Journal of Experimental Botany, Vol. 52, No. 357, pp. 715-723,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
In vitro control of floral transition in tomato (Lycopersicon esculentum Mill.), the model for autonomously flowering plants, using the late flowering uniflora mutant
Laboratoire de Cytogénétique, Département de Biologie, Université catholique de Louvain, Croix du Sud, 13-5, B-1348, Louvain-la-Neuve, Belgium
Received 3 July 2000; Accepted 10 October 2000
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Abstract |
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In vitro control of floral transition in tomato (Lycopersicon esculentum Mill.), the model plant for autonomously flowering species has been investigated using the late flowering mutant uniflora (uf). Apices collected from truly vegetative plants were cultivated on solid media supplemented with different combinations of growth regulators and chemicals. Several chemical factors implicated in the promotion of floral transition of the uf mutant have been identified: sucrose, cytokinins and nitrogenous nutrients have all to be supplied at optimal concentrations. In contrast, gibberellic acid was found to be inhibitory. These results are discussed in relation to knowledge accumulated on the nature of the flowering signals circulating, at floral transition, in other plants, especially in photoperiodic species. This study suggests that tomato could constitute an adequate model to investigate the genetic and physiological control of floral transition and contribute in unravelling pathways which are constitutively regulating this important step of plant life cycle.
Key words: Cytokinins, floral transition, nitrogen, sucrose, tomato.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Flowering transition is a major event in the plant life cycle that has to be precisely timed for reproductive success. Both physiological and genetical studies have revealed the complexity of the mechanisms that tightly control the apical meristem switch from vegetative to reproductive growth (Bernier et al., 1993
; Weller et al., 1997; Levy and Dean, 1998).
By the analysis of mutants affected in their flowering time (Koornneef et al., 1998), multiple genes implicated in the transition to flowering of Arabidopsis thaliana have been identified. They have been categorized in at least four separate pathways, either promotive or inhibitory of flowering, depending on their phenotypes under various environmental conditions and on the basis of genetic epistasis experiments (Koornneef et al., 1991). Two of these pathways co-ordinate flowering with environmental conditions, namely daylength and vernalizing temperatures. They operate in plants that use these cues that regularly fluctuate within the year. Two other pathways control flowering constitutively; the floral repression pathway and the autonomous promotion pathway which co-ordinate flowering with the developmental stage of the plant. Several of the implicated genes have been cloned (Koornneef et al., 1998; Levy and Dean, 1998; Piñeiro and Coupland, 1998; Reeves and Coupland, 2000), but although much progress has been made, important questions still remain to be answered.
Several studies revealed that there is a link between the autonomous pathway and both the vernalization and long-day (LD) promotion pathways (see references in Reeves and Coupland, 2000) and that all three pathways converge to regulate the expression of LEAFY (LFY) a gene that acts within the floral primordium to promote the expression of genes that specify floral organ identity (Parcy et al., 1998). FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) have also been shown recently to act downstream of the autonomous and LD promotion pathways (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000). Some of the first questions that emerge concern the connection between flowering-time and floral meristem identity genes and the biochemical nature of the floral inductive signals which control the expression of the latter. In A. thaliana, gibberellins, which are known to promote flowering, were found to activate LFY transcription (Blázquez et al., 1998; Blázquez and Weigel, 2000).
A second important task is to define how A. thaliana flowering genes correspond to genes that regulate floral transition in other species. Pnueli et al. postulated that the same genes are involved in the flowering control of different plants, but that the physiological context of these genes alter their expression pattern (Pnueli et al., 1998). Levy and Dean established a list of possible orthologues implicated in flowering time control from A. thaliana, pea, sugar beet, barley, and wheat (Levy and Dean, 1998). All these plants are vernalization-responsive, quantitative LD plants and it appears that information coming from short-day (SD) plants as well as from autonomously flowering plants is essentially lacking although it could be postulated that these plants, at least, share with all other species the pathways that constitutively affect flowering time.
