Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

The Role of Water Uptake in the Transition of Recalcitrant Seeds from Dormancy to Germination

In recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) maintaining a high water content during winter, dormancy is determined by the presence and influence of the seed coat, while the axial organs of the embryos excised from these seeds are not dormant. Such axial organs were capable for active water uptake and rapid fresh weight increase, so that their fresh weights exceeded those in intact seeds at the time of radicle protrusion. Fructose plays an essential role in the water uptake as a major osmotically active compound. ABA interferes with the water uptake by the axial organs and thus delays the commencement of their growth. The manifestation of seed response to ABA during the entire dormancy period indicates the presence of active ABA receptors and the pathways of its signal transduction. The content of endogenous ABA in the embryo axes doubled in the middle of dormancy period, which coincided with a partial suppression of water uptake by the axes. During seed dormancy release and imbibition before radicle protrusion, the level of endogenous ABA in axes declined gradually. Application of exogenous ABA can imitate dormancy by limiting water absorption by axial organs. Fusicoccin A (FC A) treatment neutralized completely this ABA effect. Endogenous FC-like ligands were detected in the seed axial organs during dormancy release and germination. Apparently, endogenous FC stimulates water uptake via the activation of plasmalemmal H⁺-ATPase, acidification of cell walls, their loosening, and turgor pressure reduction. FC can evidently counteract the ABA-induced suppression of water uptake by controlling the activity of H⁺-ATPase. It is likely that, in dormant intact recalcitrant seeds, axial organs, maintaining a high water content, are competent to elevate their water content and to start their preparation for germination under the influence of FC when coat-imposed dormancy becomes weaker.     

Maturation and germination of Phaseolus vulgaris embryonic axes in culture

In this study of embryo development in Phaseolus vulgaris L., we found that immature embryonic axes placed in culture show a growth lag before germinating. The length of this lag phase varies according to axis age at excision, but is not affected by transfer to fresh medium, alteration of sucrose concentration between 0.5 and 2%, or whether the culture medium is liquid or agar-solidified. The lag phase was shortened by both actinomycin D and cordycepin treatment, and by treatment with 10^-5 to 10^-6 M benzyladenine. The effect of abscisic acid (ABA) varied with concentration: below a certain level, it had no effect on the lag phase, but above that level it inhibited, germination. This threshold concentration was 10^-7 M for 20-d-old axes but increased to 10^-5 M by the time the axes were 32 to 34 d old. To determine whether the axes were continuing their embryonic development during the lag phase, we tested them for desiccation-tolerance and for synthesis of phaseolin, a seed storage protein which is specific for embryos of P. vulgaris. The ability to germinate after rapid desiccation was acquired by axes at 26 d past anthesis; when axes younger than this were placed in culture, they developed desiccation-tolerance during the lag phase of growth, indicating that they were continuing embryonic maturation. Phaseolin was present in isolated axes, although at lower levels than in cotyledons. It accumulated during axis development in parallel with total protein, staying at about 1% of total protein content. When isolated immature axes were pulsed with ³H-or ¹⁴C-amino acids, they incorporated label into phaseolin, shown by precipitation with anti-phaseolin antibody. Isolated axes from mature seeds, however, did not synthesize detectable amounts of phaseolin. Immature axes cultured in vitro for a period of one to several days continued synthesizing phaseolin until the day prior to visible germination. Treatment of cultured axes with ABA increased the amount of precursor amino acids incorporated into protein, but had a small or no effect on the relative proportion of phaseolin synthesized. We conclude that P. vulgaris axes in culture continue to develop embryonically for a period of time which seems to be under intrinisc control by the axis. This contrasts with precocious germanation, a pattern of embryo behavior seen in many other species. When such embryos are excised from seeds while immature and placed in culture, they switch promptly from embryo development into germination. If ABA or water stress is responsible for preventing precocious germination, it may be that a high level of ABA is maintained or synthesized internally by embryonic axes of Phaseolus, while in other embryos the maternal environment supplies ABA and/or causes water stress.Abbreviations ABA Abscisic acid - BA benzyladenine     

Activation of acid growth in germinating horse chestnut seeds

The validity of the acid-growth hypothesis is proved for the case of cell elongation initiation in germinating seeds of horse chestnut (Aesculus hippocastanum L.), the embryo axes of which are known to extend during the first stages of germination only by cell elongation. During seed imbibition, H⁺-ion excretion was firstly low; it increased several times prior to radicle emergence and was maintained at a high level during growth initiation and further cell elongation. Cell wall acidification and radicle emergence were enhanced in the presence of 0.02 mM fusicoccin, thus indicating the involvement of the plasma membrane H⁺-ATPase in the execution of acid growth. The presence of this enzyme and its activator (14-3-3 protein) in microsomal fractions obtained from radicles and hypocotyls of the embryo axes during and after initiation of cell elongation was demonstrated immunochemically. It is supposed that the initiation of cell elongation at early germination occurs via the activation of the plasma membrane H⁺-ATPase and results in the acidification of cell walls, leading to their higher extensibility, in accordance with the hypothesis of acid growth.     

