Thermodormancy in Celery Seeds and its Removal by Cytokinins and Gibberellins

Imbibition of celery (Apium graveolens L.) seeds at 32°C for up to 96 h lowered the upper temperature limit for germination. If this high temperature treatment was given in the light, these seeds germinated slightly earlier than those treated in the dark although the final percentage germination was similar for both treatments. The inhibitory effect of the high temperature treatment was completely removed by allowing the seeds to imbibe in a mixture of the gibberellins A₄ and A₇ (GA_4/7) and partially removed by the cytokinin N⁶-benzylaminopurine (BA). GA_4/7 was less effective when added before rather than after the high temperature treatment, whereas the opposite was true of BA. At constant temperatures more GA_4/7 was required to promote germination as the temperature was raised but addition of BA reduced the concentration of GA_4/7 required. A model is proposed for the control of celery seed germination by light and temperature through the action of endogenous cytokinins and gibberellins.     

Evidence for the accumulation of a germination inhibitor during progressive thermoinhibition of seeds of celery (Apium graveolens L.)

The germination of seeds of celery (Apium graveolens L.) becomes progressively thermoinhibited on incubation in the dark at high temperatures, the inhibitory temperature being dependent on the cultivar used. In two high-dormancy cultivars of celery, the production of germination inhibitors in seeds incubated in the dark at 26°C gradually increased over a 7-day period. Inhibitor production was measured by incubating seeds of the low-dormancy cultivar Florida 683 in homogenates of the thermoinhibited seeds of the high-dormancy cultivars and recording germination either in the light or with the gibberellins A₄ and A₇ (GA_4/7) in the dark. Most Florida 683 seeds which failed to germinate in the homogenates after 15 days were induced to germinate by addition of N⁶-benzyladenine (BA). The presence of BA in addition to GA_4/7 throughout incubation in the dark completely overcame the inhibitory effects of homogenates. This indicates that thermoinhibition of celery seeds is associated with the accumulation of a germination inhibitor which interacts with cytokinins. This does not appear to be abscisic acid (ABA) since ABA levels in thermoinhibited seeds were lower than in untreated seeds and did not increase with duration of high temperature treatment.Abbreviations ABA Abscisic acid - BA N⁶-benzyladenine - GA_4/7 a mixture of the gibberellins A₄ and A₇ - HTP high-temperature pretreatment     

Possible control of gibberellin-induced release of temperature-dependent primary dormancy in seeds of celery (Apium graveolens L.) by transmembrane ion fluxes

The temperature-dependent primary dormancy of cv Florida 683 celery seeds in darkness was broken by GA_4/7 (2 × 10^-4 M) alone but other growth regulators such as BA, ethephon or daminozide were necessary to break dormancy of cv Lathom Blanching seeds in the presence of GA_4/7 at this concentration. Although AgNO₃ partially inhibited both the ethephon- and BA- induced germination of cv Lathom Blanching seeds in the presence of GA_4/7 in the dark it did not affect the promotive action of daminozide. Ethephon did not overcome the inhibitory action of high concentrations of AgNO₃ in the light. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) did not inhibit the germination of cv Lathom Blanching seeds induced by growth regulators in the dark or in the absence of growth regulators in the light. Fusicoccin (FC) did not break celery seed dormancy unless applied in the presence of GA_4/7. Germination of cv Lathom Blanching celery seeds treated with GA_4/7 at 16°C in the dark was inhibited by the K⁺ ionophore benzo-18-crown-C-6 (18-C-6) and in the presence of Ca²⁺ by the Ca²⁺ ionophore A23187; the 18-C-6 inhibition was reversed by BA.It is concluded that the involvement of gibberellin in celery seed dormancy is not dependent on endogenous ethylene and is directly or indirectly controlled through the action of other hormones on transmembrane ion fluxes.     