It is with these ideas in mind, that a study of flowering control in tomato which is classically considered as being the model for autonomously flowering plants was started. So far, floral transition in tomato has been poorly investigated, probably because it occurs very early after germination, when the third leaf is expanding (Kinet and Peet, 1997), making its manipulation difficult, especially by treaments that are expected to advance flowering.
In the present work, the facilities offered by the uniflora (uf) mutant which exhibits an extended vegetative phase in winter conditions (Dielen et al., 1998) were exploited. Data are reported of experiments aimed at defining the influence of various chemicals on flowering behaviour of in vitro-cultured shoot apical meristems that have been isolated from true vegetative plants. The aim of this study was to identify physiological signals implicated in the reproductive switch of the meristem of a species in which flowering time is under the control of autonomous promotion and repression pathways and to detect, in comparison with plants in which flowering is environmentally controlled and that have been extensively investigated, similarities and/or discrepancies which could condition the use of tomato as a model plant to approach the pathways that constitutively control flowering time. Tomato is an ideal material for physiological and molecular genetic investigations (Kinet and Peet, 1997). It is easy to cultivate and is amenable to various horticultural manipulations, including grafting or cutting. A large number of genes have been described and assigned to specific locations among the 12 chromosomes, and numerous monogenic mutants are available (Rick and Chetelat, 1993).
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Materials and methods |
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Plant material
Seeds of the uniflora (uf) mutant of tomato (Lycopersicon esculentum Mill.), were initially obtained from the Tomato Genetics Resource Centre (University of California, Davis, USA). The uf mutant was first described in 1967 by Fehleisen (Fehleisen, 1967
); it produces single normal and fertile flowers, instead of classical inflorescences (Dielen et al., 1998). The seeds were germinated in a peat compost into growing rooms at a mean temperature of 24–25 °C.
In vitro culture conditions
Aerial parts of 1-week-old seedlings were surface-sterilized for 5 min with 3% (w/v) calcium hypochlorite containing 0.1% (v/v) Tween 20 and washed four times with sterile distilled water. Shoot apical buds (meristems with 3–4 true leaf primordia) were aseptically excised under a Zeiss stereo microscope and transferred into 25x200 mm glass test tubes containing 25 ml of different media solidified with 0.25% gelrite (Phytagel, Sigma). The basal medium used consisted of macro- and microelements of Murashige and Skoog (Murashige and Skoog, 1962), vitamins as in B5 medium (Gamborg et al., 1968) and 3% sucrose (w/v). Depending on the objective of the experiment performed, this medium was supplemented with growth regulators (different cytokinins and gibberellic acid (GA3)) at various concentrations. The influence of sucrose and ammonium nitrate concentrations was also investigated in specific experiments. pH was adjusted to 5.7–5.8 before adding gelrite and autoclaving for 20 min at 120 °C. A single explant was placed upright on the medium in each culture tube and was incubated in a growth cabinet at a mean temperature of 23.5 °C and under a 12 h photoperiod given at an irradiance of 170 µmol m-2 s-1. These light conditions were selected because they can be considered as being in between summer and winter conditions and are thus suspected of causing an intermediate flowering response, thus allowing an investigation into the effects of treatments that either promote or inhibit flowering. Cultures were scrutinized once a week to evaluate plant growth.
Morphological analysis
After 7 weeks of culture, plants were dissected under the stereo microscope to evaluate their development by counting the total number of leaves initiated by the shoot and recording the state of the apical meristem, either vegetative or floral. A meristem was classified as ‘floral’ when the first sepal primordium of the unique flower that characterizes the uf reproductive structure was visible. It was referred to as ‘vegetative’ when there was no apparent reproductive morphogenesis. The presence or absence of a basal callus and of roots was also registered.
Each experiment was repeated at least twice with 10 explants per treatment. Pooled results of the differents experiments are presented.
Statistical analysis
Statistical analysis (ANOVA I) at the 5% level was performed on the number of leaves initiated below the flower. Differences between means were evaluated for significance by using the Scheffe F-test.
