In vitro flower bud formation in tobacco: interaction of hormones

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

Effects of the developmental state of the tissue on the competence for flower bud regeneration in pedicel explants of tobacco

The competence of pedicel explants of tobacco (Nicotiana tabacum L. cv Samsun) to regenerate flower buds in response to auxin was manipulated by preincubating excised tissues in the absence of auxin. When exposed to 1 micromolar 1-naphthaleneacetic acid, these tissues formed fewer buds than controls that were not preincubated. The number of buds eventually formed correlated with the 1-naphthaleneacetic acid concentration in the tissue 6 hours after the start of hormone application. The internal concentrations in pretreated explants were lower than in tissues that were not pretreated due to diminished uptake per milligram fresh weight and increased hormone conjugation. The change in the developmental state induced by auxin deprivation had a dual effect on bud regeneration: (a) the pretreatment caused fewer buds to be formed at any 1-naphthaleneacetic acid concentration tested, and (b) a higher auxin concentration in the medium was required to get a maximum bud number on precultured explants. An increase of the 1-naphthaleneacetic acid concentration in the medium led to an elevated hormone level in freshly cut as well as in preincubated tissues. It was concluded that the developmental state of the tissue directly affects the maximum number of buds that can be regenerated. Apart from that there is an indirect effect exerted via modulation of the ratio between external and internal auxin concentration. The change in this ratio can be compensated for by an adjustment of the auxin concentration in the medium.     

Isolation of <Emphasis Type="Italic">HAG1</Emphasis> and its regulation by plant hormones during in vitro floral organogenesis in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

Floral organs have been successfully induced from the regenerated floral buds of Hyacinthus orientalis L. by precisely controlling exogenous hormones in the medium. Under high concentrations of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. To understand the molecular mechanism of hormone-regulated flower development, a MADS-box gene, HAG1, which is homologous to AGAMOUS (AG) in Arabidopsis, was isolated from the floral tissues of Hyacinthus. Overexpression of HAG1 in Arabidopsis created flower phenotypes resembling those of the apetala2 mutant and AG transgenic Arabidopsis plants. Furthermore, the HAG1 expression pattern was similar to that of AG, confirming that HAG1 is the ortholog of AG in Hyacinthus. HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin, which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAG1 mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals. Our data support the hypothesis that hormone-regulated HAG1 activity is required for the induction of floral buds and the determination of floral organ types during the regeneration of floral buds.     

Induction of flower bud formation in vitro by dihydrozeatin

Flower stalk explants of tobacco cultured on a medium with an auxin and cytokinin regenerate flower buds within 14 days. The optimal medium concentrations of dihydrozeatin (DHZ) and benzyladenine (BA) were both 1 μM. The presence of DHZ in the culture medium was only essential during an initiation period of 7 days, whereas BA was needed only during the first 4 days. The difference in length of the initiation period is neither explained by the unequal uptake rates of the cytokinins nor by differences in their conjugation. At the medium concentration optimal for bud formation, the internal concentration of DHZ was two to three times the internal concentration of BA, which could be attributed to faster uptake of DHZ. It is concluded from the combined data that DHZ is less active in inducing flower bud formation than BA and that the exogenous cytokinins play only a role during the initiation phase of bud regeneration.     

Induction of flower bud formation in vitro by dihydrozeatin

Flower stalk explants of tobacco cultured on a medium with an auxin and cytokinin regenerate flower buds within 14 days. The optimal medium concentrations of dihydrozeatin (DHZ) and benzyladenine (BA) were both 1 M. The presence of DHZ in the culture medium was only essential during an initiation period of 7 days, whereas BA was needed only during the first 4 days. The difference in length of the initiation period is neither explained by the unequal uptake rates of the cytokinins nor by differences in their conjugation. At the medium concentration optimal for bud formation, the internal concentration of DHZ was two to three times the internal concentration of BA, which could be attributed to faster uptake of DHZ. It is concluded from the combined data that DHZ is less active in inducing flower bud formation than BA and that the exogenous cytokinins play only a role during the initiation phase of bud regeneration.     