Relationship between Position on the Parent Plant and Dormancy Characteristics of Seeds of Three Cultivars of Celery (Apium graveolens)

Germination and seedling emergence studies were made on seeds harvested from four different umbel positions of three cultivars of celery (Apium graveolens L.). Although heavier seeds were produced from primary umbels than from other umbels, these were less viable as measured by the germination percentage at I8°C in the light. However, germination of viable seeds from quaternary umbels was lower than that of seeds from primary umbels at 18°C in the dark when incubated with GA₄, (2 × 10 ^?4M) and seed from secondary and tertiary umbels tended to be intermediate in response. All viable seeds germinated when N⁶-benzyladenine (10^?2M) was used in combination with GA₄. Seeds from quaternary umbels of two of the cultivars had a lower high-temperature limit for germination in the dark than did seeds from other umbels. In glasshouse experiments the emergence of viable ‘quaternary’ seeds of these cultivars was higher than that of ‘primary’ seeds. Under these conditions the time to 50% of the final emergence as determined after 42 days was similar for seeds from all umbel positions within each cultivar. In two varieties seedling weights were greater from seeds of primary as compared to quaternary umbels, and in general, the largest seedlings arose from the heaviest seeds and the smallest from the lightest seeds in all three cultivars.     

Interactions between Abscisic Acid, Gibberellins and Cytokinins in Grand Rapids Lettuce Seeds Germination

Experiments with Grand Rapids lettuce seeds (Lactuca sativa L.) maintained in darkness or irradiated with red light have shown that the inhibition of germination induced by low concentrations of ABA (2, 4, 6 μM) could be overcome by gibberellins (GA₃ or GA₄). The same results were obtained, although to a lesser extent, under the influence of two out of the four cytokinins tested (K and BAP) for seeds maintained in darkness. To suppress the block induced by higher concentrations of ABA (for example 8 μM), it was necessary to apply a cytokinin (K, BAP, Z or 2iP) and a gibberellin (GA₄ or GA₃) simultaneously, or a cytokinin following a red light treatment. Experiments conducted in darkness in which ABA (8 μM) was applied together with a cytokinin (BAP) and a gibberellin (GA₄) showed that the gibberellin and the cytokinin played similar roles towards each other and towards ABA.     

Responses of dormant heather (Calluna vulgaris) seeds to light, temperature, chemical and advancement treatments

Two seed lots of Calluna vulgaris were obtainedfrom English Nature (seed of Cornish provenance) (EN) and John ChambersWildflower Seeds (JCWS). In laboratory tests, under continuous light untreatedseeds of both seed lots were partially dormant at temperatures between14–35 °C, but JCWS seeds were more deeply dormant thanENseeds. The optimum temperature for germination for both lots was ca 18°C. Germination of EN seeds was much lower in the dark than inthe light at all temperatures; JCWS seeds did not germinate in the dark. In thelight at 22 °C, dormancy of both seed lots was broken whenseeds were incubated in GA_4/7 solution(2×10^–4 M). Dormancy ofJCWSseeds at 22 °C in the light was broken when seeds wereincubated in four different smoke solutions but more so when used incombinationwith GA_4/7. Soaking seeds for 4h insmoke/GA_4/7solutions before sowing improved both the speed andpercentage germination in pot experiments on a mist bench in the glasshouse byat least 10-fold. Soaking with GA_4/7 alone produced a 5-fold increasein germination but seedlings were more etiolated than with thesmoke/GA_4/7 mixtures. A seed advancement treatment modified from thatused commercially on sugar beet seeds also promoted germination in bothlaboratory and glasshouse tests. This entailed soaking seeds in 0.2% thiramsuspension for 4h followed by incubation in excess solution at 22°C for 4 days. This treatment was not as effective as thesmoke/GA_4/7 seed soaks.     

