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.     

Immobilization of cells of Nicotiana tabacum L. on polyphenyleneoxide coated with poly-L-lysine

Cells of Nicotiana tabacum L. cv. Wisconsin 38 were immobilized on poly (2,6-dimethyl)-p-phenyleneoxide in powder form (Sorfix) coated with poly-L-lysine (molecular weight 40 000 daltons). The dependence of cell immobilization on the amount of bound polyL-lysine was estimated.Abbreviations MW molecular weight - dwt dry weight - fwt fresh weight     

In vitro induction of stigma-like and style-like structures from receptacle tissue in Nicotiana tabacum L.

Stigma-like and style-like structures were induced from the receptacle tissue in tobacco (Nicotiana tabacum L.). The presence of kinetin was necessary to induce these structures. The structures have a form similar to and the same function as normal stigmas and styles.     

Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the in vivo/in vitro technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Micromanipulation of male and female gametes ofNicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture

This research is part of an attempt to establish an in vitro fertilization system in tobacco to aid in understanding mechanisms of fertilization. Fusions of isolated male and female gametes were induced in a polyethylene glycol solution. Fusion appears similar to that in maize. One nuclear division of both an unfertilized egg cell and a synergid was induced in KM8p medium with 1 mg/l 2,4-dichlorophenoxyacetic acid in a microchamber culture; one cellular division of the egg cell was also induced in the same medium in solid-drop culture. The osmolality of suspension culture feeder cells was critical for the development of these cells. These results indicate that in vitro fertilization is possible in tobacco, which would be the first such system in dicots.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - PEG Polyethylene glycol     

Quantitative differences in sperm cells and organelles of tobacco (Nicotiana tabacum L.) grown under differing environmental conditions

In flowers grown at warm temperatures in environmental chambers and at cooler temperatures in the greenhouse, eight parameters of the sperm-cell organization of Nicotiana tabacum were examined during sperm cell maturation using serial ultrathin sectioning, transmission electron microscopy and quantitative cytology. Despite employing the same seed source, and similar soil and nutrient conditions, the surface area and volume of the cell, the nucleus and the chondriome were larger in flowers grown in growth chambers under warmer controlled conditions, whereas the number of plastids appeared to be the same, or slightly higher, in flowers grown under cooler greenhouse conditions. These results suggest that environmental conditions may influence the quantity of cytoplasmic organelles, including mitochondria and plastids, thus potentially influencing the likelihood of male cytoplasmic inheritance.     

RNA,proteins and polyamines during gametophytic and androgenetic development of pollen in Nicotiana tabacum and Datura innoxia

Variations in polyamines, proteins and RNA during in vivo gametogenesis and in vitro androgenesis in Datura innoxia and in Nicotiana tabacum were studied. Spermidine was the major polyamine during gametogenesis in both species. Marked differences in proteins, RNA and polyamines were evident during meiosis and at the first haploid mitosis. In Nicotiana an unknown amine (X₆₀) appears at the beginning of the first haploid mitosis. At the same time a rapid increase in the concentrations of RNA and proteins is observed. In Datura, at the time of the first haploid mitosis there is large increase in amine and RNA levels followed by an arginine peak. During androgenesis, putrescine and spermidine were the major polyamines in both species. In Nicotiana during androgenesis an unknown amine (X₈₁) was observed together with putrescine and spermidine. This unknown compound peaks during the developmental stages of embryogenesis. In Datura androgenic induction was marked by an arginine peak followed by an increase in the putrescine and spermidine levels associated with maximum RNA. These biochemical events are tentatively correlated with structural changes during pollen development. The significance of these results is discussed in relation to the role of polyamines during gametogenesis and androgenesis.     

Studies on the relationship between ploidy level,morphology, the concentration of some phytohormones and the nicotine concentration of haploid and doubled haploid tobacco (Nicotiana tabacum L.) and NICA plants

As compared to doubled haploid plants of the same origin, haploid tobacco plants are characterized by narrow leaves and in these leaves the endogenous concentration of gibberellins was considerably higher than in doubled haploids. This higher GA activity is almost entirely due to elevated levels of polar gibberellins. The same leaf shape as in haploids could be induced by GA₃ sprays to doubled haploids. A similar leaf shape was also observed on tissue culture derived so called NICA plants displaying the morphology of tobacco plants as described by Dudits et al. (1987) from whom the plant material was obtained as a gift. Here, in the leaves of a special strain with narrow lamina again a much higher gibberellin activity was detected than in the leaves of plants of the original tobacco strain. Histochemical determination of the relative DNA content indicated that leaves of NICA were chimaeras containing 1C cells besides cells with higher C values. Obviously, haploidy is somehow related to the endogenous gibberellin activity in tobacco plant material with consequences on the morphological appearance of 1n plants. Comparing some haploid and doubled haploid strains in tissue culture and pot and field experiments in several years apparently the genotype of the plant material is more significant for nicotine concentration than the ploidy level.Abbreviations DW dry weight - FW fresh weight - LSI leaf shape index     