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Results |
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When starting this work, in vitro studies using apical buds of tomato as explant were few. Following earlier recommendations (Koppel and Butenko, 1992
; Koppel, 1994), media containing a cytokinin and a GA, namely benzyladenine (BA) at 3.5 µM and GA3 at 0.58 µM were used in initial experiments. A puzzling observation was that auxin supply to the nutritive medium was not required for floral transition although it is known that this hormone has essential roles in cell activity regulation. This was probably due to the fact that auxin was required at a low level as suggested by the flower-promoting effect of reducing auxin availability in different experimental systems (Bernier, 1988). The explants used had 3–4 small leaf primordia which probably sufficed to meet auxin requirements for the floral transition process to occur. Another important observation was that in all experiments performed it was not possible to alter the single flower phenotype of the uf mutant. This result suggests that UF is a major gene implicated in maintenance of inflorescence meristem identity in tomato.
Effect of sucrose concentration
The medium used initially to investigate the influence of sucrose concentration on the fate of the tomato shoot contained macro- and microelements (Murashige and Skoog, 1962), vitamins as in B5 medium (Gamborg et al., 1968) and BA and GA3 (Koppel and Butenko, 1992; Koppel, 1994).
Increasing sucrose concentration above 30 g l-1 resulted in a decrease in the rhythm of leaf initiation and in the percentage of explants producing roots (Table 1). Vitrification was partial on the medium containing 45 g l-1 sucrose and affected all explants at 90 g l-1. Plants on this latter medium were very short with filiform leaves. All the media caused the basal formation of a callus.
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0.05).The percentage of flowering plants was maximal on medium with 30 g l-1 sucrose while shoots remained at the vegetative stage when sucrose was supplied at concentrations from and above 60 g l-1 that strongly impaired growth. The number of leaves under the flower had a tendency to decrease when the sucrose concentration increased, but the differences were not statistically significant due to the high variability and the reduced number of explants that flowered.
In all further experiments, sucrose at a 30 g l-1 concentration was used.
Effect of GA3 and BA
The development of control plants on growth regulator-free medium strongly depended on explant ability to produce roots: when adventitious roots were formed and had direct contact with the medium, plants developed normally, otherwise they remained stunted.
Media supplemented with 3.5 µM BA alone usually resulted in the production of plants that developed a callus at their base and produced a short multishoot system. Root formation was inhibited compared with control plants (Table 2||.
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On the medium containing 0.58 µM GA3, plants always developed one single elongated shoot and callogenesis never occurred. All but one plant produced a well-developed root system. The addition of BA to the GA3 medium reduced plant height and root production while stimulating callogenesis at the base of the stem. Whichever the medium, plants produced approximately the same number of leaves (Table 2
), indicating that variations in stem length were due to differences in stem internode extension.
On control medium, plants remained vegetative while the highest flowering percentages were recorded in the presence of the cytokinin alone (Table 2). No one plant flowered on the GA3 containing medium and an inhibitory effect of this growth regulator upon floral transition was revealed by the fact that it strongly reduced the promotive action of the cytokinin (Table 2). GA3 similarly inhibited the flowering occurring on a kinetin (K)-supplemented medium (results not shown). In all treatments, the number of leaves initiated before the formation of the flower was rather low (less than 12) and no significant differences were found.
These data clearly indicate that cytokinin is required for the floral transition of the uf mutant. In contrast, GA3 is inhibitory and will be withdrawn from the medium in the further experiments.
Effect of different cytokinins
Shoot formation and total leaf number were uniform in plants derived from media supplemented with 3.5 µM K, 3.5 µM BA and 3.5 µM isopentenyladenine (IPA) (Fig. 1A, B, D). Several shoots were usually produced per explant and a callus always developed at the base of the stem. A root system was present on twice as many plants growing on the K or the IPA medium than on the BA medium (Table 3). Plants growing on the medium supplemented with 3.5 µM zeatin (Z) were rather different (Fig. 1C). A basal callus developed, root formation never occurred and numerous shoots (up to 18) with short internodes were produced, conferring a bushy habit to the explants. Almost all leaves were vitrified and the total number of leaves on the longest shoot was also reduced compared with the plants growing on the other three cytokinin media (Table 3).