Induction of Secondary Dormancy in Chenopodium bonus-henricus L. Seeds by Osmotic and High Temperature Treatments and Its Prevention by Light and Growth Regulators

Factors controlling the establishment and removal of secondary dormancy in Chenopodium bonus-henricus L. seeds were investigated. Unchilled seeds required light for germination. A moist-chilling treatment at 4 C for 28 to 30 days removed this primary dormancy. Chilled seeds now germinated in the dark. When chilled seeds were held in the dark in −8.6 bars polyethylene glycol 6000 solution at 15 C or in water at 29 C a secondary dormancy was induced which increased progressively with time as determined by subsequent germination. These seeds now failed to germinate under the condition (darkness) which previously allowed their germination. Continuous light or daily brief red light irradiations during prolonged imbibition in polyethylene glycol solution at 15 C or in water at 29 C prevented the establishment of the secondary dormancy and caused an advancement of subsequent germination. Far red irradiations immediately following red irradiation reestablished the secondary dormancy indicating phytochrome participation in “pregerminative” processes. The growth regulator combination, kinetin + ethephon + gibberellin A₄+A₇ (GA₄₊₇), and to a relatively lesser extent GA₄₊₇, was effective in preventing the establishment of the secondary dormancy and in advancing the germination or emergence time. Following the establishment of the secondary dormancy by osmotic or high temperature treatments the regulator combination was relatively more active than light or GA₄₊₇ in removing the dormancy. Prolonged dark treatment at 29 C seemed to induce changes that were partially independent of light or GA₄₊₇ control. The data presented here indicate that changes during germination preventing dark treatment determine whether the seed will germinate, show an advancement effect, or will become secondarily dormant. These changes appear to be modulated by light and hormones.     

Responses of florence fennel (Foeniculum vulgare azoricum) seeds to light,temperature and gibberellin A4/7

The percentage and the rate of germination of seeds of three varieties of Florence fennel was higher in the dark than in the light, the high temperature cut-off points being between 27.2 and 29.4°C. The optimum temperature for germination was between 20 and 25°C. Seeds of all three varieties responded to incubation in solutions containing gibberellin A_4/7 mixture (GA_4/7; Regulex), giving higher germination in the light at temperatures from 20 to 30°C. Seeds steeped for 4 h at 25°C or for 24 h at 5°C in GA_4/7 solutions gave a higher percentage and increased rate of emergence as compared with untreated dry seeds, when sown in compost at 25°C; steeping in water alone was also beneficial. In general, drying the treated seeds before sowing reduced the rate but sometimes increased the percentage of germination as compared with seeds sown when still moist. Seeds harvested from secondary umbels of var. Zefa fino germinated better both in the light and dark than those taken from primary umbels.Abbreviations GA_4/7 a commercial mixture of the gibberellins A₄ and A₇ (trade name Regulex)     

Enhancement of Gibberellin Activity by Ethylenediaminetetraacetic Acid in Celery Seeds and Embryoless Barley Seeds

The phytochrome-controlled dormancy of celery seeds (Apium graveolens L.) can be broken by exogenously applied growth regulators. Gibberellins, especially the mixture of GA₄ and GA₇ appeared to be the primary stimuli for germination of darkincubated seeds. However, concentrations less than 1 mM were ineffective in replacing the light requirement in one celery cultivar (cv.Utah), and in another cultivar (cv.Lathom Blanching), concentrations above 1 mM were insufficient. The addition of ethylenediaminetetraacetic acid (EDTA) to gibberellins, increased markedly their activity and reduced by several orders of magnitude the gibberellin concentrations needed to stimulate a light treatment. This synergistic activity is similar to the effect of benzyladenine (BA) or Succinic acid-2,2-dimethylhydrazide (SADH) when added to gibberellins. In a diverse plant system, the mixture of gibberellin with EDTA increased significantly the amount of reducing sugars released by embryoless barley seeds.     

A potential role for endogenous microflora in dormancy release,cytokinin metabolism and the response to fluridone in Lolium rigidum seeds