Dynamics of carbohydrates in the embryo axes of horse chestnut seeds during their transition from dormancy to germination

It was shown that the content of carbohydrates and their composition in embryo axes of horse chestnut seeds changed as seeds acquired a capability of dormancy release and germination. Sucrose prevailed among carbohydrates, comprising to 150–160 mg/g dry wt. During the first half of the seed imbibition time, oligosaccharides, namely raffinose and stachyose, degraded, whereas the contents of glucose and fructose were very low. The second half of the imbibition period (until radicle protrusion) was characterized by a cessation of oligosaccharide breakdown and accumulation of monosaccharides. Carbohydrate balance showed that the contribution of oligosaccharide breakdown to sucrose and monosaccharide accumulation was rather small, and monosaccharides accumulated mostly at the expense of sucrose gradually coming from cotyledons during imbibition. The trend of carbohydrate metabolism in imbibing axial organs was similar during the entire period of a seed dormancy release in the course of stratification. A readiness for the commencement of these processes during the entire dormancy period implies that carbohydrate conversions in embryo axes are not a trigger for a dormancy release. Monosaccharide accumulation in embryo axes before radicle protrusion produces an increase in the osmotic pressure, as compared to that provided by sucrose, by approximately 20%. Recalcitrance of the horse chestnut seeds is discussed in relation to the role of carbohydrates and other endogenous osmotica in the establishment of osmotic properties.     

The effect of abscisic acid on the growth and storage of germinating rape (Brassica napus L.) seed dried following selection on the basis of a newly-emerged radicle

Abscisic acid at 1 × 10^–4M concentration controlled the progress of the emerging radicle from germinating rape seeds and apparently restricted it to the dehydration tolerant phase. ABA treatment during germination followed by washing reduced the deleterious effects of drying the seeds following selection based on a newly-emerged radicle. Furthermore, the longevity of these low-moisture-content germinating seeds at a range of temperatures was improved by the ABA treatment. The viability of the treated seeds stored at –20°C was maintained for up to 100 days.     

Effect of root length on epicotyl dormancy release in seeds of Paeonia ludlowii,Tibetan peony

Background and Aims

Epicotyl dormancy break in seeds that have deep simple epicotyl morphophysiological dormancy (MPD) requires radicle emergence and even a certain root length in some species. However, the mechanisms by which root length affects epicotyl dormancy break are not clear at present. This study aims to explore the relationship between root length and epicotyl dormancy release in radicle-emerged seeds of Tibetan peony, Paeonia ludlowii, with discussion of the possible mechanisms.

Methods

Radicle-emerged seeds (radicle length 1·5, 3·0, 4·5 and 6·0 cm) were incubated at 5, 10 and 15 °C. During the stratification, some seeds were transferred to 15 °C and monitored for epicotyl–plumule growth. Hormone content was determined by ELISA, and the role of hormones in epicotyl dormancy release was tested by exogenous hormone and embryo culture.

Key Results

Cold stratification did not break the epicotyl dormancy until the root length was ≥6 cm. The indole-3-actic acid (IAA) and GA₃ contents of seeds having 6 cm roots were significantly higher than those of seeds with other root lengths, but the abscisic acid (ABA) content was lowest among radicle-emerged seeds. GA₃ (400 mg L⁻¹) could break epicotyl dormancy of all radicle-emerged seeds, while IAA (200 mg L⁻¹) had little or no effect. When grown on MS medium, radicles of naked embryos grew and cotyledons turned green, but epicotyls did not elongate. Naked embryos developed into seedlings on a mixed medium of MS + 100 mg L⁻¹ GA₃.

Conclusions

A root length of ≥6·0 cm is necessary for epicotyl dormancy release by cold stratification. The underlying reason for root length affecting epicotyl dormancy release is a difference in the GA₃/ABA ratio in the epicotyl within radicle-emerged seeds, which is mainly as a result of a difference in ABA accumulation before cold stratification.     

