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.     

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.     

Isolation and characterization of mRNAs accumulated during in-vitro flower bud formation

The development of vegetative and generative buds on thin-layer expiants of tobacco (Nicotiana tabacum L. cv. Samsun) has been studied at the level of translatable mRNA to detect changes in the mRNA population during bud initiation and differentiation, and several quantitative differences were found. By differential screening of a cDNA library obtained from flower-bud-regenerating explants we have isolated a group of six cDNA clones representing genes that are preferentially expressed during in-vitro flower bud formation. Nucleotide sequence analysis of one of these cDNAs, pAP8, showed that the most likely open reading frame has some typical characteristics of, and homology with, extensin-like genes. Northern blot analysis and in-situ hybridization suggest a specific role for these extensin-like genes in flower bud initiation on tobacco pedicel explants.     

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non-reproductive tobacco plants was tested on a hormone-free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR- and TL-DNA from Agrobacterium rhizogenes 1855 root-inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone-free medium, indoleacetic acid alone (1 μ M ), kinetin alone (1 μ M ), and most abundantly on indoleacetic acid plus kinetin (1 μ M each). No flower formation was observed on indolebutyric acid plus kinetin (10 μ M and 0.1 μ M , respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone-free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.
The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non-reproductive plants.     

Hormonal Regulation of Pedicel Abscission in Begonia Flower Buds

In order to analyse the hormonal regulation of flower bud shedding in Begonia, levels of indoleacetic acid (IAA), abscisic acid (ABA) and ethylene were determined in buds and pedicels. The translocation and metabolism of ¹⁴C-labeled IAA in pedicel segments were also studied. In a monoecious Begonia fuchsioides hybrid, abscising male flower buds contain about 1% of the IAA present in non-abscising female flowers. In a male Begonia davisii hybrid, the seasonal variation in bud drop coincides with changes in the IAA content of the buds, while also the release of IAA from the bud to the pedicel is hampered. Abscission zones of these pedicels always contain abscission promoting ethylene concentrations. The tissue is prevented from responding with abscission by IAA from the flower buds. The buds also contain ABA but without influencing abscission considerably. Pretreatment with ethylene or ABA does not affect IAA transport in pedicel segments. The rate of this transport is 4–6 mm × h^–1:; the capacity increases with the transverse area. In young segments, IAA is decarboxylated and also otherwise metabolized.     

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     

Opening of Iris flowers is regulated by endogenous auxins

Flower opening in Iris (Iris × hollandica) requires elongation of the pedicel and ovary. This moves the floral bud upwards, thereby allowing the tepals to move laterally. Flower opening is requires with elongation of the pedicel and ovary. In cv. Blue Magic, we investigated the possible role of hormones other than ethylene in pedicel and ovary elongation and flower opening. Exogenous salicylic acid (SA) and the cytokinins benzyladenine (N6-benzyladenine, BA) and zeatin did not affect opening. Jasmonic acid (JA) and abscisic acid (ABA) were slightly inhibitory, but an inhibitor of ABA synthesis (norflurazon) was without effect. Flower opening was promoted by gibberellic acid (GA₃), but two inhibitors of gibberellin synthesis (4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate, AMO-1618; ancymidol) did not change opening. The auxins indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) strongly promoted elongation and opening. An inhibitor of auxin transport (2,3,5-triodobenzoic acid, TIBA) and an inhibitor of auxin effects [α-(p-chlorophenoxy)-isobutyric acid; PCIB] inhibited elongation and opening. The data suggest that endogenous auxins are among the regulators of the pedicel and ovary elongation and thus of flower opening in Iris.     

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.     

Polar transport of 1-naphthaleneacetic Acid determines the distribution of flower buds on explants of tobacco

Upon addition of 1-naphthaleneacetic acid (1-NAA) and benzylaminopurine, flower buds developed on explants from flower stalks of Nicotiana tabacum L. cv Samsun cultured in vitro. At low concentrations of 1-NAA, buds emerged mainly at the basal edge, whereas at high concentrations they developed on the remaining surface. The optimum concentrations for the two groups of buds were 0.45 micromolar and 2.2 micromolar, respectively, and the shapes of the concentration versus response curves were similar. The level of benzylaminopurine in the medium affected neither the shape nor the optimum concentration of these curves. The distribution of the buds over the explants was shown to be caused by polar auxin transport, leading to accumulation at the basal side. First, in the presence of the inhibitors 2,3,5-triiodobenzoic acid and 1-naphthylphthalamic acid, both groups of buds had the same optimum concentration of 1 micromolar 1-NAA. Second, after 6 hours of culture applied 1-NAA had accumulated in the basal part of the explant. In the presence of 1-naphthylphthalamic acid, no transport or accumulation of applied 1-NAA occurred.     

Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: Role of hormones in different stages

The in vitro development of flower buds was studied on tissue explants of epidermis and subepidermal cortex from the flower stalks of Nicotiana tabacum L. cv. Samsun. The number of flower buds formed depended mainly on cytokinin concentration. Auxin acted as a modifier in a complex way. In early development, NAA at 1 μ M decreased the number of buds initiated and delayed bud emergence. At a later stage, auxin promoted bud outgrowth at the same concentration. Optimal results were obtained when explants were first incubated at low auxin concentration for 3–5 days and subsequently transferred to an elevated auxin level. Physiological processes that lead to flower bud initiation start very soon after the onset of incubation. This was inferred from experiments whereby explants were first cultured at an inductive cytokinin concentration and then transferred to a non-inductive hormone level.     

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.     

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.     

Tissue 11In vitro Bud Formation on Explants from the Inflorescence of Nicotiana tabacum L.

Croes, A. F., Creemers-Molenaar, T., van den Ende, G., Kemp,A. and Barendse, G. W. M. 1985. Tissue age as an endogenousfactor controlling in vitro bud formation on explants from theinflorescence of Nicotiana tabacum L.—J. exp. Bot. 36:1771–1779. The in vitro formation of generative buds was studied on explantsfrom flower and fruit stalks and from internodes of the floralramifications of tobacco. A floral gradient was found to existalong the axis of the branch. The gradient concerns the numberof flower buds formed in vitro and is present in both typesof tissues. The number of flower buds is greater on tissuesfrom the apical than from the basal portion of the branch. Thecapacity to generate these buds is largely determined by tissueage at the moment of the excision. Consequently, the gradientmoves along the axis during the outgrowth of the inflorescence. The alternative possibility that some apex-derived stimuluspredetermines the morphogenetic capacity of the tissue priorto excision is excluded by the observation that the gradientremains virtually unaltered if the apex is removed one weekbefore the onset of culturing. Auxin affects the floral gradient Increasing the auxin concentrationin internode tissue culture causes a steeper gradient of flowerbud generation by almost completely abolishing bud formationon older tissues. Key words: Auxin, flower buds, gradient, tissue culture, tobacco     

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.     

Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA

Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.     

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.     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

Auxin Regulation of Flower Bud Formation in Tobacco Explants

Smulders, M. J. M, Janssen, G. F. K, Croes, A. F., Barendse,G. W. M., and Wullems, G. J. 1988. Auxin regulation of flowerbud formation in tobacco explants.—J. exp. Bot. 39: 451–459. The auxin 1-naphthaleneacetic acid (NAA) induces flower budformation in tobacco explants at concentrations between 0.1and 10 mmol m^–3. This regeneration process is completedin 14 d. The presence of NAA in the medium is required onlyduring the first 4 d of culture. In this period the explantstake up NAA at a very high rate, which is proportional to theconcentration in the medium. Due to the high uptake from a limitedvolume, the external concentration diminishes and, as a consequence,the uptake rate declines rapidly. A negative correlation wasfound between medium volume and the NAA concentration optimalfor bud formation. It is concluded that it is not the concentrationbut the dose of hormone taken up during the induction periodwhich determines the number of flower buds formed. Key words: Auxin, NAA, development, tissue culture     

诸葛菜花柄切段和花托离体培养中直接长出小花

在ＭＳ附加ＢＡ的几种培养基上首次从诸葛菜花柄切段和花托部位直接诱导出小花及花枝开花。花发育不正常,无花粉。该花花茎、叶柄、子房等的切段培养仍能再生出小枝,说明成花诱导态是稳定的,不可逆的．     

Expression of ipt gene controlled by an ethylene and auxin responsive fragment of the LEACO1 promoter increases flower number in transgenic Nicotiana tabacum

Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO1_{0.821 kb}-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO1_{0.821 kb}-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO1_{0.821 kb} promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO1_{0.821 kb} promoter fragment. The LEACO1_{0.821 kb}-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit.