Spermidine and flower-bud differentiation in thin-layer explants of tobacco

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Comparison of de-novo flower-bud formation in a photoperiodic and a day-neutral tobacco

Regeneration of flower buds in thin tissue layers from pedicels of photoinduced short-day (SD) tobacco, Nicotiana tabacum L. cv. Maryland Mammoth, is described. Up to seven flower buds per explant were obtained in a medium containing Murashige and Skoog's macro- and microclements, 100 mg/l myoinositol, 0.1 mg/l thiamine-HCl, 6% glucose, 5 M N⁶-benzylaminopurine, and 0.5 M -naphthaleneacetic acid. Usually some vegetative buds were also formed in the pedicel thin tissue layers. Thin tissue layers from other positions in the induced SD tobacco regenerated vegetative buds only. A comparative study with a day-neutral (DN) tobacco, Samsun, showed that the capacity to form de-novo flower buds was more localized and less strongly determined in photoperiodic than in the DN tobacco. The differences between the photoperiodic and DN tobaccos in flower-bud regeneration capacity are thus quantitative and not qualitative. The basis for this quantitative difference is not known, but may depend on factors controlling production of floral stimulus (florigen) and competency of cells to respond to florigen, and-or stability of the determined state to form flower buds in vitro.Abbreviations BAP N⁶-benzylaminopurine - DN day-neutral - GA₃ gibberellic acid - LD long-day - MM Maryland Mammoth - NAA -naphthaleneacetic acid - SD short-day     

Influence of leaves and roots on meristem development inNicotiana tabacum L. cv. Wisconsin 38

The terminal, apical shoot meristem ofN. tabacum cv. Wisconsin 38 normally differentiates into a flower after producing 30 to 40 nodes. The influence of leaves and roots on the regulation of flowering was evaluated by counting the number of nodes produced after removal of leaves or the induction of adventitious roots. Leaf removal has no effect on the number of nodes produced before flower formation. Root induction significantly increases the number of nodes produced before flower formation. The plant behaves as if it were measuring the number of nodes between the meristem and the roots as a means of regulating meristem conversion from vegetative to floral differentiation.     

Quantitative studies of bud initiation in cultured tobacco tissues

After transferring leaf, pith, and stemcortex tissues ofNicotiana tabacum L. cv. Havana 425 from a complete medium containing auxin and cytokinin to an inductive medium with auxin deleted, there is lag phase of approx. 14d followed by a linear phase in which the rate of bud initiation is constant. The incidence of buds formed is very low, approx. one bud per 10³ or 10⁴ cells. Statistical analysis of the distribution of buds among explants and subcloning experiments provide evidence that the paucity of buds results from neither negative interactions among bud forming centers nor a paucity of cells with the potential for organogenesis. Our results are consistent with the hypothesis that the frequency of bud initiation is determined by the availability of competent cells, by position effects, or by a combination of both mechanisms.     

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. Havana 425 becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10^–3 per cell generation, was 100–1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10^–10 M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.     

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.     

Regeneration of tepals,stamens and ovules in explants from perianth of Hyacinthus orientalis L. importance of explant age and exogenous hormones

Regeneration of tepals, stamens and ovules from perianth explants of Hyacinthus orientalis L. in different developmental stages could be controlled by means of exogenous hormones. Perianth explants in a relatively early stage of development were competent for differentiation of tepals on Murashige and Skoog (MS) medium supplemented with 2 mg·1^-1 N⁶-benzylaminopurine (BAP) or zeatin and 0.1 mg·1^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Perianth explants in a later stage of development regenerated stamens and ovules, and marked difference was observed in the activity of BAP and zeatin in this regard. Zeatin stimulated more strongly stamen formation, while BAP enhanced ovule formation. Thus, stamens were formed when the explants were cultured for four months on medium with 2 mg·1^-1 BAP and 0.1 mg·1^-1 2,4-D and then transferred to medium with 0.2 mg·1^-1 zeatin and 0.005 mg·1^-1 1-naphthaleneacetic acid. On the other hand, differentiation of ovules occured in explants cultured for two weeks on the former medium and then transferred to medium with 0.1 mg·1^-1 BAP and 0.01 mg·1^-1 2,4-D. Although ovule formation could also be obtained with 2 mg·1^-1 BAP alone, it was substantially enhanced by the presence of 0.1 mg·1^-1 2,4-D in the medium in the early stages of culture. The results demonstrate the importance of both the developmental stage of the source organ from which explants are excised and of the hormone composition of the medium for the regeneration of different floral organs by perianth explants of Hyacinthus.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Orientation of cellulose microfibrils in cortical cells of tobacco explants