Endogenous abscisic acid during the germination of chick-pea seeds

Over the past twenty years many studies have been undertaken to elucidate the regulation of seed germination. Abscisic acid (ABA) and the gibberellins (GAs) are the hormones proposed to control this process, the first by inhibiting and the second by inducing germination. It has been proposed that a high water potential increases the growth potential of the embryo, presumably permitting the production or activation by GA of the cell wall hydrolases and thus decreasing the yield threshold of the endosperm close to the radicle tip. A low water potential, e.g., imbibition in an osmoticum. imposes a stress on cell metabolism, by reducing the turgor of the radicle cells, and there is a decrease in growth potential. Exogenous ABA also causes a decline in growth potential of the radicle: however, the actions of low water potential in preventing germination are not mediated through an increase in ABA in the seeds. In the present paper an attempt is made to asses the role of ABA and polyethylene glycol (PEG) in the germination of chick-pea (Cicer arietinum L.) seeds. The endogenous ABA of chick-pea seeds was purified by reversed-phase HPLC and quantified by GC-ECD. The variations in the ABA levels in the embryonic axes and the cotyledons were studied during 120 h. of imbibition. The highest ABA level in the embryome axes was found at 18 h. coinciding with an increase in fresh weight and a high germination percentage. ABA was not detected in the cotyledons during incubation which probably indicates that the hormone is more involved in the active growth of the embryonic axes itself than in the mobilization process of the reserves. When seeds were treated with different PEG-cycles. PEG delayed germination, reduced the fresh weight of embryonic axes, and retarded the onset of ABA synthesis. It is concluded that endogenous ABA is related to the onset of germination and the growth of the embryonic axis. In addition, there is no correlation among the different PEG-cycles and the level of ABA and germination. Germination was related more to the water conditions inside the embryo's cells than to ABA levels.     

Uptake and Effect of Abscisic Acid during Induction and Progress of Radicle Growth in Seeds of Chenopodium album

In the seeds of Chenopodium album L. visible phenomena preceding the final protrusion of the radicle enable a clear distinction between the induction and the progress of growth inside the covering structures. The light-dependent induction of radicle growth is not inhibited by exogenously applied abscisic acid (ABA). Experiments with 1-¹⁴C-ABA ruled out a lack of penetration of the hormone. However, ABA does inhibit the growth of the radicle before final protrusion. This inhibition and the uptake of 1-¹⁴C-ABA are enhanced at lower pH values, indicating absorption of the undissociated molecule. The uptake of labeled hormone strongly increases during the growth of the radicle. This increase is not merely a reflection of extra water uptake. Seeds of different degrees of dormancy contain equallly low levels of endogenous ABA. Much higher levels of ABA in the seeds were obtained by exogenous application of the hormone but these levels stills do not prevent the breaking the dormancy by light. It is concluded that ABA has no function in the regulation of dormancy in C. album seeds.     

Cold stratification and growth of radicles of loblolly pine (Pinus taeda) embryos

The mechanism of germination enhancement by cold stratification was examined in seeds of loblolly pine ( Pinus taeda L. ), Removal of the seed coat permitted elongation of radicles from unstratified embryos, but both rates of germination and radicle elongation were increased by stratification. Radicles of both stratified and unstratified embryos excised from the megagamethophyte elongated only when in contact with solid incubation media supplemented with sucrose. Stratification of embryos either in the presence or absence of the megagametophyte resulted in similar enhancement of radicle elongation. Elongation rates of radicles were increased after stratification independent of sucrose concentration, and changes in sucrose content in the megagamethophyte during stratification or incubations subsequent to stratification were insufficient to regulate radicle growth. Our results support the hypothesis that the embryos of pine seeds perceive the low temperature stimulus directly and this stimulus results in a growth potential increase in the embryonic axes. We propose that this growth potential increase enables the embryos to overcome the mechanical restraint of the seeds coats and to germinate.     

Hormonal Complex and Ultrastructure of Maturing Aesculus hippocastanum Seeds

The content and temporal changes in the endogenous IAA, cytokinins, gibberellin-like compounds (GLC), and ABA were determined during horse chestnut (Aesculus hippocastanum L.) seed development (the stages of embryo axis development, its active growth, and storage compound deposition). The active growth of the embryo was characterized by the highest amounts of free phytohormones. Later, by the end of seed maturation, we observed the accumulation of the bound forms of IAA and ABA and a trend to a decrease in the content of free IAA, zeatin, and GLC (butanol fraction). The electron-microscopic examination of the embryo from the mature seed demonstrated that some structural components of the cytoplasm were similar in the cells of embryo axes and cotyledons. During the entire period of maturation, the embryo cells preserved native vacuoles and protein bodies were not formed. Thus, the structure of cotyledonary and axial cells and the distribution of free and bound phytohormones in the horse-chestnut seeds are similar to those in maturing seeds characterized by exogenous dormancy.     

Kinetics of dormancy release and the high temperature germination response in Aesculus hippocastanum seeds

The kinetics of primary dormancy loss were investigated in seeds of horse chestnut (Aesculus hippocastanum L.) harvested in four different years. Freshly collected seeds from 1991 held for up to 1 year at temperatures between 2C and 42C exhibited two peaks in germination (radicle growth), representing a low temperature (2-8°C) and a high temperature response (31-36°C). Germination at 36°C generally occurred within 1 month of sowing, but was never fully expressed in the seedlots investigated. At low temperatures (2-8°C), germination started after around 4 months. Generally, very low levels of termination were observed at intermediate temperatures (11-26°C). Stratification at 6°C prior to germination at warmer temperatures increased the proportion of seeds that germinated, and the rate of germination for all seedlots. Within a harvest, germination percentage (on a probit scale) increased linearly with stratification time and this relationship was independent of germination temperature (16-26°C). However, inter-seasonal differences in the increases in germination capacity following chilling were observed, varying from 0.044 to 0.07 probits d^-1 of chilling at 6°C. Increased sensitivity to chilling was associated with warmer temperatures during the period of seed filling. The estimated base temperature for germination, Tb, for newly harvested seeds varied slightly between collection years but was close to 25°C. For all seedlots, Tb decreased by 1°C every 6 d of chilling at 6°C. This systematic reduction in Tb with chilling ultimately facilitated germination at 6°C after dormancy release.     

Deep simple epicotyl morphophysiological dormancy in seeds of two Viburnum species, with special reference to shoot growth and development inside the seed

Background and Aims

In seeds with deep simple epicotyl morphophysiological dormancy, warm and cold stratification are required to break dormancy of the radicle and shoot, respectively. Although the shoot remains inside the seed all winter, little is known about its growth and morphological development prior to emergence in spring. The aims of the present study were to determine the temperature requirements for radicle and shoot emergence in seeds of Viburnum betulifolium and V. parvifolium and to monitor growth of the epicotyl, plumule and cotyledons in root-emerged seeds.

Methods

Fresh and pre-treated seeds of V. betulifolium and V. parvifolium were incubated under various temperature regimes and monitored for radicle and shoot emergence. Growth of the epicotyl and cotyledons at different stages was observed with dissecting and scanning electron microscopes.

Key Results

The optimum temperature for radicle emergence of seeds of both species, either kept continuously at a single regime or exposed to a sequence of regimes, was 20/10 °C. GA₃ had no effect on radicle emergence. Cold stratification (5 °C) was required for shoot emergence. The shoot apical meristem in fresh seeds did not form a bulge until the embryo had grown to the critical length for radicle emergence. After radicle emergence, the epicotyl–plumule and cotyledons grew slowly at 5 and 20/10 °C, and the first pair of true leaves was initiated. However, the shoot emerged only from seeds that received cold stratification.

Conclusions

Seeds of V. betulifolium and V. parvifolium have deep simple epicotyl morphophysiological dormancy, C_1bB (root)–C₃ (epicotyl). Warm stratification was required to break the first part of physiological dormancy (PD), thereby allowing embryo growth and subsequently radicle emergence. Although cold stratification was not required for differentiation of the epicotyl–plumule, it was required to break the second part of PD, thereby allowing the shoot to emerge in spring.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

Abscisic acid relationships in the chill-related dormancy mechanism in apple seeds

Abscisic acid (ABA) levels in seeds from three cultivars of apple (Malus domestica Borkh.) which have substantially different chilling requirements were investigated by gas chromatography mass-spectrometry selected ion monitoring (GCMS-SIM) during stratification. The ABA content of dormant unchilled seeds was similar in the three cultivars, suggesting no relationship between the chilling requirement of those seeds and their ABA status. That chilling is not related to ABA changes during stratification was confirmed by warm (20°C) and cold (5°C) stratification experiments. ABA content dropped rapidly and nearly identically under both temperature regimes, but only cold stratification promoted germination. The decline in ABA during stratification was due in large part to leaching from the seed coat and nucellar membrane; the ABA content of the embryo remained nearly constant. The radicle in intact seeds stratified at 5°C began growing 20–30 days after the ABA in the seed coat and nucellar membrane had nearly disappeared. Radicle growth did not occur in unchilled seeds, even though ABA had leached from them as well. It is possible that the leaching of ABA from the seed allows certain promotive forces to develop, but if so, these can develop only at chilling temperatures. Studies were also conducted on 2-trans ABA relationships to apple seed dormancy, but no association was evident.Report No. 12, Department of Fruit and Vegetable Science, Cornell University.     

