Abscisic acid controls dormancy development and bulb formation in lily plantlets regenerated in vitro

Plantlets of lily regenerated in vitro from scale explants consist of scales and leaves from which the base of the petiole has swollen to a scale. Fluridone, an inhibitor of ABA-synthesis, applied during culture in vitro, inhibited the swelling of the petioles and promoted leaf formation. At high fluridone concentrations (10 or 33μ M ), swelling was completely blocked, and plantlets consisted of leaves only. Addition of ABA during the regeneration in vitro had the opposite effect and resulted in plantlets with scales only. When applied simultaneously with fluridone, ABA nullified the effect of fluridone. This demonstrates that bulb formation in lily is under the control of ABA. Lily plantlets regenerated in vitro on scale explants at 20 or 25°C were harvested after 11 weeks, and the leaves were removed from the bulblets. The bulblets were dormant and required a cold treatment to achieve rapid emergence after planting in soil. Fluridone added during the culture in vitro prevented the development of dormancy, and the bulblets did not require a cold treatment. The effect of fluridone was nullified by simultaneous addition of ABA. Bulblets harvested after 6 weeks of culture at 20°C had not yet developed dormancy. Bulblets regenerated at 15°C were only slightly dormant. In both types of bulblets, it is unlikely that the lack of dormancy was due to low ABA-levels since addition of ABA did not affect the dormancy status. These data indicate that the level of endogenous ABA and an unknown additional factor play major roles in the development of dormancy.     

The development of dormancy in bulblets of Lilium speciosum generated in vitro

We have studied the effect of various in-vitro conditions on dormancy of bulblets generated on scale explants of Lilium speciosum Thunb. cv. Rubrum nr. 10. The bulblets were harvested after 11 weeks of culture. Dormancy was measured by determining the percent emergence in soil of viable, non-cold-treated bulblets. A study of the physical conditions showed that temperature had a strong effect on the induction of dormancy (15°C induced hardly any dormancy; 25°C induced a high level of dormancy), whereas short or long day and light or dark had no effect. Of the medium components, a low concentration of sucrose (1 gl^–1 or less) or a high concentration of gibberellic acid (1 mg 1^–1) reduced the level of dormancy. Application of various concentrations of abscisic acid, 6-benzylaminopurine, -naphthaleneacetic acid, indole-3-acetic acid, 2,3,5-triiodobenzoic acid or a Murashige and Skoog macro- and microelement mixture did not affect the dormancy status.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog macro- and microelements - NAA -naphthalene-acetic acid - TIBA 2,3,5-triiodobenzoic acid     

The development of dormancy in bulblets of Lilium speciosum generated in vitro. II. The effect of temperature

Upon harvest, lily ( Lilium speciosum Thunb. cv. Rubrum) bulblets generated in vitro under standard conditions (11 weeks at 20°C) were dormant and needed a cold treatment prior to planting. During culture in vitro at 20°C, the bulblets proceeded through three phases: (1) at first they were non–viable and non-dormant (up to 5 weeks), (2) then viable and non-dormant (5–9 weeks) and (3) finally viable and dormant (from 9 weeks onwards). At 15°C, the bulblets became viable but did not develop dormancy, even after protracted culture. The results suggest that the development of dormancy depends upon an accumulation of'heat units'occurring at temperatures higher than 15°0. At 25°C, the succession of the three phases occurred more rapidly than at 20°C and heat units were accumulated more rapidly. During the third period, the chilling requirement increased showing that heat units continued to be accumulated during this period.
Dormancy connotes an arrest of growth. In lily bulblets, however, the number of scales continued to increase after the induction of dormancy at 20 or 25°C. Many of the scales initiated before the onset of dormancy were formed by swelling of a petiole, whereas, after the onset of dormancy, all scales were formed directly from a primordium. We conclude that the development of dormancy corresponds to a switch in the development of the primordium. Thus, after the induction of dormancy the primordium lost the ability to become a leaf and always developed into a scale.     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

Effects of endogenous ABA levels and temperature on cedar (Cedrus libani Loudon) bud dormancy in vitro