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All four cytokinins promoted floral transition and the flowering percentages were in the same order of magnitude, ranging from 55 to 67. The percentage of flowering plants was higher for K than for the other three cytokinins whereas the number of leaves produced below the flower was significantly lower with K (Table 3
). K was thus selected and used in the further experiments.
Effect of kinetin concentration
Normal plants developed on media supplemented with the three lower K concentrations (Table 4). In contrast, internode extension was inhibited by high K levels, leading to the production of bushy explants which were almost deprived of roots and slowly initiated leaves which were frequently vitrified.
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A large callus developed at the base of the stem in all conditions. Flowering did not occur on the two media containing either the lowest or the highest K concentration. It was maximal when cytokinin was provided at 3.5 µM which also caused the earliest flowering response as revealed by the number of leaves initiated before the production of the floral bud (Table 4
). In further experiments, the K concentration was therefore kept at 3.5 µM.
Effect of nitrogen supply
In all conditions, explants developed a callus. It was very small on the N-deprived medium. Decreasing NH4NO3 concentration compared with the normal supply of the MS medium reduced plant growth and caused chlorosis (results not shown). Root production was inconsistently affected by N concentration (Table 5||.
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The flowering percentage increased when NH4N03 concentration decreased from 16.5 g l-1 to 8 g l-1, reaching 100% on this latter medium. Lower NH4NO3 contents greatly reduced flowering (Table 5
). Except for the single and very early flowering plant on the N-free medium, there was no significant difference between treatments in the number of leaves produced before floral structure initiation (Table 5). Thus, for the first time and in all three repetitions of this experiment, a half-strength NH4NO3 concentration in the MS macrosalts, in addition to K and sucrose at optimal concentrations, caused flowering of all uf plants.
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Discussion |
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Controlling floral transition in an autonomously flowering species
This study resulted in the design of an experimental system that allows the in vitro control of floral transition in tomato. Conditions are defined that either totally prevented flowering or caused the early production of the reproductive structure in all explants. This is the first report of a complete control of the switch from vegetative to reproductive development in a species which is usually considered to be a model for plants which exhibit the autonomous flowering habit.
The chemicals involved in the floral signalling pathway
Several chemical factors, implicated in the promotion of floral initiation of the uf tomato mutant were identified which are also considered to be constituents of the floral stimulus or floral signalling pathway in many other photoperiodic species (Bernier et al., 1993; Kinet, 1993; Corbesier et al., 1998). These are namely sucrose, cytokinins and nitrogenous nutrients which have to be supplied at optimal concentrations.
Sucrose has long been recognized as a floral promoting compound. Work done in vitro demonstrated that culture media intended to produce flowers contain higher sucrose levels than media used to grow plants vegetatively (Dickens and van Staden, 1988) and treatments or plant manipulations which result in increasing assimilate supply to the apex promote floral transition in various species. This observation constituted the basis of the nutrient diversion hypothesis (Sachs, 1977).
In these experiments, 30 g l-1 was found to result in a higher flowering percentage, although medium containing 15 g l-1 gave very similar data. In further experiments (data not shown), using media deprived of GA3 and supplemented with 3.5 µM K, the flowering percentage was strongly reduced (10%) on a medium containing 7.5 g l-1 sucrose compared with media with 15 or 30 g l-1 where percentages of flowering explants were, respectively, 55% and 59%. Since higher sucrose levels were found to impair growth and inhibit flowering (Table1), these results indicate that sucrose is required at an optimal concentration.
