Cytokinin Habituation in Juvenile and Flowering Tobacco

The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38.Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.     

Flower Formation in Excised Tobacco Stem Segments; II. Reversible Removal of IAA Inhibition by RNA Base Analogues

The RNA base analogues, 2-thiouracil, 6-azauracil and 8-azaguanine incorporated singly into the medium, increased the number of floral buds in excised stem segments of Nicotiana tabacum variety Wisconsin No. 38 cultured in vitro. Combined treatments with 2 and 3 base analogues were even more effective. The effects were prevented by the corresponding natural counterparts, uracil, uridine, and guanosine respectively. These nucleic acid constituents added to cultures without base analogues did not affect the number of floral buds formed. In stem segments from the lower internodes treatments with the analogues effected a transition from vegetative to floral bud formation, thus in a sense removing the floral gradient as defined by Chouard and Aghion.     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Floral gradient in flowering tobacco in relation to free amino acids

By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those from the tobacco plants in full blooming or fruiting stages. Analysis of free amino acid levels revealed the acropetal gradient of Pro in flowering tobacco stem. L-Pro. L-Trp. D,L-Met and L-Arg were respectively added into the culture medium for testing their influence on floral bud formation from tobacco pedicel segments. Only L-Trp evidently enhanced the floral bud neoformation.     

Carbon Dioxide and Flowering in Pharbitis nil Choisy

The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO₂ concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO₂, while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO₂, however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO₂ concentrations which formed floral buds at stem node 3 or 4. At high CO₂ concentrations, floral buds did not form until stem node 6 or 7. Both high CO₂ concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.     

The Role of Auxins in Root-Knot Nematode-Induced Growth on Excised Tobacco Stem Segments

Root-knot nematodes, Meloidogyne incognita, induced lumps of callus tissue on the cambial surfaces of peeled tobacco stem segments cultured in vitro. Except for a layer 1 to 3 cells thick, callus was limited to the basal ends of control segments. Indole-3-acetic acid (IAA) applied in agar blocks to the centers of stem segments, when it had any effect on the cambial surface, induced streaks of callus extending from the blocks toward the basal ends of the segments. IAA in agar blocks also increased callus growth at the basal ends of the segments, increased the growth of pith on the undersides of the segments, promoted root initiation, but inhibited bud initiation. Nematodes produced none of these effects, nor did they change the type of organs induced by various concentrations of IAA in the medium. Callus tissue did grow on the cambial surface of stem segments surrounding agar blocks containing 2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport. Paraffin sections showed that the nematodes were confined to the callus tissue on the cambial surfaces of the segments. Except for occasional syncytia and areas of cell division, nematode-induced callus was composed of thin-walled, irregularly shaped cells arising from the cambium. Differences between the responses of tobacco stem segments to root-knot nematodes and IAA-agar blocks indicate that auxins were not freed from the plant tissue nor secreted by the nematodes. Instead, it is suggested that nematodes enabled the tissue to retain and use endogenous auxins that otherwise would have been transported to the basal ends of the segments.     

Overexpression of Two PsnAP1 Genes from Populus simonii × P. nigra Causes Early Flowering in Transgenic Tobacco and Arabidopsis

In Arabidopsis, AP1 is a floral meristem identity gene and plays an important role in floral organ development. In this study, PsnAP1-1 and PsnAP1-2 were isolated from the male reproductive buds of poplar (Populus simonii × P. nigra), which are the orthologs of AP1 in Arabidopsis, by sequence analysis. Northern blot and qRT-PCR analysis showed that PsnAP1-1 and PsnAP1-2 exhibited high expression level in early inflorescence development of poplar. Subcellular localization showed the PsnAP1-1 and PsnAP1-2 proteins are localized in the nucleus. Overexpression of PsnAP1-1 and PsnAP1-2 in tobacco under the control of a CaMV 35S promoter significantly enhanced early flowering. These transgenic plants also showed much earlier stem initiation and higher rates of photosynthesis than did wild-type tobacco. qRT-PCR analysis further indicated that overexpression of PsnAP1-1 and PsnAP1-2 resulted in up-regulation of genes related to flowering, such as NtMADS4, NtMADS5 and NtMADS11. Overexpression of PsnAP1-1 and PsnAP1-2 in Arabidopsis also induced early flowering, but did not complement the ap1-10 floral morphology to any noticeable extent. This study indicates that PsnAP1-1 and PsnAP1-2 play a role in floral transition of poplar.     