Axillary and apical buds of in-vitro-propagated cuttings of Cedrus libani are unable to burst at 24 °C, but this inhibition was overcome at 30 °C. Here we have used cedar microcuttings to investigate whether the levels of endogenous hormones vary with bud dormancy and temperature. We analysed the levels of abscisic acid, indole-3-acetic acid, zeatin, isopentenyladenine and their major metabolites using HPLC purification and fractionation of the samples coupled to an ELISA method for hormonal quantitation involving several antibodies elicited against each hormonal family. Abscisic acid levels in microcuttings with dormant buds were higher than those in microcuttings with growing buds. At 24 °C, needles accumulated more abscisic acid than at 30 °C. In addition, when needles were removed, but growth release was achieved at 24 °C. Abscisic acid supplied at 30 °C induced the formation of dormant buds. These results suggest that abscisic acid accumulation in the needles can explain the bud dormancy of cedar microcuttings at 24 °C. Received: 14 November 1997 / Revision received: 16 January 1998 / Accepted: 5 May 1998     

Development of dormancy in different lily genotypes regenerated in vitro

Dormancy development in four Lilium genotypes,L. speciosum, Star Gazer, C. King and Snow Queenregenerated in vitro was compared. Major factorsinfluencing dormancy development were the same for different genotypes andespecially L. speciosum and Star Gazer, that are closelyrelated, reacted similarly. Temperature was the main factor in dormancyinduction and breaking. The range of temperatures that induced dormancy and thelevel of dormancy that developed differed per genotype. In Star Gazer, dormancydeveloped gradually but in Snow Queen, dormancy developed very fast. Thereactions to temperature, reflected the climate in the area of origin. Abscisicacid deepened the level of dormancy induced by temperature but had no effectunder non-inductive temperature conditions. When abscisic acid synthesis wasblocked, no dormancy developed. Dormancy in all genotypes was broken by coldincubation for severalweeks. The cold requirement of the genotypes differed in line with the naturalwinter conditions in their habitat. The effect of hormones on dormancy breakingwas also investigated. A gibberellin treatment of 24 h brokedormancy in L. speciosum, Star Gazer and Snow Queen.     

Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.     

毛百合的组织培养和快速繁殖

以毛百合鳞片切块为外植体,置入不同激素配比的培养基中,观察和比较切块的近轴面、远轴面、侧面分化小鳞茎、芽和根的情况,并通过不同土质、不同温度移栽实验,选出试管苗移栽成活的最好方法。     

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.     

Involvement of abscisic acid in bulb dormancy of Allium wakegi Araki. II. A comparison between dormant and nondormant cultivars

The content of abscisic acid (ABA) in bulbs of two Allium wakegi Araki cultivars, Kiharabansei No. 1 (dormant type) and Ginoza (nondormant type), was similar and changed similarly during the development and storage of the bulbs. It increased during bulb development, reached a maximum shortly after bulb harvesting, and gradually decreased during bulb storage. The bulbs of Kiharabansei No. 1 showed dormancy correlated with the change in ABA content, but those of Ginoza did not show significant dormancy throughout the experimental period. The ABA content in the buds of dormant bulbs of Kiharabansei No. 1 did not change after planting of bulbs, but that of nondormant bulbs of Ginoza planted on the same day rapidly decreased after planting. Application of ABA to bulbs delayed sprouting of both cultivars, but dormant bulbs of Kiharabansei No. 1 had higher sensitivity to ABA than the bulbs of Ginoza or the bulbs of Kiharabansei No. 1 partly released from dormancy. These results suggest that the decrease in the ABA content after planting (watering) and low sensitivity to ABA are correlated with the nondormancy of Ginoza.     

