibit a state of dormancy that is caused by factors residing partly in the endosperm and partly within the embryo.     

Indirect effect of abscisic acid on the induction of secondary dormancy in lettuce seeds

During temporary incubation at 25°C in buffered solutions (pH 4.0) of abscisic acid (ABA) seeds of lettuce ( Lactuca sativa L. cv. Olof) lost the red-light initiated ability to germinate in buffer. The development of secondary dormancy required an inhibitory ABA content in the seeds during a number of days. A temporary incubation in ABA during 24 h met these requirements only if the solution was about 100-fold more concentrated than during continuous incubation. Studies with 2-¹⁴C-ABA showed that the amount of ABA which had penetrated in 24 h was reduced by a factor 100 within 3 to 4 days during subsequent incubation in buffer. Both leaching and metabolic changes were involved in the reduction process. The nature of the metabolic products remained obscure. A shift to 2°C after incubation in ABA prevented the induction of secondary dormancy, but inhibited ABA metabolism. ABA did not interfere with the induction rate of secondary dormancy, and it was not required to maintain the state of dormancy. The sole function of ABA was the non-specific inhibition of germination, which indirectly facilitated the development of an ABA independent secondary dormancy. – The level of endogenous ABA was compared to the amount of ABA found in the embryo during and after incubation in ABA solutions marked with 2-¹⁴C-ABA. The level of endogenous ABA in air-dry seeds (0.11 ng/mg dry weight) corresponded to the minimal level at which penetrated ABA inhibited germination. This level had to be present at least during 4 to 5 days to inhibit the effect of red light. Since endogenous ABA was quickly reduced upon imbibition, a regulatory function of endogenous ABA in the inhibition of red light induced germination can be ruled out. A function in the temporary inhibition of dark germination and, consequently, in the development of secondary light irresponsiveness cannot be excluded, however.     

Combinational dormancy in seeds of Sicyos angulatus (Cucurbitaceae,tribe Sicyeae)

Seeds with a water‐impermeable seed coat and a physiologically dormant embryo are classified as having combinational dormancy. Seeds of Sicyos angulatus (burcucumber) have been clearly shown to have a water‐impermeable seed coat (physical dormancy [PY]). The primary aim of the present study was to confirm (or not) that physiological dormancy (PD) is also present in seeds of S. angulatus. The highest germination of scarified fresh (38%) and 3‐month dry‐stored (36%) seeds occurred at 35/20°C. The rate (speed) of germination was faster in scarified dry‐stored seeds than in scarified fresh seeds. Removal of the seed coat, but leaving the membrane surrounding the embryo intact, increased germination of both fresh and dry‐stored seeds to > 85% at 35/20°C. Germination (80–100%) of excised embryos (both seed coat and membrane removed) occurred at 15/6, 25/15 and 35/20°C and reached 95–100% after 4 days of incubation at 25/15 and 35/20°C. Dry storage (after‐ripening) caused an increase in the germination percentage of scarified and of decoated seeds at 25/15°C and in both germination percentage and rate of excised embryos at 15/6°C. Eight weeks of cold stratification resulted in a significant increase in the germination of scarified seeds at 25/15 and 35/20°C and of decoated seeds at 15/6 and 25/15°C. Based on the results of our study and on information reported in the literature, we conclude that seeds of S. angulatus not only have PY, but also non‐deep PD, that is, combinational dormancy (PY + PD).     

Seed dormancy and germination of the European Chaerophyllum temulum (Apiaceae), a member of a trans-Atlantic genus

BACKGROUND AND AIMS: The European Chaerophyllum temulum and two North American Chaerophyllum species have a trans-Atlantic disjunct distribution. This work aimed to resolve requirements for dormancy break and germination of C. temulum seeds and to compare dormancy traits with those of the two North American congeners. METHODS: Phenology of germination and embryo growth was studied by regularly exhuming seeds sown in natural conditions. Temperature requirements for embryo growth, breaking of dormancy and germination were determined by incubating seeds under controlled laboratory conditions. Additionally the effect of GA(3) on germination was tested to determine the specific dormancy type. KEY RESULTS: In natural conditions, embryo growth starts in early winter. Seedlings emerge in late winter shortly after the embryos reached the critical ratio for embryo length to seed length (E : S) of approx. 0.95. Growth of the embryo only occurs during a prolonged incubation period at 5 degrees C. After stratification at 5 degrees C, which breaks physiological and morphological dormancy, seeds can germinate at a wide range of temperatures. GA(3) did not substitute for cold stratification in seeds placed at 23 degrees C. CONCLUSIONS: Chaerophyllum temulum has deep complex morphophysiological dormancy. This dormancy type differs considerably from that of the two North American congeners.     

Involvement of ABA in induction of secondary dormancy in barley (Hordeum vulgare L.) seeds

At harvest, barley seeds are dormant because their germination is difficult above 20 degrees C. Incubation of primary dormant seeds at 30 degrees C, a temperature at which they do not germinate, results in a loss of their ability to germinate at 20 degrees C. This phenomenon which corresponds to an induction of a secondary dormancy is already observed after a pre-treatment at 30 degrees C as short as 4-6 h, and is optimal after 24-48 h. It is associated with maintenance of a high level of embryo ABA content during seed incubation at 30 degrees C, and after seed transfer at 20 degrees C, while ABA content decreases rapidly in embryos of primary dormant seeds placed directly at 20 degrees C. Induction of secondary dormancy also results in an increase in embryo responsiveness to ABA at 20 degrees C. Application of ABA during seed treatment at 30 degrees C has no significant additive effect on the further germination at 20 degrees C. In contrast, incubation of primary dormant seeds at 20 degrees C for 48 and 72 h in the presence of ABA inhibits further germination on water similarly to 24-48 h incubation at 30 degrees C. However fluridone, an inhibitor of ABA synthesis, applied during incubation of the grains at 30 degrees C has only a slight effect on ABA content and secondary dormancy. Expression of genes involved in ABA metabolism (HvABA8'OH-1, HvNCED1 and HvNCED2) was studied in relation to the expression of primary and secondary dormancies. The results presented suggest a specific role for HvNCED1 and HvNCED2 in regulation of ABA synthesis in secondary seed dormancy.     

Changes in protein synthesis during stratification and dormancy release in embryos of sugar maple (Acer saccharum)

Protein synthesis in dormant embryos of sugar maple ( Acer saccharum ) was investigated in seeds stratified at 4°C or incubated at 15°C. Seeds stratified at 4°C germinated after 27 days; seeds incubated at 15°C failed to germinate. Stratification increased the embryo's capacity for protein synthesis by day 11 as measured by in vivo incorporation of [³⁵S]-methionine into purified protein. At 4°C protein synthesis in the embryonic axis rose in a linear fashion prior to germination, whereas in cotyledons it increased until day 20 and then declined. Analysis of radiolabelled proteins by two-dimensional gel electrophoresis revealed that the levels of specific proteins were altered by temperature, primarily in the cotyledons. Several proteins were expressed in the cotyledons at 15°C but were absent in unstratified embryos and in embryos stratified at 4°C. That is, the expression of these proteins was repressed during stratification and release from dormancy. Levels of other proteins in the cotyledons declined at 4°C during stratification. We suggest that one or more of these proteins may be associated with the inhibition of growth of the embryonic axis imposed by the cotyledons.