Isoperoxidases in Epidermal Layers of Tobacco and Changes during Organ Formation in vitro

The content and pattern of soluble isoperoxidases were determined in epidermal explants taken from different internodes of tobacco plants in the vegetative and floral states. There were qualitative and quantitative differences in the isoperoxidases, with a decrease in content and fewer bands being observed acropetally, i.e., in going from the base of the stem towards the apex. Epidermal explants from floral branches were grown in in vitro culture, with various media moditications, to form de novo floral or vegetative buds, roots or callus. Changes in soluble isoperoxidases were followed electrophoretically in relation to these varying morphogenetic pathways. In each of them, the number of bands increased on both the anodic and cathodic sides with time in culture. Compared to each other these four morphogenetic programmes were different in their peroxidase zymograms, mainly through varying kinetics in the development of activity of the isoenzymes. The changes observed during root and vegetative bud formation agree with previously published data, and the changes during floral bud formation agree with those observed in vivo.     

The Maryland mammoth allele reduces floral stimulus activity in stem piece explants of Nicotiana tabacum (Solanaceae)

The response of axillary buds to floral stimulus activity in stem pieces was examined in two near-isogenic cultivars of tobacco that differ in the recessive maryland mammoth (mm) allele, which confers short-day behavior. All axillary buds from day-neutral plants assayed on six-internode stem pieces made few nodes (less than 20) before flowering, while axillary buds from plants homozygous for mm assayed on six-internode stem pieces either did not flower in noninductive conditions or made many nodes before flowering in inductive conditions. About 80% of day-neutral axillary buds grafted onto day-neutral stem pieces did not respond to floral stimulus in stem pieces, indicating that the floral stimulus in stem pieces is ephemeral. In other graft combinations, the proportion of axillary buds that did respond to floral stimulus in stem pieces was substantially reduced from the 20% of day-neutral buds on day-neutral stem pieces that responded. These results indicate that the mm allele probably reduces both the amount of floral stimulus activity in stem pieces and the competence of axillary buds to respond.     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro I. Effects of Mineral Nutrients and Sugars

Internodal segments of Torenia fournieri Lind. were culturedon various media to investigate chemical factors influencingin vitro flowering. The elimination or dilution of ammoniumnitrate from Murashige and Skoog's medium increased the formationof adventitious buds which subsequently differentiated floralbuds. The dilution of mineral salts in Murashige and Skoog'smedium enhanced adventitious bud formation, but did not influencethe ratio of cultures with floral buds to those with adventitiousbuds. Among various media tested, in vitro floral bud formationand development of Torenia was best on a medium having 1/5 ofthe mineral salts and no NH₄NO₃. Eighty-seven percent of thecultures produced floral buds on this medium. Using this medium,the effects of various sugars were also examined. Increasingthe concentration of sucrose in the medium (up to 60 g/liter)increased the rate of cultures with floral buds, and stimulatedthe development of floral buds led to anthesis. (Received January 17, 1981; Accepted February 21, 1981)     

A Developmental Pattern of Flowering in Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp.: Effects of Bud Position and Gibberellin