Endogenous Cytokinins in Flower Bud Forming Explants of Tobacco

The accumulation of endogenous cytokinins was studied in pedicelexplants of tobacco (Nicotiana tabacumL.) during regenerationof flower buds in vitro. Maximal bud formation was induced onmedia containing 1.0 mmol m^–3 of benzyladenine or dihydrozeatin.No buds were formed in the absence of cytokinin. The levelsof dihydrozeatin, zeatin, and the corresponding ribosides weredetermined in explants cultured in the presence or absence ofcytokinin by means of a competitive ELISA technique. In explantsincubated without a cytokinin, only the dihydrozeatin concentrationincreased significantly during the first day of incubation anddecreased during the second day. No increase was observed inexplants incubated in the presence of benzyladenine. The concentrationof dihydrozeatin in these bud-forming explants was only 10 to15% of the concentration built up in explants cultured on dihydrozeatininstead of benzyladenine. This suggests that the endogenouscytokinins only play a minor role in the regeneration of flowerbuds in vitro. Key words: cytokinin, flower bud development, tissue culture, tobacco     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

The accumulation and metabolism of plant growth regulators during organogenesis in cultures of thin cell layers of Nicotiana tabacum

The accumulation and metabolism of exogenously applied and endogenously produced auxins and cytokinins were studied in relation to organogenesis in thin cell layers of Nicotiana tabacum L. It was shown that, in order to obtain maximal flower bud formation, both exogenous auxin and cytokinin needed to be present during the first 4 days of culture (to the formation of a subepidermal meristematic zone) whereas cytokinins needed to be present for at least 4 days more (until formation of organogenic centres). Explants taken from floral branches have higher endogenous indole-3-acetic acid (IAA) levels compared with explants from the basal part of the stem which form only vegetative buds. This might be related to a different IAA metabolism in these two types of explants as was shown by the different accumulation of exogenously applied IAA. Both 'floral' and 'vegetative' cells layers contained comparable amounts of zeatin riboside (ZR) as their major cytokinin. Free bases, zeatin (Z) and dihydrozeatin [(diH)Z], given exogenously, were largely metabolised to their respective ribosides. The observation that Z was less effective than (diH)Z in the induction of flower buds could be related to (diH)ZR apparently not being a substrate for cytokinin oxidase.     

Cytokinins and flower bud formation in vitro in tobacco: role of the metabolites

Explants from flower stalks of Nicotiana tabacum L. were cultured on different cytokinins to induce flower bud formation. All cytokinins tested except zeatin and zeatin-riboside induced the same maximal number of flower buds. Benzyladenine, benzyladenosine, and dihydrozeatin were the most active compounds whereas isopentenyladenosine and isopentenyladenine acted at a 20-fold higher concentration. These data suggest that the active cytokinins bind to the same receptor with different affinities. The presence of benzyladenine in the medium was necessary only during the first 2 days of culture (initiation period). The equilibrium between benzyladenine and its conjugates (the riboside, glucoside, and nucleotides) after a 4-day pulse was independent of the benzyladenine concentration whether it was inductive or noninductive for bud formation. The level of all derivatives was proportional to the benzyladenine concentration in the medium. Isopentenyladenine was used as a competitive inhibitor of benzyladenine conjugation. Isopentenyladenine concentrations that were too low for bud formation led to a synergistic increase in bud number when applied together with benzyladenine. Isopentenyladenine decreased benzyladenine uptake and conjugation. In spite of the lower uptake, the concentration of free benzyladenine inside the explants was higher in the presence of isopentenyladenine than in its absence whereas the concentration of the 7-glucoside of benzyladenine was lower. It was concluded that the free cytokinin base is the main active compound.     

Role of ethylene in auxin-induced flower bud formation in tobacco explants

The effect of ethytene on in vitro flower bud formation in thin-layer explants from tobacco pendicels ( Nicotiana tabacum L. cv. Samsun) was studied Endogenous ethylene production was stimulated by l-minocyclopropanc-l-carhoxylic acid (ACT), and inhibited by aminoethoxyviny lglycine (AVG). resulting in higher and lower ethylene accumulation. respectively. In the presence of an elevated ethylene concentration, the number of flower buds formed after 7 days of culture in explants was increased, compared with the control. Treatment with AVG or with AgNO₃ which blocks ethylene action resulted in decreased bud numbers after 7 days of culture. A different effect of ethylene was visible after 14 days of culture, when regeneration was complete. Treatment with AgNO₃ led to more bud regeneration, and increasing ethylene concentrations to lower bud numbers. The endogenous production of ethylene was enhanced by high concentrations of 1-naphthaleneacetic acid (NAA).
The inhibitory effect of applied ethylene was almost 100% in explants cultured at low concentrations of NAA (below 1 μ M ). but hardly visible at high concentrations (4.5 μ M ). As a consequence, the optimal NAA concentration shifted to a higher value in the presence of ethylene. These results are interpreted as a reduction in tissue sensitivity to auxin and in regenerative capability by ethylene. The effect of ethylene on auxin action is not exerted at the level of hormone concentration. Neither NAA uptake nor conversion to conjugates was effected by ethylene.     