Background and Aims Dormancy in Lolium rigidum (annual ryegrass) seeds can be alleviated by warm stratification in the dark or by application of fluridone, an inhibitor of plant abscisic acid (ABA) biosynthesis via phytoene desaturase. However, germination and absolute ABA concentration are not particularly strongly correlated. The aim of this study was to determine if cytokinins of both plant and bacterial origin are involved in mediating dormancy status and in the response to fluridone.Methods Seeds with normal or greatly decreased (by dry heat pre-treatment) bacterial populations were stratified in the light or dark and in the presence or absence of fluridone in order to modify their dormancy status. Germination was assessed and seed cytokinin concentration and composition were measured in embryo-containing or embryo-free seed portions.Key Results Seeds lacking bacteria were no longer able to lose dormancy in the dark unless supplied with exogenous gibberellin or fluridone. Although these seeds showed a dramatic switch from active cytokinin free bases to O-glucosylated storage forms, the concentrations of individual cytokinin species were only weakly correlated to dormancy status. However, cytokinins of apparently bacterial origin were affected by fluridone and light treatment of the seeds.Conclusions It is probable that resident microflora contribute to dormancy status in L. rigidum seeds via a complex interaction between hormones of both plant and bacterial origin. This interaction needs to be taken into account in studies on endogenous seed hormones or the response of seeds to plant growth regulators.     

Variation in seed dormancy and germination among populations of Silybum marianum (Asteraceae)

Milk thistle (Silybum marianum) is a medicinal plant; however, lack of consistency in past dormancy studies has hindered propagation of this species from seeds. We tested the germination responses of freshly harvested and after-ripened (stored for 2 and 7 months; 25°C at 50% relative humidity) seeds from three populations (P1, P2 and P3) in Iran at varying constant or alternating temperatures, with or without GA₃ and in light and continuous darkness. No germination occurred in freshly harvested seeds incubated at any condition without GA₃ application, indicating that all the seeds were dormant. Seeds from P1 and P2, which developed under relatively dry, warm conditions, germinated over a wider range of temperatures after 2 months of dry storage, indicating type 6 of non-deep physiological dormancy (PD). Seeds from P3, which developed under relatively wet, cool conditions, incubated at constant temperatures (especially on GA₃), exhibited an increase in maximum temperature for germination, indicating type 1 of non-deep PD. Light improved germination of after-ripened seeds, and GA₃ application substituted for the light requirement for germination. This is the first report that environmental conditions during seed development may be correlated with differences in the type of non-deep PD. We conclude that milk thistle seeds are positively photoblastic and photodormant and the germination responses of after-ripened seeds from different populations are different under darkness. Therefore, the impacts of genetic differences and maternal effects on the induction of dormancy during seed development should be considered in attempts to domesticate this medicinal plant.     

Comparative effects of gibberellins and an N-substituted phthalimide on seed germination and extension growth of celery (Apium graveolens L.)

The N-substituted phthalimide AC 94377 (1-(3-chlorophthalimido)-cyclohexanecarboxamide) was equally effective as a mixture of the gibberellins A₄ and A₇ (GA_4/7) in breaking dormancy and stimulating germination of celery seeds when either was used in combination with ethephon or daminozide as a seed soak. Whereas seedlings emerging from GA_4/7-treated seeds became etiolated in comparison with those from untreated seeds, those from AC 94377-treated seeds showed normal development. Preharvest sprays of gibberellic acid (GA₃) increased the height of mature plants in comparison with untreated controls by about 16 per cent whereas AC 94377 was ineffective. The yield from GA₃-treated plots was about 10 per cent greater than that from AC 94377-treated plots.     

Changes in endogenous cytokinins of celery (Apium graveolens L.) seeds following an osmotic priming or growth regulator soak treatment

Celery seeds were less thermoinhibited when dried back after a seed soak treatment with the gibberellins A₄ and A₇ (GA_4/7) plus ethephon (G+E) or an osmotic priming treatment in the light with polyethylene glycol (PEG). At temperatures between 18 and 25° in the dark, 50 percent of the PEG-treated seeds germinated after 3 days whereas G+E-treated seeds required 7 days and untreated seeds did not germinate at all.Irrespective of treatment, dry control and dried-back, treated seeds contained very little detectable cytokinin activity. However, when such seeds were imbibed for 18 h in the dark and then analysed immediately with the soybean callus bioassay, less cytokinin activity was detected in both G+E and PEG seeds than in the untreated seeds. In particular, cytokinins with the HPLC properties of zeatin and its riboside were decreased in G+E seeds and virtually absent from PEG seeds. Conversely, extracts from PEG and to a lesser extent G+E seeds contained activity which chromatographically resembled cytokinin glucosides whereas this was absent from untreated seeds.     

