Mature seeds of Helianthus annuus L. exhibit dormancy that is eliminated during storage in dry conditions. In vitro culture of immature embryos isolated at different times after anthesis showed that the youngest embryos are able to germinate, but within the third week after pollination, dormancy progressively affected most of the embryos. A radioimmunoassay showed that the endogenous abscisic acid (ABA) level, which increased sharply in the first half of the development period, fell at precisely the moment when embryo dormancy became established. An application of fluridone, before the increase of ABA level, prevented both ABA synthesis and development of embryo dormancy. Applied later, after the rise of the ABA level, fluridone could not prevent embryo dormancy development. Dormancy thus appears to be dependent on ABA synthesis but not concomitant with its accumulation; it must therefore be induced by ABA during maturation. Furthermore, a preincubation in water allowed dormant embryos to germinate. This acquisition of germinability could not be directly related to a leaching of free ABA. Possible effects of this treatment are discussed.The classic concept of the hormone balance theory supposed that induction, maintenance, and release of seed dormancy were regulated by the simultaneous action of promotive and inhibitory hormones. Recent work with hormone deficient mutants of Arabidopsis thaliana and Lycopersicon esculentum led Karssen and Groot (8) to a revision of this theory. In particular, it appears that ABA is responsible for the onset of dormancy in developing seeds but that it does not control the maintenance of dormancy in mature seeds. However, the authors noted that this conclusion might be valid for all species with so-called 'coat-imposed dormancy' but that direct evidence is missing to invoke the same hormonal control for embryo dormancy.Our first objective, then, was to look for the involvement of ABA in the onset of the dormancy of an embryo. Our plant material, Helianthus annuus seeds, exhibited at harvest a dormancy which could be eliminated by storage in dry conditions, and in vitro culture showed that embryos themselves were dormant. Furthermore, in this species, the lack of dormancy shown by a nondormant white mutant strain was correlated by Wallace and Haberman (16) (17), it may be suggested that the lack of dormancy must be correlated to the lack of ABA synthesis. This species appeared to be a good material to try to modify the physiological behavior of the embryo through an alteration of ABA synthesis. So from this perspective, we applied to the seeds during their development a solution of fluridone, a pyridinone inhibitor of carotenoid biosynthesis, in order to decrease their endogenous ABA levels. This approach allowed us to demonstrate the responsibility of ABA in the induction of the dormancy of the embryo.On the other hand, these dormant embryos could be germinated after a preincubation in water. This treatment, which evokes the leaching of an inhibitor, was imposed to discuss...

In vitro cultures showed that the proximal buds isolated from a rose (Rosa hybrida L. cv. Ruidriko Vivaldi®) stem were endodormant. Growth and a high percentage of bud break could be observed when cultures were treated with fluridone, an inhibitor of carotenoid synthesis. Flow cytometry determination of nuclear DNA content revealed that cell cycle activity of endodormant buds was arrested in the G 1 phase. Upon culture, the large decrease in bud ABA content was responsible for the progress from G 1 to G 2 phase whatever the culture medium. However, in control culture, neither cell division nor leaf primordium initiation could be observed and cells appeared stably arrested in G 2 . By contrast, with fluridone, an additional ABA decrease was observed resulting from an inhibition of its synthesis inside the bud. New leaf primordia were initiated and many figures of mitosis could be observed, indicating that intense activity of cell division occurred after DNA replication. Therefore, the results indicate that, as long as ABA was synthesized inside the buds, cell cycle was arrested in G 2 phase and buds remained dormant. Continued in situ ABA biosynthesis appears, therefore, to be required for the maintenance of bud dormancy.

When applied to young nondormant embryos of sunflower (Helianthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy.The involvement ofABA in the initiation ofseed dormancy has been the subject of many recent studies using either mutants deficient in or insensitive to the hormone (4-6) or inhibitors of synthesis, particularly fluridone (3,11,12 embryos isolated at various times after pollination to exogenous ABA. It seemed necessary to distinguish clearly between the physiological consequences of the presence of exogenous ABA in the culture medium and those associated with a genuine induction effect, that is to say those produced by transient application of the hormone and which subsequently persist in its absence. Furthermore, we studied the consequences of an application of fluridone to isolated dormant embryos on their physiological behavior, their endogenous ABA levels and their responsiveness to exogenous ABA. MATERIALS AND METHODS Plant MaterialPlants of sunflower (Helianthus annuus cv Mirasol) were grown in the fields during summer. In September, flowers of male sterile plants were manually pollinated by pollen harvested on small female sterile flowers (seeds generously provided by S.A. Cargill). Flowers were tagged and harvest took place at different times after pollination.For each age, 25 embryos, i.e. a sample considered as sufficient to be representative of the population, were used for in vitro culture and extraction. All the experiments were carried out two or three times in two different years (1989)(1990).

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