Involvement of abscisic acid (ABA) in bulb dormancy of Allium wakegi Araki I. Endogenous levels of ABA in relation to bulb dormancy and effects of exogenous ABA and fluridone

Allium wakegi plants exposed to long days (LD, 14 h-photoperiod) developed bulbs, which were dormant from the 30th to the 125th day of LD, but those grown under natural short days (SD) did not develop bulbs. The contents of abscisic acid (ABA) in both whole bulbs and buds of the bulbs increased in LD, reaching a maximum at the 60th day of LD and decreasing thereafter, but those in basal leaf sheaths (this part corresponds to a bulb after bulb development) and buds did not increase in SD. The ABA content was related to the depth of bulb dormancy. Application of 500 M ABA to bulbs for 24 h significantly delayed sprouting, but that of 5 or 50 M ABA had little or no effect. Application of 25 or 125 M fluridone to the soil just before exposure to LD bleached new expanding leaves and reduced bulb size, but had no effect on the development of bulb scales that characterize bulb formation. The bulbs formed under such conditions sprouted earlier than those of control plants. The levels of endogenous ABA in bulbs, buds of the bulbs, leaf blades, and roots were reduced by fluridone application. These results indicate that ABA plays an important role in bulb dormancy of Allium wakegi.     

脱落酸和赤霉素调控种子休眠与萌发研究进展

种子的休眠与萌发是高等植物生长发育进程中非常重要的环节,是维系物种繁衍的重要过程。而激素在这一过程中扮演着非常重要的角色。而在这个过程中脱落酸(abscisic acid,ABA)和赤霉素(gibberellin GA)发挥着尤其重要的作用。本文综述了当前对复杂分子网络的理解,这些分子网络涉及脱落酸和赤霉素在调节种子休眠和萌发中的关键作用,其中含AP2结构域的转录因子起着关键作用。     

Conditions of induction of secondary dormancy in apple after breaking of primary dormancy by anaerobiosis and low temperature

Dormant embryos of Pyrus malus L. cv. Golden delicious, isolated from the fruits at harvest time or after a few months storage at 10 to 15°C, were kept under anaerobic conditions in order to eliminate primary dormancy. Germination tests were then carried out at different temperatures, using three modes of culture depending on the nature of the contact between the embryo and the medium. In CM the distal part of the two cotyledons was immersed in the medium. In RM only the embryonic axis was immersed. In C/2M the embryo was placed flat on the medium, the radicle and the external surface of one cotyledon being in contact with it.
Results showed that primary dormancy was released progressively depending on the duration of the anaerobic treatment. After a treatment of 11 or 13 days the last symptoms of primary dormancy were only apparent when germination tests were carried out at high temperatures (26–30°C) or in CM mode of culture.
When the embryos were kept at 4°C during 3 months inside the fruits, subsequent germination was inhibited at high temperature and in CM mode of culture. When the embryos were kept under anaerobic conditions (7 days) after the chilling treatmem inside the fruits, germination was no longer inhibited. It is concluded that the inhibition of germination at high temperature and in CM mode of culture is due to the persistence of traces of primary dormancy. Therefore, these conditions do not seem to induce secondary dormancy in apple embryos.
After elimination of primary dormancy by anaerobiosis. only application of (±) abscisic acid (3.8 and 19 μM) inhibited germination. These results support the idea that ABA is an important factor in the induction of dormancy. However, the question remains whether this secondary embryo dormancy has the same characteristics as the original primary dormancy.     

Changing sensitivity to light and nitrate but not to gibberellins regulates seasonal dormancy patterns in Sisymbrium officinale seeds

Seeds of Sisymbrium officinale (L.) Scop, that are buried under natural conditions in soil pass annually through a seasonal pattern of changes in dormancy. Dormancy is broken in autumn-winter and re-induced in summer. To elucidate dormancy regulation in this species under natural conditions, a detailed analysis of the changes in sensitivity to some relevant germination factors was carried out Germination data fitted as logistic dose response curves showed that sensitivity to light and nitrate, both indispensable stimuli for germination of this species, varied with the seasons. Patterns of shifts in requirement for light and nitrate were remarkably similar. Sensitivity increased when both primary and secondary dormancy were alleviated, and it was reversed during induction of secondary dormancy. During alleviation of primary dormancy in spring 1991, the fluence response curves exhibited a biphasic character with responses occurring both in the very-low-fluence-range and in the low-fluence-range. The nitrate dose response data could all be fitted as monophasic curves, although responses might have occurred in two distinct ranges as well. From interpretation of curve parameters, it is postulated that dormancy is regulated by changes in the number of phytochrome and nitrate receptors, in shifts in the binding characteristics of the receptors and/or in shifts in the response chain initiated by the ligand-receptor interaction. Somewhere in this response chain, biosynthesis of gibberellins (GAs) is stimulated. By use of the GA biosynthesis inhibitor tetcyclasis, it was indirectly proven that the capacity to synthesize GAs indeed varied with the seasons. Sensitivity to GAs gradually increased from burial onwards and was not particularly related to changes in dormancy. Thus, except for the first few months of burial, GA sensitivity may not be regarded as a limiting factor in controlling dormancy in this species.     

兰州百合鳞茎发育及低温解除休眠过程中内源激素的变化

以兰州百合为试材,研究了鳞茎发育过程中以及2、6、10℃条件下保湿贮藏101 d内母鳞茎与新鳞茎中内源激素的变化。结果表明：鳞茎发育过程中内源ABA含量以及母鳞茎的GA₃与ZR含量增加,而内源IAA含量以及新鳞茎的GA₃与ZR含量下降。低温贮藏期间,母鳞茎与新鳞茎的GA₃、IAA含量均有升高过程,而ABA含量呈下降趋势;新鳞茎的ZR含量呈下降趋势,母鳞茎的ZR含量也有升高过程。低温处理初期的34 d内,内源激素变化最为显著。不同贮藏温度相比较,ABA含量差异不大,GA₃含量随温度升高而下降。在富含淀粉的新鳞茎中,GA₃和ABA表现出极显著的负相关关系,而在淀粉含量较低的母鳞茎中GA₃和ABA无相关性。通径分析结果表明,母鳞茎与新鳞茎的物质代谢机制不同,母鳞茎的物质变化受内源GA₃的调控,新鳞茎主要是ABA作用的结果。     

Accumulation and leakage of abscisic acid during embryo development and seed dormancy in wheat

Seed dormancy develops latein embryogenesis after a period of potential prematuregermination and has been associated with levels ofabscisic acid (ABA) in, and sensitivity to, ABA ofembryos. In wheat (Triticum aestivum L.)embryos, there are two peaks in levels of ABA duringdevelopment: the first occurs 25 days afterpollination (DAP) and the second from 35 to 40 DAP. The first peak of ABA appears to be associated withthe development of the embryo's sensitivity to ABAsince such sensitivity was altered in seeds on earsthat were incubated in a solution of ABA from 15 and20 DAP. In the embryos of Kitakei wheat, a line thatexhibits dormancy, the second peak, at around 35 DAP,was more prolonged in comparison to Chihoku, anon-dormant line. The results support the proposedinvolvement of ABA in the formation and maintenance ofseed dormancy during middle and late embryogenesis. When developing embryos were incubated in water,embryonic ABA leaked out from the embryos, inparticular between 30 and 40 DAP. Prematuregermination observed between 30 and 40 DAP might berelated to such leakage of ABA from embryos.     

Antagonistic effects of abscisic acid and gibberellic acid on the breaking of dormancy of Fagus sylvatica seeds

Study of the factors involved in the dormancy of Fagus sylvatica seeds shows that such dormancy is due partly to the seed coats and partly to endogenous factors. Seed coat removal accelerates both the release from dormancy and the effects of the other treatments that abolish it. The dormancy of these seeds is eliminated by cold treatment at 4°C over a period longer than 8 weeks, and exogenous application of abscisic acid (ABA) reverses the effects of low temperature, the seeds remaining in an ungerminated state. Additionally, ABA reduces protein synthesis but slightly increases RNA synthesis, which suggests its involvement in the synthesis of RNAs related to this process. In vitro translation of the RNAs isolated from these seeds shows that ABA delays the disappearance of at least 2 polypeptides (of ca 22 and 24 kDa), which are abundant in dormant seeds and under conditions that prevent the release from dormancy, but which disappear under treatments that abolish it. Exogenous application of gibberellic acid (GA₃) proved to be efficient in breaking the dormancy of these seeds and in substituting for cold treatment as well as in antagonizing the effects of ABA on the synthesis of both DNA and proteins. GA₃ also accelerates the disappearance of the two polypeptides abundant in dormant seeds and in ABA-treated seeds. These findings suggest that both ABA and GA₃ could be involved in the regulation of nucleic acid and protein metabolism during dormancy, acting antagonistically in these processes and, specifically, in the regulation of the synthesis of the two proteins that appear to play a role in the maintenance of dormancy in these seeds.     