The restricted flowering of colored cultivars ofZantedeschia is a consequence of developmental constraints imposed by apical dominance of the primary bud on secondary buds in the tuber, and by the sympodial growth of individual shoots. GA₃ enhances flowering inZantedeschia by increasing the number of flowering shoots per tuber and inflorescences per shoot. The effects of gibberellin on the pattern of flowering and on the developmental fate of differentiated inflorescences along the tuber axis and individual shoot axes were studied in GA₃ and Uniconazole-treated tubers. Inflorescence primordia and fully developed (emerged) floral stems produced during tuber storage and the plant growth period were recorded. Days to flowering, percent of flowering shoots and floral stem length decreased basipetally along the shoot and tuber axes. GA₃ prolonged the flowering period and increased both the number of flowering shoots per tuber and the differentiated inflorescences per shoot. Activated buds were GA₃ responsive regardless of meristem size or age. Uniconazole did not inhibit inflorescence differentiation but inhibited floral stem elongation. The results suggest that GA₃ has a dual action in the flowering process: induction of inflorescence differentiation and promotion of floral stem elongation. The flowering pattern could be a result of a gradient in the distribution of endogenous factors involved in inflorescence differentialtion (possibly GAs) and in floral stem growth. This gradient along the tuber and shoot axes is probably controlled by apical dominance of the primary bud. Online publication: 7 April 2005     

Ectopic expression of a poplar APETALA3-like gene in tobacco causes early flowering and fast growth

A MADS-box gene, designated PtAP3, was isolated from a floral bud cDNA library derived from Populus tomentosa. Analysis by multiple alignments of both nucleotide and amino acid sequences, together with phylogenetic analysis, revealed that PtAP3 is an ortholog of Arabidopsis AP3. Analysis of RNA extracts from vegetative and reproductive tissues of P. tomentosa by RT-PCR indicated that PtAP3 is expressed in roots, stems, leaves and vegetative and floral buds. Notably, the expression of PtAP3 fluctuated during floral bud development between September and February with differences between male and female buds. In the former, a gradual down-regulation during this period, interrupted by a slight up-regulation in December, was followed by a sharper up-regulation on February. In developing female floral buds, expression was stable from September to November, sharply up-regulated in December, and then gradually down-regulated until February. The functional role of PtAP3 was investigated in transgenic tobacco plants. Of 25 transformants, nine displayed an earlier flowering phenotype compared with the wild type plants. Furthermore, transgenic tobacco had faster growth and more leaves than untransformed controls. The traits proved to be heritable between the T0 and T1 generations. Our results demonstrate a regulatory role of the PtAP3 gene during plant flowering and growth and suggest that the gene may be an interesting target for genetic modification to induce early flowering in plants.     

Growth Promotion and Flowering Induction in Mango (Mangifera indica L. cv “Ataulfo”) Trees by Burkholderia and Rhizobium Inoculation: Morphometric, Biochemical, and Molecular Events

Inoculation of mango trees with Burkholderia caribensis XV and Rhizobium sp. XXV led to mango growth promotion (dry biomass increased in root 89 %, stem 34 %, leaves 51 %, and foliar area 53 %), floral fate (floral buds 100 %), and increased number of flowers (100 %). Nitrogen content in leaves was similar in inoculated and noninoculated trees, around 1.4 % (optimal condition for floral induction). The total foliar nitrogen content increased significantly (56 %) when the microbial inoculation treatment was applied. In addition, the initial content of sucrose, glucose, and fructose in leaves was higher in the microbial inoculation treatment trees but decreased during the evaluated period. The sucrose content in the noninoculated trees presented similar dynamics compared to the microbial inoculation treatment trees, but glucose and fructose showed increased values compared to those of the microbial inoculation treatment. FLOWERING LOCUS T (FT) expression profiles normalized to ACTIN showed similar dynamics but different expression levels: RQ values of 0.03 and 0.05 for noninoculated and microbial inoculation treatments, respectively. In addition, FT expression profiles in microbial inoculation, normalized to the noninoculated treatment, showed an increased FT expression dynamic over time (up to RQ = 2.2), although a drastic decrease in the last sampling date, when all trees presented developed panicles and flowers, was observed. This FT upregulation was in accordance with the flowering induction in that treatment. Temperature had an important influence on mango flowering induction, which was observed for a 1-month period (~10 °C at night and 20 °C during the day). Bud growth that occurred during that period generated mixed and floral buds depending on the exposure time to these inductive temperatures, less than 2 weeks and more than 3 weeks, respectively. Data indicate that inoculation of mango trees with plant growth-promoting rhizobacteria (associated with this crop) is a potential alternative way to promote growth and induce flowering in mango, greatly reducing the high economical costs and environmental contamination associated with traditional agricultural practices.     