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations — all given with respect to the longitudinal axis of the cell —, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.Abbreviations CMF cellulose microfibril - MT microtubule We thank Professor M.M.A. Sassen and Dr. G.W.M. Barendse (Department of Experimental Botany, University of Nijmegen, Nijmegen, The Netherlands) for helpful discussions and Mrs. A. Kemp for her assistance in the ethylene experiments.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10⁵ copies in the genome, can escape de-novo methylation.Abbreviation AzaC 5-azacytidine This project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic. We thank Ms. Libue Jedliková, Ms. Hana Suchánková, and Ms. Emilie Koudelková for technical assistance.     

Metabolism of myo-Inositol and growth in various sugars of suspension-cultured tobacco cells

Tobacco (Nicotiana tabacum L.) cells were cultured in a liquid medium which contained sucrose as a source of carbon and energy. Various cell-wall constituents and wall precursors (L-arabinose, D-xylose, D-galactose, D-mannose, D-glucuronate, myo-inositol) were added to cells growing in this medium to by-pass possible rate-limiting steps in the relevant metabolic pathways. None of these compounds stimulated growth as measured by increase in fresh weight; myo-inositol did cause a slight increase and L-arabinose a decrease in dry weight accumulation compared to controls grown on sucrose only. Although myo-inositol was not needed for rapid growth, tracer level amounts of [2-³H]myo-inositol were rapidly absorbed and metabolized. Label was incorporated into the uronide and pentose residues of cell walls and exocellular polysaccharide.     

DPX-3778 promotes proliferation of tobacco callus in vitro

DPX-3778, the triethanolamine salt of 3-(p-chlorophenyl)-6-methoxy-s-triazine-2,4(1H,3H) dione, at concentrations of 0.124–2.48 M enhanced ca. 4-5-fold the proliferation of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus cultured in the presence of indole-3-acetic acid and kinetin, and retarded its senescence.     

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     

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.Abbreviations ELISA enzyme-linked immunosorbent assay - Fg immunoglobulin - kDa kilodalton     

Amino-acid transport into cultured tobacco cells

Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca²⁺ for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not the result of exchange diffusion. Arginine uptake over a concentration range of 2.5 M to 1 mM was characterized by simple Michaelis-Menten kinetics with a K_m of 0.1 mM and a V_max of 9,000 nmol g^-1 fresh weight h^-1. Transport was inhibited by several compounds including carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, N,N-dicyclohexylcarbodiimide, and N-ethylmaleimide. Inhibition by these compounds was not the result of increased efflux resulting from membrane damage. A variety of amino acids and analogs, with the exception of D-arginine, inhibited transport, indicating that arginine transport was mediated by a general L-aminoacid permease. Competition experiments indicated that arginine and lysine exhibited cross-competition for transport, with K_i values similar to respective K_m values. Arginine transport and low-affinity lysine transport are probably mediated by the same system in these cells.Abbreviations BTP Bis Tris Propane - CCCP Carbonylcyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - DTT Dithiothreitol - NEM N-ethylmaleimide - MES 2(N-morpholino)ethanesulfonic acid - TCA trichloroacetic acid This paper is the third in a series on amino-acid transport into cultured tobacco cells. For parts I and II, see Harrington and Henke (1981) and Harrington et al. (1981)     

Role of Exogenous Hormones in Inducing Cells in different Positions of Perianth Explants to Regenerate Flower Buds in Hyacinthus orientalis

Role of the exogenous hormone in inducing different position cells of perianth explants of hyacinth to regenerate flower buds was studied. Experiments showed that (1) Exogenous hormones are necessary for inducing cells of the perianth explant to regenerate the flower buds; (2) Only cytokinine alone could induce the regeneration of the flower buds, the auxin was not necessary; (3) Exogenous hormones in different concentrations could induce cells in the different parts of the perianth explants to differentiate the flower buds: 6-BAP or zeatin 2 mg/L alone could induce cells located at the lower part of the perianth to differentiate flower buds. Combination of 6-BAP or zeatin 2 mg/L and 2, 4-D 0.1 mg/L was advantageous to cells located middle part of the perianth to regenerate the flower buds. Combination of 6-BAP or zeatin 2 mg/L and 2, 4-D 1.0 mg/L could promote cells located at the upper part of the perianth to differentiate flower buds.     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid