Studies in seed dormancy

Summary The dormancy of freshly harvested hazel seeds appears to be induced by inhibitors occuring mainly in the testa and pericarp. Although d abscisic acid may not be one of the natural inhibitors involved, d,l abscisic acid has been shown to strongly inhibit the germination of hazel seeds, probably through its antagonism towards the action of gibberellin. Dry storage of hazel nuts causes a deeper state of dormancy (secondary dormancy) to be superimposed on the primary dormancy. It is suggested that secondary dormancy consists of a block to gibberellin synthesis. The essential effect of chilling intact hazel seeds, which is the natural means of breaking their dormancy, may be to activate the mechanism for gibberellin synthesis, the subsequent synthesis of gibberellin being thought to occur at the germination temperature (20°C) and not at the chilling temperature (5°C).     

Studies in seed dormancy

Summary When dormant hazel seeds were subjected to six weeks chilling at 5° C their subsequent transfer to 20° C resulted in the accumulation of gibberellin (GA) followed by germination. In the presence of either phosphon D or -chlorethyltrimethylammonium chloride (CCC) at 20° C there was inhibition of both GA accumulation and germination, a finding consistent with the hypothesis that GA biosynthesis is a necessary prerequisite for the germination of chilled hazel seeds. As abscisic acid showed a strong inhibition of germination but had little effect on GA accumulation it is presumed not to have affected GA biosynthesis but to have inhibited GA action. These conclusions were supported by experiments in which the interaction of exogenous GA₃ with growth retardants and ABA was tested on the germination of chilled hazel seeds. Experiments in which the embryonic axes and cotyledons of chilled seeds were incubated separately at 20° C established that GA biosynthesis de novo occurred in the embryonic axis and indicated that in the intact seed some of the GA would have been translocated to the cotyledons. The isolated cytoledons showed no GA biosynthesis de novo but gave some release of GA from one or more bound forms.Abbreviations ABA abscisie acid - AMO 1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine carboxylate - CCC -chlorethyltrimethylammonium chloride - GA gibberellin, phosphon - D tributyl-2,4-dichlorobenzylphosphonium chloride - TLC thin-layer chromatography     

Effect of Growth Retardants on α-Amylase Production in Germinating Barley Seed

The effect of growth retarding compounds, (2-chloroethyl)trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (AMU-1618), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D) and N-dimethylamino succinamic acid (B-995) on α-amylase production in germinating barley seed was studied. Seeds were germinated in growth retardants in presence and absence of gibberellic acid (GA₃). CCC, AMO-1618 and Phosfon D inhibitedα-amylase production in germinating seed and the effect was reversed by GA₃ Phosfon D and AMO-1618 were stronger inhibitors of α-amylase production than CCC. CCC was by far the strongest inhibitor of all the other analogs tested. B-995 was comparatively only slightly inhibitory. The results reported here, when viewed in light of the results of other workers, provide good evidence that CCC, AMO-1618 and Phosfon D inhibit α-amylase production by inhibiting the synthesis of gibberellin or gibberellin-like hormone(s) during germination of barley seed. Consistent with other reports, B-995 possibly acts by other mechanism (s).     

Hormonal Regulation of Seed Dormancy in Hazel (Corylus avellana L.) and Beech (Fagus sylvatica L.)

Dormancy in seeds of hazel (Corylus avellana L.) and beech (Fagussylvatica L.) has been studied with special reference to changesin growth-promoting and inhibiting substances during after-ripening.About 12 weeks at low temperature and under moist conditionsis necessary for complete after-ripening. Gibberellic acid,kinetin, and thiourea stimulate germination in dormant seedsbut have no effect on nuts with the pericarp intact. Gibberellin‘D’ is ten times more active than gibberellic acid.Bio-assays, following chromatographic fractionation of seedextracts, have revealed no significant changes in the concentrationsof auxins and inhibitors during after-ripening. Dwarf maize-leafsection assays have revealed low concentrations of gibberellin-likesubstances in purified extracts of chilled, dormant hazel seedbut no gibberellin activity in extracts of dormant seed. Gibberellinsare present in both dormant and germinating beech seeds butthere appear to be differences in the chromatographic patternof activity. The possible role of endogenous gibberellins inthe after-ripening process is discussed.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Wound healing in potato tuber tissue: phosphon inhibition of developmental processes requiring protein synthesis