DPX 1840-induced Organogenesis in Xanthi-nc Tobacco Plants

The synthetic growth regulant DPX 1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5, 1-a]isoindol-8-one) induced callus growth and subsequent tissue differentiation on cut surfaces of decapitated Xanthi-nc tobacco plants (Nicotiana tabacum). Callus formation and organogenesis induced by DPX 1840 depended on the presence of leaves. The adventitious meristems developed into either vegetative or flowering shoots. Pedicels that bore single flower buds developed two abscission zones that caused the buds to abscise before anthesis. The various morphological and physiological processes affected by DPX 1840 suggests that this growth regulator affects the endogenous hormonal distribution and/or activity.     

Hormonal Profile in Vegetative and Floral Buds of Azalea: Levels of Polyamines, Gibberellins, and Cytokinins

The floral transition includes a complex system of factors that interact and involve various biochemical signals, including plant growth regulators. The physiological signals involved in the control of the floral transition have been sparsely studied and mainly in plant species whose genetics are poorly known. In this work, the role of polyamines, gibberellins, and cytokinins was investigated by analyzing their endogenous content in vegetative and floral buds of azalea. The results showed that there is a clear distinction between floral and vegetative buds with respect to the levels of these plant hormones, with floral buds containing higher amounts of conjugated polyamines, gibberellins (GAs) from the non-13-hydroxylation pathway (GA₉, GA₇, and GA₄), and cytokinins (particularly isopentenyl-type species), and vegetative buds containing higher amounts of free polyamines and gibberellins from the early 13-hydroxylation pathway and fewer cytokinins. In conclusion, there is a specific pattern of endogenous hormone profiles in both vegetative and floral bud development in azalea, which may be relevant for future research on the control of flowering by exogenous hormone applications.     

Floral activity in solutions of deoxyribonucleic Acid extracted from tobacco stems

For Nicotiana tabacum cv. Wis. 38 plants, the capabilities of solutions containing DNA, extracted from either homogenates of stems in a floral state or nuclei of stems in a vegetative state, to effect flowering of vegetative plants have been studied. Previous work indicates that the DNA from homogenates of stems in a floral state is mainly nuclear. If DNA solutions are supplied to axillary buds of vegetative plants and if the axillary buds are defoliated every 4th day for 12 days, the buds supplied a solution of DNA from stems in a floral state initiate flowers under noninductive conditions, and the buds supplied a solution of DNA from stems in a vegetative state remain vegetative. Heating and rapidly cooling a solution of DNA from stems in a floral state enhances its floral activity. Heating and cooling a DNA solution also results in novel flowers showing up in many treated plants. Novel flowers are more striking in the offspring than in the parents. The capabilities of heated-cooled DNA solution to initiate flowers in noninductive conditions and to cause novel flowers are eliminated completely by treating (before heating and cooling) the DNA solution with deoxyribonuclease. Heated-cooled solutions of DNA extracted from nuclei of either vegetative stems or vegetative leaves contain no floral activity.     

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD₆₀₀) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD₆₀₀ = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.     

Photoperiodic studies on growth and development of Impatiens balsamina L.

Summary In the short-day plant Impatiens balsamina it was found that, while floral buds are initiated with 3 short-day (SD) cycles, at least 8 such cycles are required for flowering. The numbers of floral buds and open flowers bear a linear relationship with the number of SD cycles. The induced floral buds revert to vegetative growth unless the plants receive the minimum number of SD cycles needed for flowering, this reversion occurring in a basipetal direction. The rate of extension growth of the stem increases with increasing numbers of SD cycles. The high rate is maintained longer in plants receiving 32 or more SD cycles, but the subsequent fall is also steeper in these plants than in plants receiving less inductive cycles. Senescence also occurs in these plants and appears to be related to the magnitude of reproductive development and the high rate of extension growth.