The use of rootless mutants for the screening of spontaneous androgenetic and gynogenetic haploids in Nicotiana tabacum: evidence for the direct transfer of cytoplasm

Summary In crosses between a homozygous rootless mutant line of Nicotiana tabacum used as female and other Nicotiana tabacum lines, androgenetic haploids can be directly selected by their ability to form plantlets with a normal rooting system, whereas hybrid plants are killed few weeks after sowing. These androgenetic plants have the nucleus of the male parent into the cytoplasm of the female parent. In crosses where the homozygous rootless mutant line is used as a pollen donor, gynogenetic haploids can also be directly selected. Haploids can therefore be derived from male sterile plants using this approach. A generalization of this system for direct cytoplasm transfer and for the screening of spontaneous haploids in dicotyledons is proposed.     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Shade Tobacco Yield Loss and Globodera tabacum tabacum Population Changes in Relation to Initial Nematode Density

Field microplot experiments were conducted from 1987 to 1992 to determine the relationship between fresh weight leaf yield of shade tobacco (Nicotiana tabacum) and initial density of Globodera tabacum tabacum (encysted J2 per cm³ soil). Initial nematode densities of 0.1 to 1,097 J2/cm³ soil were negatively correlated with leaf yield, total shoot weight, and normalized plant height 5 to 6 weeks after transplanting (r = -0.73, -0.73, and -0.52, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to the yield and shoot weight data. The model described leaf yield losses of < 5 % for initial nematode densities of less than 100 J2/cm³ soil. Densities above 100 J2 resulted in yields decreasing exponentially to a maximum yield loss of >40% at 500 to 1,000 J2/cm³ soil. A similar initial density tolerance threshold relationship was observed for total shoot weight. No threshold effect was evident for standardized plant height, which was a poor predictor of leaf yield. Globodera tabacum tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.73).     

Somatic embryogenesis in Nicotiana tabacum L.: induction by thidiazuron of direct embryo differentiation from cultured leaf discs

Summary Induction of somatic embryogenesis by different growth regulators was examined in leaf disc cultures of Nicotiana tabacum L. Direct differentiation of somatic embryos occurred on media supplemented with naphthaleneacetic acid (NAA) and N⁶-benzylaminopurine (BAP). Thidiazuron (N-phenyl-N-1, 2,3,-thiadiazol-5-ylurea; TDZ) not only substituted for the most effective NAA-BAP combination but also induced a higher frequency of somatic embryogenesis. Regenerated somatic embryos were capable of developing into plants.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - NAA Naphthaleneacetic acid - TDZ N-phenyl-N-1,2,3,- thiadiazol-5-ylurea     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

CO2 enrichment in vitro. Effect on autotrophic and heterotrophic cultures of Nicotiana tabacum (var. Samsun)

Summary Plantlets of Nicotiana tabacum (var. Samsun) were grown under CO₂ enriched air supplied by a Warburg buffer. Growth of all plant parts was enhanced. The maximum growth increase was found for roots (120%).Addition of 30 g.l^-1 sucrose in the medium resulted in a three time faster growth. However, the effect of CO₂ enrichment was still positive in these conditions, although less pronounced than in autotrophic cultures.     

Role of Ca2+ in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.)

Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to determine how different application rates of calcium may influence this metabolic process. Increased calcium in the nutrient solution in which tobacco plants were grown considerably reduced the foliar concentration of phenolic compounds. Calcium clearly exerted a positive influence on the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase) involved in the metabolism of the phenolics. High dosages of calcium (5 mM) promoted more oxidation than synthesis of these compounds, thus explaining the lower concentration of the phenolics.     

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm^-1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm^-1 DC pulses, each of 50 s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages of protoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca²⁺ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca²⁺ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l^-1, the cytochalasins B and D, and Ca²⁺ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca²⁺ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaCl₂ treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F_2a, lecithin and CaCl₂.2H₂O treatments. The implications of the results are discussed.Abbreviations AC alternating current, approx.-approximately - BA benzylaminopurine, cv.-cultivar - DC direct current, diam.-diameter - FDA fluorescein diacetate - MS Murashige & Skoog (1962) - NAA napthaleneacetic acid - PCM protoplast culture medium - PIM protoplast isolation medium - PPM protoplast purification medium - rpm revolutions per minute - SD(n) standard deviation of a variate - SEM standard error of the mean     

Localization of myosin on sperm-cell-associated membranes of tobacco (Nicotiana tabacum L.)

Summary Actomyosin interactions are reportedly the principal mechanism for the transport of nonmotile sperm cells of flowering plants inside the pollen tube and inside the embryo sac. Myosin has been demonstrated on the generative cell (the predecessor of sperm cells), although it is unclear from previous studies whether myosin is located directly on the plasma membrane of the male germ cells or on the external plasma membrane of the pollen cell that surrounds them. Immunogold scanning electron microscopy was used to localize myosin on isolated tobacco sperm cells, with and without associated membranes. When present, the pollen tube plasma membrane surrounding the sperm cells was labeled by an antimyosin antibody, as were pollen tube cytoplasmic organelles. Negligible labeling was observed directly on the plasma membrane of the sperm cells.