Abscisic Acid Levels in Soybean Reproductive Structures during Development

Abscisic acid (ABA) concentrations and growth rates of developing soybean (Glycine max [L.] Merr. cv. Wye) seeds and pod walls were determined from anthesis to maturation using high pressure liquid chromatographic techniques. Developing soybean seeds contain up to 12,200 ng/g fresh weight of ABA compared to 330 ng/g fresh weight for pod walls. In the developing seeds ABA levels correlated with growth rates, being the highest during the most active growth period of seed enlargement, and then decreasing to less than 10 ng/g fresh weight at maturity. Higher levels of ABA were found to occur in the cotyledons and seed coats than the root-shoot axes at 21 days postanthesis. The time required for excised root-shoot axes to initiate growth in liquid culture decreased as seed development progressed and ABA levels of the seeds declined.     

Changes in germination and respiratory potential of embryos of dormant Grand Rapids lettuce seeds during long-term imbibed storage,and related changes in the endosperm

Grand Rapids lettuce (Lactuca sativa L.) seeds were stored in an imbibed state for up to two years. Embryos dissected from stored seeds showed a progressive loss with time in their ability to germinate on polyethylene glycol (PEG) solutions. Little germination of dissected embryos from one-month imbibed seeds occurred on-6 bar PEG but only after four months of storage did the dissected embryos fail to germinate on-4 bar PEG. After two years storage 30% of dissected embryos still were able to germinate on-2 bar PEG. This loss of germination potential, which may be a symptom of the development of an embryo dormancy, could be reversed by N⁶-benzyladenine (BA) and red light (R) applied together or separately to dissected embryos. Two weeks of chilling of 12-month imbibed seeds restored sensitivity to R and a 48-h BA pretreatment prior to R resulted in germination rates similar to those of seeds emerging from primary dormancy. There was loss of embryo control of endo--mannanase activity after two weeks of storage even though the endosperms themselves retained their capacity for enzyme synthesis for six more weeks. Eventually, then, endo--mannanase synthesis is not possible because of inherent changes in both the embryo and endosperm, although each tissue undergoes changes at its own rate. Oxygen uptake by embryos dissected from two-month imbibed seeds did not increase to the same extent as embryos dissected from freshly imbibed seeds. In intact seeds germinating from a skotodormant state, oxygen uptake increased at a time coincident with radicle protrusion, but did not achieve the levels of uptake of those seeds germinating from a primary dormant state. The decline in uptake of oxygen by secondary dormant seeds is the result of a lowered respiratory capability of the embryo itself, rather than of changes in permeability of the surrounding structures.Abbreviations BA N⁶-benzyladenine - Pfr active (far-redabsorbing) form of phytochrome - R red light - PEG polyethylene glycol     

Dissecting seed dormancy and germination in Aquilegia barbaricina,through thermal kinetics of embryo growth

• Threshold‐based thermal time models provide insight into the physiological switch from the dormant to the non‐dormant germinating seed.
• This approach was used to quantify the different growth responses of the embryo of seeds purported to have morphophysiological dormancy (MPD) through the complex phases of dormancy release and germination. Aquilegia barbaricina seeds were incubated at constant temperatures (10–25 °C) and 25/10 °C, without pre‐treatment, after warm+cold stratification (W+C) and GA₃ treatment. Embryo growth was assessed and the time of testa and endosperm rupture scored. Base temperatures (T_b) and thermal times for 50% (θ₅₀) of embryo growth and seed germination were calculated.
• W+C enabled slow embryo growth. W+C and GA₃ promoted rapid embryo growth and subsequent radicle emergence. The embryo internal growth base temperature (T_be) was ca. 5 °C for W+C and GA₃‐treated seeds. GA₃ treatment also resulted in similar T_b estimates for radicle emergence. The thermal times for embryo growth (θ_e50) and germination (θ_g50) were four‐ to six‐fold longer in the presence of GA₃ compared to W+C.
• A. barbaricina is characterised by a multi‐step seed germination. The slow embryo growth during W+C reflects continuation of the maternal programme of development, whilst the thermal kinetics of both embryo and radicle growth after the removal of physiological dormancy are distinctly different. The effects of W+C on the multiphasic germination response in MPD seeds are only partially mimicked by 250 mg·l^?1 GA₃. The thermal time approach could be a valid tool to model thermal kinetics of embryo growth and radicle protrusion.