Germination studies on three critically endangered endemic angiosperm species of the Kashmir Himalaya,India

The critically endangered and perennial alpine endemic angiosperms, namely, Aquilegia nivalis, Lagotis cashmeriana and Meconopsis latifolia inhabit such habitats in the Kashmir Himalaya that are characterised by short growing season and heavy snow cover for about 3–4 months during winter season. The seeds of these species under natural conditions experience a long period of pre-chilling during winter prior to their germination in following spring season. Taking cue from such a requirement, present study investigated the effect of chilling and exogenous application of growth hormones, NO₃ ⁻ and NH₄ ⁺ on total percent germination of otherwise deep-dormant seeds of these species, under alternate light/dark and continuous dark light regimes. Prolonged pre-chilling followed by treatment of seeds with different doses of GA₃ had a pronounced stimulatory effect on the total germination percentage in all the three species. In fact, highest germination percentage in A. nivalis was recorded only when pre-chilled seeds were treated with 1.5 mM GA₃ under alternate light/dark conditions. Likewise, germination of pre-chilled seeds of L. cashmeriana with ruptured seed coats was improved when treated with various concentrations of GA₃. Seed germination in M. latifolia was also favourably influenced by treatment of pre-chilled seeds with GA₃ or nitrogen applied either as NO₃ ⁻ or as NH₄ ⁺ under alternate light/dark conditions. Treatment of seeds with kinetin (6-furfuryl-aminopurine) had no significant influence on germination percentage in any of the three species. Thus, prolonged chilling of seeds followed by their treatment with GA₃ under alternate light/dark conditions are the requirements necessary for seed germination in these species.     

Gibberellins in seeds of Arabidopsis thaliana: biological activities,identification and effects of light and chilling on endogenous levels

The activities of several gibberellins in stimulating germination of wild-type and GA-deficient gal seeds of Arabidopsis thaliana were compared. Of the six compounds tested GA₄ and GA₇-isolactone had the highest activity and GA₇ and GA₉ the lowest; activities of GA₁ and GA₃ were intermediate. Combined application of pure GAs presented no indications that more than one GA receptor is involved. Four GAs were identified in extracts from wild-type and GA-insensitive gai seeds by combined gas chromatography mass spectrometry: GA₁, GA₃, GA₄ and GA₉. Effects of light and chilling on levels of GA₁, GA₄ and GA₉ were studied using deuterated standards. Light increased both GA levels and germination in unchilled wild-type and gai seeds. As a result of irradiation GA levels in gai seeds were 7–10 times as high as in wild-type seeds. In the dark germination was 0%, in the light 14% of gai seeds and 95% of wild-type seeds germinated. A chilling pre-treatment of 7 days at 2°C was required to enhance further the germination of gai seeds in the light. Light did not increase GA levels of chilled seeds of either genotype; levels of GA₄ and GA₉ of chilled gai seeds, in the light were respectively 7 and 12 times lower than in non-chilled seeds, whereas the latter seeds germinated better. Slightly elevated levels of GA₄ were detected in darkness after chilling, but germination capacity was still 0%. These results strengthened the conclusion that GAs are required for germination of A. thaliana seeds, whereby GA₄ has intrinsic biological activity. However, it is unlikely that light and chilling stimulate germination primarily by increasing levels of GA. Instead GA sensitivity is a possible alternative.     

Stimulation of Empress Tree Seed Germination by Liquid Smoke

The germination of Empress tree (Paulownia tomentosa Steud.) seeds is phytochrome-controlled. Liquid smoke could not induce germination in darkness but red light irradiation of liquid smoke imbibed seeds induced a high percentage of germination. Maximum germination was achieved at liquid smoke concentration of 0.1% (v/v) when present during the imbibition phase or during the phase of phytochrome activity. The light requirement of these seeds could be completely substituted by exogenously applied gibberellins. In the presence of liquid smoke, optimal concentrations of GA₃, GA₄, and GA₉ necessary for inducing germination were several times lower than in the controls, while that of GA₇ was equally active when applied at a concentration one order of magnitude lower. The inhibitory effect of the applied growth retardants was strongly reduced and liquid smoke, in the presence of retardants, allowed light-induced germination, if applied simultaneously or after retardants treatment.     

