The effect of photoperiod on flower formation in vitro in a quantitative short-day cultivar of Nicotiana tabacum

Superficial cell layers of a quantitative short-day tobacco plant ( Nicotiana tabacum L. cv. White Burley) were excised from different parts of the inflorescence (i.e. pedicels, branch internodes, rachises), and cultured in continuous darkness, continuous light or 8 h light/16 h dark daily. The flowering response in vitro of the different types of explants was investigated with respect to the effect of light on the post-evocation phases of the flowering process and explant commitment. Treatment effect was qualitatively and quantitatively influenced by explant origin. Three morphogenic features were observed: flower neoformation, caulogenesis and rhizogenesis (the latter on rachis explants only). Under all treatments, the highest flowering potential was shown by pedicels, while the highest vegetative potential was shown by rachises. Branch internodes showed an intermediate response, but with a tendency towards caulogenesis, which probably reflects their phylogenetic origin. Thus, opposite gradients of the neoformation of flowering and vegetative buds on explants were observed under all treatments. Pedicels formed new single flowers rather than inflorescences, while rachises regenerated mainly inflorescences. In darkness, flowering was limited mostly to pedicels. Vegetative bud formation was higher than floral bud regeneration in all types of explant. Continuous light enhanced the flowering response mostly in pedicel and branch internode explants. Short days enhanced flower bud formation in vitro on all types of explant. Results with respect to microsporogenesis, flower and inflorescence anomalies observed under darkness also seem to support the existence of a quantitative photoperiodic control on floral neoformation in vitro in this plant. These results suggest that in Nicotiana tabacum cv. White Burley in vivo floral induction, initiation and development are governed by the same photoperiodic requirements.     

Induction and floral determination in the terminal bud of Nicotiana tabacum L. cv. Maryland Mammoth,a short-day plant

Floral determination in the terminal bud of the short-day plant Nicotiana tabacum cv. Maryland Mammoth has been investigated. Plants grown continuously in short days flowered after producing 31.4±1.6 (SD) nodes while plants grown continuously in long days did not flower and produced 172.5±9.5 nodes after one year. At various ages, expressed as number of leaves that were at least 1.0 cm in length above the most basal 10-cm leaf, one of three treatments was performed on plants grown from seed in short days: 1) whole plants were shifted from short days to long days, 2) the terminal bud was removed and then rooted and grown in long days, and 3) the terminal bud was removed and then rooted and grown in short days. Whole plants flowered only when shifted from short days to long days at age 15 or later. Only rooted terminal buds from plants at age 15 or older produced plants that flowered when grown in long days. Only terminal buds from plants at age 15 or older that were rooted and grown in short days produced the same number of nodes as they would have produced in their original locations while buds from younger plants produced more nodes than they would have in their original locations. Thus, determination for floral development in the terminal bud, as assayed by rooting, is simultaneous with the commitment to flowering as assayed by shifting whole plants to non-inductive conditions.Abbreviations LD long day(s) - SD short day(s) - DN dayneutral     

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.     

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.     

Zearalenone and flower bud formation in thin-cell layers of Nicotiana tabacum L.

Three lines of evidence indicated a connectionbetween zearalenone (ZEN) and flower bud formationin thin cell layer (TCL) explants of Nicotianatabacum L. cv. Samsun. (1) There were two peaks inthe endogenous ZEN level during the formation offlower buds. (2) The specific inhibitor of ZENbiosynthesis, malathion (MAL), inhibited thebiosynthesis of endogenous ZEN and at the same timeflower bud neoformation. (3) Exogenous ZEN inducedflower bud neoformation.     

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.     

Timing of morphological and histological development in premeiotic anthers of Nicotiana tabacum cv. Xanthi (Solanaceae)

The tobacco stamen has been the object of many developmental studies, and the organ has more recently become a model for molecular genetic studies of anther differentiation. However, the spatial and temporal details of cellular differentiation of early anther development have never been thoroughly characterized. In the present study, the age of 15 tobacco flowers from plants grown under constant light and temperature was estimated using growth analysis. Prior to tissue fixation for light microscopy, moulds of stamen and anther primordia were made with a dental impression polymer so morphological and histological observations could be made on each tissue sample. Flower ages spanned an 8-d interval during which petal and stamen initiation occurred, and sporogenous cells reached the leptonema stage of meiosis. The initial development of the tetrasporangiate anther shape largely preceded periclinal division of archesporial initials. Anatomically, periclinal divisions in the hypodermal ∗∗∗(l₂) layer were observed before archesporial initials began to divide. These data indicate differences in the cellular basis of tobacco anther development compared to earlier clonal analyses of Datura. The pattern of mitotic cell division associated with microsporangial development suggested modal peaks in division over time. The ability to estimate developmental time in the tobacco anther has implications for future studies directed at understanding mechanisms of anther evolution via heterochrony.     

The fission yeast mitotic activator cdc25 and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun

Here, the tobacco (Nicotiana tabacum) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene Spcdc25 was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time to flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. The higher sucrose treatments enabled WT flowering; two-thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and fewer leaves compared with wild type. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.     

A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.     

Response of shape in vitro cultures of shape Nicotiana tabacum L. to copper stress and selection of plants from Cu-tolerant callus

In Nicotiana tabacum L. var. BEL W3 copper (Cu) at concentrations higher than 50 μM significantly inhibited callus growth and shoot regeneration. After 5–6 months of culture only a few morphogenic callus lines survived in the presence of 100 μM Cu. These calluses showed the capacity to grow and regenerate shoots through successive subcultures on medium containing 100 μM Cu. The 100 μM Cu-tolerant shoots, in comparison to regenerated control shoots, formed roots only when cultured in the presence of 100 μM Cu. From five independent Cu-tolerant callus lines in a culture period of 4–5 months more than 50 plants (defined ‘tolerant’) able to grow in presence of 100 μM Cu were obtained. These plants showed normal xylem tissue formation while control regenerated plants growing in normal Cu MS content (0.1 μM) had few xylem elements in the central cylinder. No difference as far chlorophyll content and chloroplast structure was found among Cu-tolerant and control plants. In Cu-tolerant plants, dry matter production was higher than in controls, particularly in roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

The expression of the 14D9 catalytic antibody in suspended cells of Nicotiana tabacum cultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

The effect of two protein stabilizers (polyvinylpyrrolidone [PVP] and gelatine) on growth and 14D9 yield of Nicotiana tabacum cell suspension cultures (Ab‐KDEL and sec‐Ab) was analyzed. The addition of PVP at a concentration of 1.0 g L^?1 produced the highest total 14D9 yield (biomass + culture medium) in the Ab‐KDEL line (4.82% total soluble protein [TSP]). With the addition of gelatine, the highest total 14D9 yield (2.48% TSP) was attained in the Ab‐KDEL line at 5.0 g L^?1 gelatine. When the Ab‐KDEL suspended cells were cultured in a 2‐L bioreactor, the highest 14D9 yield was 8.1% TSP at a 5% w/v inoculum size, which was the best 14D9 yield so far obtained in the platforms tested (E. coli, N. tabacum leaves and seeds, N. tabacum hairy roots, and cell suspension cultures). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1185–1189, 2014