Several aspects of wound healing in tuber tissue of potato (Solanum tuberosum var. Kennebec), known to require protein synthesis, are inhibited by 2,4-dichlorobenzyltributylphosphonium chloride (Phosphon D). Cell division was completely blocked by 60 mum Phosphon and markedly reduced by concentrations as low as 3 mum. When applied at the time of wounding, 0.25mm Phosphon completely prevented the wound-induced respiratory increase. Application at 15 hours after wounding arrested respiration at the rate present at that time. The same concentrations of Phosphon inhibited auxin-induced cell expansion of the tissue, protein synthesis as measured by the incorporation of leucine-(14)C into the trichloroacetic acid-insoluble fraction of tissue disks, and the appearance of wound-induced peroxidase isozymes. None of these inhibitory effects of Phosphon could be prevented or reversed by the application of gibberellic acid. All wound-induced processes inhibited by Phosphon are also inhibited by cycloheximide. It is suggested that inhibitory effects of Phosphon on wound healing in potato and on other developmental processes in excised plant tissues which cannot be reversed by gibberellin are due to interference with protein synthesis.     

Growth Retardants and the Germination of Light Sensitive Lettuce Seed

The effect of the dwarfing compounds chlormequat, B995, and Phosphon D on the germination of lettuce seed in darkness was found to be a depression which could be overcome in part by red light, GA, and kinetin. There was a marked statistical interaction between the growth retardants and all the mitigating treatments except B995 and GA, and light, at 30°C and GA and Phosphon D at 20°C and this implies a biological interaction at the site of action of the growth retardant. It is suggested that the action of the compounds considered is in the establishment of an ordered system since it is unlikely that there would be a common binding site for kinetin and GA.     

Differential effects of actinomycin d and cordycepin in lettuce seed germination and RNA synthesis

Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using ³H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. ³H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited ³H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of ³H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination.     

Relationships between position on the parent plant and germination characteristics of seeds of parsley (Petroselinium crispum Nym.)

The percentage germination of seeds of parsley cv. Imperial Curled was higher in the light than in the dark, the high temperature limits for germination being 30 and 28°C for light and dark respectively. At the higher temperatures, the germination rate was slower in the dark. At 30°C, treatment with a gibberellin A_4/7 mixture at 2 × 10^–4 M partially alleviated the inhibiting effect of darkness on the germination percentage. Pre-incubation of parsley seeds at 35°C in the dark for 30 h increased the rate, but decreased the percentage, of germination of seeds incubated at 15°C in the light. Germination and seedling emergence studies were made on seed harvested from four different umbel positions. Although heavier seeds were produced from primary umbels than from other umbel orders, they were less viable as measured by seedling emergence in the glasshouse. The rate of emergence was decreased with increasing umbel order i.e. with later seed development: this was reflected in subsequent seedling weights, with seedlings from quarternary umbel seeds being about half the weight of those from primary umbel seeds. The upper temperature limit for dark germination was only slightly affected by umbel order, with quarternary umbel seeds being the most thermo-inhibited.Abbreviations BA N⁶-benzyladenine - GA_4/7 a mixture of gibberellins A₄ and A₇ - SD8339 6-benzyl-amino-9-(tetrahydropyran-2-yl)-9H-purine     

Germination of Tagetes minuta L. II. Role of gibberellins

The effect of the application of gibberellins to Tagetes minuta L. achenes (seeds) was determined at both 25°C, the optimal germination temperature, and 35°C, at which temperature the achenes are thermoinhibited. Both GA₃ and GA₄₊₇ accelerated germination at 25°C. Seed germination at 25°C was inhibited by paclobutrazol, but on subsequent application of GA₄₊₇ rapid germination was induced. Following application of GA₃ or GA₄₊₇ to thermoinhibited seeds, a significantly higher final germination percentage was observed than in the distilled water control. However, endogenous gibberellin levels in germinating and thermoinhibited seeds did not differ significantly.     

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.     

RNA synthesis and the germination of light-sensitive lettuce seeds

Summary The germination of lettuce seeds is inhibited by the nucleotide base analogue 6-methylpurine. RNA synthesis has been measured during imbibition and germination as ³²P-phosphate incorporation into RNA species as fractionated by polyacrylamide gel electrophoresis. Seeds were surface sterilized and imbibed in the presence of various antibiotics. RNA preparations from lettuce seeds were coelectrophoresed with ³H-RNA prepared from bacteria to check for bacterial contamination of the seeds. There is a much higher rate of RNA synthesis in illuminated, germinating seeds as compared to dark, non-germinating seeds. This difference does not develop until after 12 hours of imbibition at 27°, which is the time of onset of germination and radicle growth.This investigation was supported by a contract from the United States Department of Agriculture (No. 616-15-3). Journal paper of the Purdue Agriculture Experiment Station.     

Embryo Development in Ripe Seeds of Eranthis hiemalis and its Relation to Gibberellic Acid

The ripe seeds of Eranthis hiemalis (L.) Salisb., the winter aconite, contain undeveloped embryos. At 20–25°C the embryos grow only little, and the seeds do not germinate. Rapid embryo development starts if the seeds, after 3 weeks of “after-ripening” at 20–25°C, are placed at low temperature, 3–4°C; germination then takes place after 2–3 months, Embryo development without germination occurs when the seeds are placed in gibberellic acid solutions at 20–25°C. Embryo development is inhibited at low temperature by the specific inhibitor of gibberellin biosynthesis, 2-chlorethyl cholin chloride, but is restored by the simultaneous addition of gibberellic acid. It is suggested that one early effect of the cold is to bring about a synthesis of gibberellin.     

Action of ferric and aluminium ions on the dormancy breakage of <Emphasis Type="Italic">Stylosanthes humilis</Emphasis> seeds

Dormancy of scarified seeds of Stylosanthes humilis was broken by acidic Al³⁺ and Fe³⁺ solutions. Fe⁺³-stimulated seeds exhibited a high activity of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and produced great amounts of ethylene, which showed correlated with the germination process. In addition, specific inhibitors of ethylene biosynthesis and action largely depressed the Fe³⁺-stimulated germination, leading to the conclusion that the ion broke dormancy by triggering ethylene production by the seeds. By contrast, inhibitors of ethylene biosynthesis and action did not impair germination of Al³⁺-stimulated dormant seeds. Moreover, ethylene production and activity of ACC oxidase of Al³⁺-treated seeds was substantially decreased by inhibitors of ethylene biosynthesis, but germination kept large. Together these data suggest that ethylene biosynthesis was not required in the chain of events triggered by Al³⁺ leading to dormancy breakage. Methyl viologen (MV), a reactive oxygen species-generating compound, broke dormancy of seeds to the same extent as Al³⁺ did. Germination of both Al³⁺- and MV-stimulated dormant seeds was inhibited by sodium selenate, an antioxidant compound; selenate, however had no effect on germination of Fe³⁺-stimulated seeds. Together these data indicate that the mechanisms underlying the germination of Al³⁺- and Fe³⁺-treated seeds are not the same.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

The Effects of Kinetin and Gibberellin on the Germination of Dehusked Seeds of Indica and Japonica Rice (Oryza sativaL.) under Anaerobic and Aerobic Conditions

The effects of kinetin and gibberellin were examined under anaerobicconditions (0% oxygen) and aerobic conditions (20% oxygen) onthe germination of dehusked seeds of indica and japonica ricecultivars that had been harvested at different times duringthe formation of seeds. Surjamkhi was used as a representativeof deep dormant indica cultivars and Assam IV as a less dormantindica cultivar. Sasanishiki was used as the japonica rice cultivar.Both phytohormones were applied at a concentration of 10^-3Mwhichproved to have the greatest stimulatory effect in preliminarywork at concentrations of 10^-3–10^-5M. Under aerobic conditions,inhibition of germination by dehusking of Sasanishiki seedsthat had been harvested either 30 or 60 d after anthesis wasovercome by kinetin and all seeds germinated. Complete germinationinduced by kinetin under aerobic conditions was also achievedwith the dehusked seeds of the indica rice cultivar Assam IVthat had been harvested on two occasions and of Surjamkhi thathad been harvested 28 d after anthesis. In contrast, germinationof dehusked japonica seeds stimulated by anaerobiosis was inhibitedby kinetin. The stimulatory effects of gibberellin on the germinationof indica and japonica rice seeds were observed under aerobicand anaerobic conditions. Under anaerobic conditions, the responsesof dehusked indica and japonica rice seeds to kinetin and gibberellindiffered, being negative with kinetin and positive with gibberellin.Under aerobic conditions, the stimulatory effects of kinetinon germination of dehusked seeds were greater than those ofgibberellin. Thus, treatment with kinetin appears to be usefulfor breaking the considerable dormancy commonly observed inthe dehusked seeds of indica rice. Mechanisms are proposed toexplain the stimulatory effects of these phytohormones on thegermination of dehusked seeds of indica and japonica rice underaerobic and anaerobic conditions. Rice; Oryza sativaL.; seed germination; dehusking treatment; gibberellin; indica; japonica; kinetin; oxygen; dormancy; germination inhibition; seed formation     

Effect of Combined Salt and Heat Treatments on Germination and Heat-Shock Protein Synthesis in Lentil Seeds

The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 –12 h imbibition. [³⁵S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.     

Interaction of triacontanol with gibberellin in control of lettuce seed germination

Triacontanol at concentrations from 2.3 × 10^-9 M to 2.3 × 10^-7 M did not affect the germination of lettuce ( Lactuca sativa L., cv. Grand Rapids) seeds in darkness, stimulated by light at 25°C or by benzyladenine at 31°C. Stimulation of seed germination by gibberellin A₃ (10^-5 M ) was significantly inhibited by triacontanol; the most effective concentration was 4.6 × 10^-8 M. Pulse experiments demonstrated that triacontanol was ineffective when applied later than gibberellin, whereas an inverse sequence of treatment caused an inhibition comparable to that resulting from continuous treatment of seeds with both factors. Possible interaction of triacontanol with gibberellin receptor is discussed.     

Effect of gibberellic acid and uniconazol on embryo abortion in the stenospermocarpic grape cultivars Emperatriz and Perlon

Hypothesizing that seed abortion in stenospermocarpic grapes (Vitis vinifera L.) is caused by high gibberellin levels in the seed during the first stages of its development, we studied the effect of gibberellic acid GA₃ and uniconazol (a GAs biosynthesis inhibitor) on this phenomenon. In vitro germination was analyzed in the seedless cultivars Emperatriz and Perlon, which were treated with 60 and 120 mg.^-l 1 uniconazol (5 and 15 days before bloom) and 100 mg.^-l 1 GA₃ (5 days after bloom). In addition, endogenous levels of free gibberellins in flowers and seeds of Emperatriz and Perlon were compared with their seedeed progenitor Emperador. Clusters were harvested at bloom and 20 days after bloom for gibberellin analysis and at commercial maturity for in vitro culture of the seeds. Considerable gibberellin activity was found in the three cultivars, but only small differences were detected between the seedless and the seeded genotypes. Exogenous applications of GA₃ had a deleterious effect on seed growth and on in vitro germination. Uniconazol also inhibited in vitro germination, though not affecting the total number of germinating embryos plus those rescued from non-germinating seeds. In conclusion, gibberellins do not appear to be directly involved in seed abortion of the stenospermocarpic cultivars Emperatriz and Perlon, although their participation in a more complex scenario should not be rejected, taking into account that in Perlon germination rates are positively correlated with the number of clusters per plant. Treatments with growth regulators also modified berry number per cluster, berry weight and rachis morphology. Finally, the plant source was a determinant affecting germination rates in vitro.     

Untersuchungen über die Regulation der DNS-Synthese während des Aktivitätswechsels vonAgrostemma githago-Samen

The relationships between DNA synthesis and germination capacity ofAgrostemma seeds have been studied. Protein synthesis and RNA synthesis are activated at the very beginning of imbibition, whereas DNA synthesis starts in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30° C), or aged seeds with a low germination capacity are characterized by a remarkably reduced protein synthesis. DNA synthesis is also reduced. The inhibition of protein-synthesis ofAgrostemma embryos fed with cycloheximid or actinomycin D causes a depression of DNA synthesis. These results indicate that the initiation of DNA synthesis of imbibingAgrostemma seeds depends on the synthesis of special proteins. Abscisic acid inhibits growth as well as DNA synthesis of isolatedAgrostemma embryos. Mitomycin inhibits germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also show a reduced incorporation of³H-thymidine in DNA. We suggest that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, is involved in the mechanism of afterripening of theAgrostemma seeds.