Seed dormancy in Acer: The role of abscisic acid in the regulation of seed development in Acer platanoides L.

Germinability of isolated embryos from developing fruits of Acer platanoides was high at the earliest developmental stage assessed (90 dpa), but fell subsequently and at seed maturity was very low. These observations showed an inverse correlation with changes in endogenous free abscisic acid (ABA) levels in the embryo, which were low during early ontogeny, but reached maximum levels late in development (150–160 dpa). These observations suggest the possibility that dormancy may be induced during development as a result of ABA accumulation in the embryo, an argument strengthened by the obvious inhibitory effect of added ABA on the germinability of isolated embryos. The cotyledons appear to exert an inhibitory influence on embryo germinability that may result from their free ABA content although the embryonic axis itself possesses an innate dormancy that may reflect its own free ABA content. The increased germinability of isolated embryos resulting form added kinetin serves only to emphasise the complexity of the system and the dangers of simplistic interpretation.The correlation between germinability and ABA content is not complete, however, since much of the reduction in germinability had occurred before any appreciable increase in free ABA levels in the embryo was observed. Indeed the failure of the intact seed to respond to endogenous changes in embryonal ABA levels suggests that even though free ABA in the embryo may influence embryo germinability, it has little effect in the intact seed, where the presence of an intact testa may be a more important factor.The absence of a desiccation phase in the embryo during the late stages of development suggests that the large increases in endogenous free ABA did not cause dormancy by inhibiting water uptake, nor did they result from water stress in the embryonal tissues.     

Regulation of summer dormancy by water deficit and ABA in Poa bulbosa ecotypes

BACKGROUND AND AIMS: Survival of many herbaceous species in Mediterranean habitats during the dry, hot summer depends on the induction of summer dormancy by changes in environmental conditions during the transition between the winter (growth) season to the summer (resting) season, i.e. longer days, increasing temperature and drought. In Poa bulbosa, a perennial geophytic grass, summer dormancy is induced by long days, and the induction is enhanced by high temperature. Here the induction of summer dormancy in a Mediterranean perennial grass by water deficit under non-inductive photoperiodic conditions is reported for the first time. METHODS: Plants grown under 22/16 degrees C and non-inductive short-day (9 h, SD) were subjected to water deficit (WD), applied as cycles of reduced irrigation, or sprayed with ABA solutions. They were compared with plants in which dormancy was induced by transfer from SD to inductive long-day (16 h, LD). Responses of two contrasting ecotypes, from arid and mesic habitats were compared. Dormancy relaxation in bulbs from these ecotypes and treatments was studied by comparing sprouting capacity in a wet substrate at 10 degrees C of freshly harvested bulbs to that of dry-stored bulbs at 40 degrees C. Endogenous ABA in the bulbs was determined by monoclonal immunoassay analysis. KEY RESULTS: Dormancy was induced by WD and by ABA application in plants growing under non-inductive SD. Dormancy induction by WD was associated with increased levels of ABA. Bulbs were initially deeply dormant and their sprouting capacity was very low, as in plants in which dormancy was induced by LD. Dormancy was released after 2 months dry storage at 40 degrees C in all treatments. ABA levels were not affected by dormancy relaxation. CONCLUSIONS: Summer dormancy in P. bulbosa can be induced by two alternative and probably additive pathways: (1) photoperiodic induction by long-days, and (2) water deficit. Increased levels of endogenous ABA are involved in both pathways.