In the LD plant Sinapis alba, a dramatic increase of the flux of sucrose reaching the apex was reported to occur early after the start of an inductive treatment. This event long preceded any morphological change suggesting that it does not result from an increased sink activity of the apex and that sucrose may have a message-like role (Lejeune et al., 1993). In the SD plant Xanthium strumarium, a transient increase of sucrose export out of the leaves has been recorded during floral transition (Houssa et al., 1991) and recently cosuppression of a plasma membrane H+-ATPase isoform of Nicotiana plumbaginifolia was reported to reduce sucrose exudation from mature leaves and sugar content of apical buds and to delay flowering (Zhao et al., 2000). Investigations performed on Arabidopsis thaliana, using a starchless mutant and various photoperiodic treatments either causing flowering or maintaining the plants at the vegetative stage, also indicated that a large percentage of induced plants is always associated with a large, early, and transient increase in carbohydrate export from leaves (Corbesier et al., 1998). Sucrose availability for the aerial part of the plant was shown to have major effects in promoting flowering in A. thaliana in the dark, suppressing late flowering in mutants of both the autonomous floral promotion pathway and the photoperiodic promotion pathway (Roldán et al., 1999) and it was observed that sucrose enhanced specifically LFY promoter activity in young A. thaliana seedlings grown in vitro (Blázquez et al., 1998). Altogether, these results suggest that extra sucrose plays a signalling and essential role in floral transition.
Promotion of in vitro flowering by cytokinins has been repeatedly reported (Scorza, 1982; Dickens and van Staden, 1988) and, more recently, BA was found to be efficient on very different kinds of explants and types of plants (van der Krieken et al., 1991; Roberts et al., 1993; Das et al., 1996; Jumin and Nito, 1996; Joshi and Nadgauda, 1997; Kumar and Reddy, 1997). The results of this study are in agreement with these findings since without cytokinin (Table 2) or in the presence of a low cytokinin concentration (Table 4), flowering did not occur. Owing to the fact that high cytokinin concentrations were also inhibitory to flowering, these data clearly indicate that there is an optimal cytokinin concentration to stimulate floral transition in the uf mutant. Inhibitory effects of high cytokinin concentrations have been consistently reported for several species (Kinet et al., 1993). All cytokinins investigated have a rather similar effect upon floral transition of uf; the slightly more promotive action of K could be due to the fact that it is a synthetic molecule which is possibly less easily metabolized and thus could have more prolonged effects.
Implication of cytokinins in the control of floral transition is also suggested by the fact that changes in endogenous cytokinin levels were commonly observed during floral transition of various species and increased concentration in and/or supply to the apex appears to occur in all plant types including LDP, SDP, day-neutral and cold-requiring plants (Kinet et al., 1993). Once again, studies on S. alba are especially instructive in this respect (Bernier et al., 1993) and clearly implicate cytokinins as constituents of the floral stimulus transported in phloem sap to the apex in response to a photoperiodic treatment inducing flowering.
The nitrogen requirement for flowering is not unexpected since nitrogen is an essential element for plant growth and development. Also the observation that high nitrogen nutrition may be inhibitory to flowering is not new. More than 80 years ago, it was postulated that flowering is dependent on an increased carbon/nitrogen (C/N) ratio as compared with needs for vegetative development. This theory was originally based on data concerning reproductive development rather than floral transition but later observations suggested that it could apply to both physiological steps (Bernier et al., 1981). The importance of a high C/N ratio for in vitro flowering of Torenia fournieri (Tanimoto and Harada, 1981) and Pharbitis nil (Ishioka et al., 1991) has already been reported.
The MS medium is characterized by its high concentration in mineral salts in general and nitrogen in particular and it is known as a floral inhibitor (Dickens and van Staden, 1988). High nitrogen concentrations usually stimulate vegetative growth which then most probably competes more efficiently than reproductive development for assimilates. This view is in accordance with the nutrient diversion hypothesis (Sachs, 1977). Recently, in S. alba and A. thaliana submitted to floral inductive light treatments an increased export of amino acids in the phloem sap of both species was found at times compatible with the export from the leaves of the floral stimulus (Corbesier, 1998). Thus, it seems that in some plants amino acids are among the controlling factors of the floral transition. Nevertheless, the C/N ratio is increased markedly in both S. alba and A. thaliana at floral induction indicating that the C supply to the shoot apical meristem is more increased at floral induction than the N supply.
In addition to flowering promoters, interaction with inhibitor(s) are apparently implicated in tomato since GA3 reduced the flowering response triggered by cytokinins. Such an effect of GAs has already been reported in tomato (Mapelli et al., 1979) and, especially, in perennial species. In contrast, GAs are promoting flowering in several LD and cold-requiring rosette plants (Bernier, 1988) and it has been postulated that a GA promotive pathway exists in A. thaliana (Koornneef et al., 1998) where GAs activate LFY transcription (Blázquez et al., 1998). Blázquez and Weigel stressed that elucidating how floral meristem identity genes which are targets for the flower promoting and/or inhibiting signals integrate these regulators will contribute in understanding the divergent flowering responses among plants (Blázquez and Weigel, 2000).
A constitutive signalling pathway dowstream of pathways that control flowering time?
That similar chemicals are implicated in the shoot apex signalling process of LD, SD or autonomous plants suggests the existence of a common pathway to trigger floral transition at the meristem, which is downstream to pathways controlling flowering time and to the time they converge and become integrated ultimately to regulate the activity of floral meristem identity genes such as LFY. However, some results seem to weaken this conclusion. It was observed that the late phenotype of ft and fwa mutants, which are affected in genes involved in the promotive long-day pathway, is not rescued by sucrose either in the dark or in the light (Roldán et al., 1999). This lack of effect of exogenous sucrose could be due to the fact that FT and FWA are genes which are implicated in the control of flower meristem identity (Ruiz-Garcia et al., 1997) and are thus required in steps that are downstream of sucrose. Furthermore, these results, together with data from the literature, suggest that, in addition to the common signalling pathway, some plants may have specific requirements. As a consequence, the same regulatory signal may have either a promotive or an inhibitory effect, depending on the species. This is the case for GAs which inhibit flowering in tomato but are promotive in A. thaliana.
Obviously, the substances which would be implicated in the common signalling pathway are neither specifically nor exclusively involved in the flowering process: they all have a critical role in sustaining cell activity and proliferation. They have each to be supplied within a defined range of concentrations, a fact that was consistently reported either in in vitro or in in vivo studies aimed at investigating the effect of exogenous applications of chemicals upon flowering (Bernier et al., 1981; Kinet, 1993). This requirement of plants to flower at optimal levels of different promoters indicates that rather than reaching minimal concentrations in various compounds, it is the establishment of an adequate balance between these chemicals at the meristem which is critical. It has long been known that different auxin/cytokinin concentration ratios may have divergent morphogenetic effects in vitro, stimulating either root or bud initiation, and supporting the idea that morphogenesis in plants is dependent on adequate concentration ratios of ‘trivial’ compounds rather than on the production of specific morphogens.
In autonomously flowering plants, the balance between regulating chemicals could be under the control of genes constitutively co-ordinating flowering with developmental stage of the plant by affecting correlations between organs and thus the equilibrium between production sites and distribution within the organism of promoting and/or inhibiting signals. Grafting, cutting, topping and leaf excision, all experimental manipulations which alter plant organ balance, were shown to modify strongly flowering time in the uf mutant (Dielen et al., 1998; unpublished results).
In conclusion, these results thus indicate that tomato could constitute an adequate model to investigate the genetic control of floral transition and contribute in unravelling pathways which are constitutively regulating this important step of plant life cycle.
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Acknowledgments |
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We thank Dr C Périlleux, University of Liège, Belgium for critical reading of the manuscript. This work was supported by the Fonds National de la Recherche Scientifique (FNRS) of Belgium (‘Crédit aux chercheurs 1995–1996’ and ‘Fonds de la Recherche Fondamentale et Collective 1997–1998’) and by the Université catholique de Louvain (‘Fonds Spécial de Recherche 1995–1999’). One of us (VD) is grateful to the UCL for the award of a research fellowship.
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Notes |
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1 To whom correspondence should be addressed. Fax: +32 1047 3435. E-mail: dielen{at}bota.ucl.ac.be
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Abbreviations |
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BA, benzylaminopurine; GA3, gibberellic acid; GA(s), gibberellin(s); IPA, isopentenyladenine; K, kinetin; LD, long day; MS, Murashige and Skoog; SD, short day; Z, zeatin..
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