Differential effects of camptothecin and interactions with plant hormones on seed germination and seedling growth

Effects of camptothecin, a naturally occurring alkaloid, on seed germination varied from promotive to inhibitory, depending on the species used. It markedly inhibited seedling root growth but its inhibition of hypocotyl growth varied among species. Camptothecin inhibited GA₃-induced dark germination of lettuce (Lactuca sativa L.) seeds and hypocotyl elongation of seedlings. In contrast to ABA, the camptothecin inhibition of GA₃-induced germination could not be overcome by cytokinin. When seeds were germinated at 29C with a 0.5 h light treatment, little or no germination occurred in the camptothecin treatment, but addition of cytokinin overcame this inhibition.     

Growth regulatory effect of three benzimidazole fungicides on the germination of celery (Apium graveolens) seeds

Seeds of four celery cultivars did not germinate when incubated in buffered solutions in the dark at 22 °C. This high-temperature-induced dormancy was broken with either of the systemic fungicides benomyl or BAS 3460 F (mainly methyl-2-benzimidazole carbamate) when used in combination with a mixture of the gibberellins A₄ and A₇ (GA_4/7). The fungicide thiabendazole was ineffective in these tests.     

Interaction between a plant-derived smoke extract,light and phytohormones on the germination of light-sensitive lettuce seeds

Plant-derived smoke extracts mimics the effect of red light on germination in light-sensitive lettuce seeds and partially overcomes the inhibitory effect of far-red light. Interaction between a smoke extract and gibberellins, cytokinins, abscisic acid and ethephon was investigated. Smoke acted synergistically with GA₃ and increased the sensitivity of the lettuce seeds to ABA. It seems likely that smoke affects membrane permeability or receptor sensitivity rather than influencing the phytochrome system of these seeds.Abbreviations R red light - FR far-red light - SM smoke extract     

Response of Amaranthus retroflexus L. seeds to gibberellic acid, ethylene and abscisic acid depending on duration of stratification and burial

Freshly harvested, dormant seeds of Amaranthus retroflexus were unable to germinate at 25 and 35 °C. To release their dormancy at the above temperatures, the seeds were stratified at a constant temperature (4 °C) under laboratory conditions or at fluctuating temperatures in soil or by outdoor burial in soil. Fully dormant, or seeds stratified or buried (2006/2007 and 2007/2008) for various periods were treated with exogenous gibberellic acid (GA₃), ethephon and abscisic acid (ABA). Likewise, the effects of these regulators, applied during stratification, on seed germination were determined. The results indicate that A. retroflexus seed dormancy can be released either by stratification or by autumn–winter burial. The effect of GA₃ and ethylene, liberated from ethephon, applied after various periods of stratification or during stratification, depends on dormancy level. GA₃ did not affect or only slightly stimulated the germination of non-stratified, fully dormant seeds at 25 and 35 °C respectively. Ethylene increased germination at both temperatures. Seed response to GA₃ and ethylene at 25 °C was increased when dormancy was partially removed by stratification at constant or fluctuating temperatures or autumn–winter burial. The response to GA₃ and ethylene increased with increasing time of stratification. The presence of GA₃ and ethephon during stratification may stimulate germination at 35 °C. Thus, both GA₃ and ethylene can partially substitute the requirement for stratification or autumn–winter burial. Both hormones may also stimulate germination of secondary dormant seeds, exhumed in September. The response to ABA decreased in parallel with an increasing time of stratification and burial up to May 2007 or March 2008. Endogenous GA_n, ethylene and ABA may be involved in the control of dormancy state and germination of A. retroflexus. It is possible that releasing dormancy by stratification or partial burial is associated with changes in ABA/GA and ethylene balance and/or sensitivity to these hormones.