Variation in nuclear DNA content of isonicotinic acid hydrazide-resistant cell lines and mutant plants of Nicotiana tabacum

Summary Isonicotinic acid hydrazide (INH)-resistant lines of Nicotiana tabacum have been maintained in callus culture for six years and mutant plants have been regenerated from a number of these lines. This study examines variations in DNA content in nuclei of several of these callus cultures, regenerated plants, and secondary callus from the regenerated plants. The lines selected for study include three easily regenerated lines (I 21, I 24, and I 9) and two lines of poor regenerating capacity (I 1 and I 18). Two of the regenerating lines eventually led to fertile plants and the third produced only sterile plants. In general, the range of total nuclear variability was not as high as anticipated from other studies of long-term tobacco callus cultures. The majority of nuclei in all the distributions were between 3 and 20 pg, and the most frequently encountered distributions concentrated in the 7–18 pg region corresponding to 2–5C by our estimate of the C value for tobacco. Distributions were not identical for plants regenerated from the same culture simultaneously, and the nuclear DNA content of secondary callus cultures from one of the plants examined did not reflect the quantitative DNA pattern of the plant from which it was derived. The greatest degree of variability and highest DNA content for individual nuclei were observed in the primary callus of the poorly- and non-regenerating lines. The variability in DNA content was not associated with the INH-resistant trait.     

Further characterization of picloram-tolerant mutants of Nicotiana tabacum

Summary A genetic and preliminary biochemical analysis has been performed on four picloram-tolerant mutants of Nicotiana tabacum that were isolated from cell cultures. The four mutations define three distinct linkage groups. Mutant seedlings incorporate radioactively labeled picloram normally and do not modify or degrade the herbicide in a manner that alters its solubility characteristics.     

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Summary Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.     

Isolation and characterization of valine-resistant mutants of Nicotiana plumbaginifolia

Summary Haploid mesophyll protoplasts of Nicotiana plumbaginifolia were mutagenized by UV-irradiation. Protoplast-derived colonies were then selected for valine resistance on a medium containing 5 or 10 mM valine. From the resistant calli, plants were regenerated. Resistance was inherited as a recessive Mendelian character in seven clones. Mutations conferring valine resistance were shown to be allelic. Protoplast-derived cells of L-valine-resistant plants were also resistant to L-threonine. Resistance to valine was based on a reduced valine uptake rate.     

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.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

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     

Isolation and characterization of Nicotiana plumbaginifolia nitrate reductase-deficient mutants: genetic and biochemical analysis of the NIA complementation group

Summary Two hundred and eleven nitrate reductase-deficient mutants (NR^–) were isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures by chlorate selection and regenerated into plant. More than 40% of these clones were classified as cnx and presumed to be affected in the biosynthesis of the molybdenum cofactor, the remaining clones being classified as nia mutants. A genetic analysis of the regenerated plants confirmed this proportion of nia and cnx clones. All mutants regenerated were found to carry monogenic recessive mutations that impaired growth on nitrate as sole nitrogen source. Mutants propagated by grafting on N. tabacum systematically displayed a chlorotic leaf phenotype. This chlorosis was therefore related to the NR deficiency. The observation of leaves with NR^– chlorotic sectors surrounded by NR⁺ wild-type tissues suggeests that an NR deficiency is not corrected by diffusible factors. Periclinal chimeras between wild-type tobacco and the NR^– graft were also observed. In this type of chimeric tissue chlorosis was no longer detectable when NR⁺ cells were in the secondmost (L2) layer, but was still detectable when NR^– cells were in the secondmost layer. The genetic analysis of nia mutants revealed that they belong to a single complementation group. However three nia mutants were found to complement some of the other nia mutants. The apoenzyme of nitrate reductase was immunologically detected in several nia mutants but not in other members of this complementation group. Some of the nia mutants, although they were NR^–, still displayed methylviologenitrate reductase activity at a high level. These data show that the nia complementation group corresponds to the structural gene of nitrate reductase. Some of the mutations affecting this structural gene result in the overproduction of an inactive nitrate reductase, suggesting a feedback regulation of the level of the apoenzyme in the wild type.     

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.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

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.     

PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies

Summary Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.     

Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Is bromodeoxyuridine resistance a consequence of cytokinin habituation in Nicotiana tabacum?

Cytokinin-habituated, as compared to nonhabituated Nicotiana tabacum cv. Havana 425 callus was shown to tolerate higher concentrations of 5-bromodeoxyuridine (BUdR) if cytokinin was added to the medium [Meins, Planta 129, 239–244 (1976)].Our aim was to clarify whether or not BUdR-resistance and cytokinin-habituation are linked characters in callus cultures of our BUdR-resistant tobacco mutant BR 37/21, obtained from Nicotiana tabacum cv. Ottawa.It is demonstrated that BUdR-resistance and cytokinin-habituation are independent characters in the case of this mutant. Non-habituated BR 37/21 callus grows as a resistant line on a medium containing BUdR at a selective concentration (30 mg/l) whereas cytokinin-habituated callus, not selected for BUdR-resistance, behaves as a sensitive line, i.e. turns brown and dies, on the same medium.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

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.     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

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.