Influence relative de la BAP et de l'IBA sur la néoformation de bourgeons et de racines sur les plantules du Biota orientalis (Cupressacées)

Relative influence of BAP and IBA on the induction of adventitious buds and roots on plantelts of Biota orientalis (Cupressaceae)
Both cytokinin (BAP) and auxin (IBA) have an influence upon the intensity of adventitious bud induction in vitro on hypocotyls of Biota orientalis .
Increase in either the concentration or the length of contact with cytokinin enhances the mean number of neoformed buds per explant, while that of auxin results in an increase of the mean number of reactive explants as well as in the nature of neoformations. Auxin contributes to a restoration of the induction when the length of contact with cytokinin is too short. However, this synergism is only observed when the contract with IBA is simultaneous or follows that with contract with BAP.
Rhizogenesis is also influenced by the auxin-cytokinin balance. An increase in the duration of contact with IBA is always followed by an increased in the mean number of explants forming roots. This organogenesis may be partially or completely inhibited if the duration of contact with cytokinin – before, simultaneously or after that with auxin- exceeds two weeks.     

Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

Flower formation in vitro in a quantitative short-day tobacco: interrelation between photoperiod and infructescence development

The flowering response of thin layers excised from branch internodes of Nicotiana tabacum cv. Maryland Catterton (quantitative short-day plant for induction) was studied under three photoperiodic treatments. The explants were excised from inflorescences bearing flowers only, flowers and green fruits, or from infructescences with green fruits only. The aim of the study was to investigate the post-inductive photoperiodic effects on in vitro flower bud formation in a quantitative short-day tobacco and the relation with infructescence development. Short days quantitatively enhanced the flower bud regeneration capacities of explants in all stages of development, both as number of explants induced to produce flowers and as mean number of flowers per explant. There was no significant difference in flower bud formation on explants of the first two stages, which produced much more flowers than those of the third stage. Observations in planta showed that, during the 20 days separating the second stage from the first stage, there was no significant difference in the number of floral buds and flowers present on the inflorescence; however, the branch internodes lengthened, as did the floral buds and flowers. During the 10 days leading to the third stage, the number of capsules did not change significantly, but a high rate of floral abscission occurred. The present results show that in Nicotiana tabacum cv. Maryland Catterton short day quantitatively controls not only the inductive step of the flowering process, but also affects the capacity to regenerate flower buds during the late post-inductive phases. The responsiveness to the photoperiodic signal decreases only when the plant exhibits only fruits.     

Uptake and metabolism of NAA and BAP in explants of tobacco in relation toin vitro flower bud formation

In vitro flower bud initiation and development depend on the presence of two hormones in the culture medium—auxin (NAA) and cytokinin (BAP). The uptake of both NAA and BAP by the explants was shown to be proportional to the concentrations supplied in the medium over a period of 4 days after the onset of culture. However, when supplied at equal concentrations for 24 h, the NAA uptake was up to 10-fold higher than the BAP uptake. Both hormones are rapidly metabolized by the explants. Nevertheless, the concentrations of free hormones inside the explants appeared to be high and in the case of NAA exceeded the concentration in the medium by more than 1 order of magnitude within 24 h. Apparently flower bud initiation in tobacco explants requires relatively high concentrations free NAA and BAP in the tissue maintained by a continuous supply in the medium. There are at present no indications that the products of hormone metabolism are directly involved in bud formation.     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

Effect of thidiazuron,light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves

The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on Lachenalia leaf tissue were investigated. The genotypes studied, showed a variation in bud formation. The hybrid explants responded differently to factorial combinations of BA and NAA. The growth regulators could not substitute for the regeneration potential of the genotype. Tissue age had a pronounced effect on regeneration potential. Young tissue formed the largest number of buds. An interaction between tissue age and genotype was detected. Cytokinin levels in young leaf tissue were higher than in older tissue. In young tissue no relationship was observed between the cytokinin level and the number of buds formed. However, in older tissue it appears as if a relatively low endogenous cytokinin level enhanced bud formation.Abbreviations BA benzyladenine - NAA naphthalene-1-acetic acid - Z zeatin - ZR ribosylzeatin