Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants

Summary A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 M CdCl₂, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.     

Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L.

Tobacco (N. tabacum cv. Xanthi) cell lines contained two forms of anthranilate synthase (AS; EC 4.1.3.27) which could be partially separated by gel-filtration chromatography. One form was resistant to feedback inihibition by 10 M tryptophan (trp) while the other form was almost completely inhibited by trp at the same concentration. Cell lines selected as resistant to 5-methyltryptophan (5MT) had more of the trp-resistant AS form. Only the trp-sensitive form was detected in plants regenerated from both normal and 5MT-resistant cell lines. Overexpression of the trp-resistant form in 5MT-resistant tobacco cells disappeared during plant regeneration but reappeared when callus was initiated from the leaves of these plants. The trp-sensitive form was localized in the particulate fraction and the trp-resistant form in the cytosol of tobacco cultured cell protoplasts. The trp-resistant form of AS from tobacco had an estimated MW of 200 000, determined by Sephacryl S-200 chromatography, compared to an estimated MW of 150 000 for the trp-sensitive form. The estimated molecular weights of AS from carrot and corn were 160 000 and 150 000, respectively. Analysis of AS activity from the diploid Nicotiana species Nicotiana otophora (chromosome number 2n=24) by high-performance liquid chromatography showed two activity peaks identical in elution time and trp inhibition characteristics to the activity from N. tabacum (chromosome No. 48). Thus the two enzyme forms found in tobacco did not appear to have originated individually from the progenitor species genomes which combined to make up the tobacco genome.Abbreviations AS anthranilate synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - HPLC high-performance liquid chromatography - 5MT D1-5-methyltryptophan - trp L-tryptophan     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Selection for salt tolerance in vitro using microspore-derived embryos ofBrassica napus cv. topas,and the characterization of putative tolerant plants

Microspore-derived embryos ofBrassica napus cv. Topas that survived salt stress, were obtained after selection against otherwise lethal doses (0.6 and 0.7%) of NaCl after mutagen treatment. A total of 10 salt-surviving embryos were obtained out of a possible 834 000 embryos that were mutagenized. One embryo out of a possible 845 000 obtained from nonmutagenized controls survived but failed to develop into a plant. Visual assessment after salt stress indicated that both the putative salt-tolerant plants and plants from control seeds behaved similarly. However, based on individual characteristics related to salt tolerance, one of the lines (PST-2) accumulated less sodium and retained more potassium, and hence was able to maintain a more favorable Na:K ratio as compared to the controls under salt stress. Also chlorophylla fluorescence induction and quenching signals indicated a high energetic state of the thylakoid membranes in PST-2 under salt stress. The other putative salt-tolerant line (PST-1) had a higher background level of proline that may have enabled it to survive salt stress during initial screening, although its later performance was no better than the control plants.     

Rapid flowering Nicotiana tabacum L.

 Selected Nicotiana tabacum Tobacco Introductions from the Nicotiana Germplasm Collection were crossed, back-crossed, and then selfed. When compared to parents, the new genotypes produce smaller plants with a shortened life-cycle that go from seed to seed in under 11 weeks. Received: 11 December 1998 / Revision accepted: 8 January 1999     

Sperm dimorphism in Nicotiana tabacum L.

Sperm cells of tobacco have been intensively studied as examples of isomorphic gametes in which major cellular and organellar parameters remain statistically indistinguishable in the two sperm cells. An examination of sperm cells late in maturation, however, displays that the sperm cell associated with the vegetative nucleus becomes statistically significantly smaller than the other sperm cell in tobacco. If late divergence occurs in the two sperms of other angiosperms, sperm dimorphism may be more prevalent than has previously been assumed and dimorphism may have a major influence on the pattern of double fertilization. Received: 15 December 2000 / Accepted: 4 May 2001     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit     

In vitro Transformation of Ovules into Rudimentary Pistils in Nicotiana tabacum L.

In our experiments with in vitro cultured young ovaries of Nicotianatabacum, var. havanensis we observed greening and extensivegrowth of the ovular primordia. Four to 6 weeks after inoculationa mass of rudimentary pistils bearing secondary ovules was tobe seen on the protruding placenta. Histological investigationproved these pistilloids not to be adventive organs arisingfrom callusing tissue, but the result of altered growth of theoriginal ovu1ar prirnordia. Though comparatively rare, reportson spontaneous ovular pistillody, including two Nicotiana species,exist in the teratological literature. However, whereas thesespon taneous ovular transformations are described as carpel-likestructures with marginally inserted secondary ovules, the invitro proliferating ovules develop into rudimentary pistilswith an early differentiating and very prominent central placenta.An interpretation of these differences is proposed.     

Sporopollenin accumulation in Nicotiana tabacum L. microspore wall during its development

Accumulation of sporopollenin components in microspore wall, its polymerization dynamics and possible participation of reactive oxygen species (ROS) in this process has been studied. For this purpose fluorescent and electron microscopy (TEM) was used. It has been determined that phenylpropanoid components of sporopollenin that form the exine accumulate in the microspore cell wall at the middle and late tetrad stages. At the late tetrad stage, they fully cover the microspore surface and accumulate abundantly in aperture areas. In accordance with this, numerous thick sporopollenin lamellae, electron-dense and acetolysis-resistant, emerge in aperture areas. Exine in the areas between apertures includes both acetolysis-resistant sporopollenin and washout components. These particular parts of the wall are intensively stained with fluorescent dye MitoSOX, which detects the presence of ROS. The staining disappeared after the treatment of microspore with superoxide dismutase, demonstrating the presence of superoxide in the exine. Superoxide easily converts to hydrogen peroxide, which can cause oxidative polymerization of sporopollenin components, leading to the formation of chemically stable biopolymer. The data obtained favor the hypothesis of ROS involvement in the formation of sporopollenin.     

Dynamic metabonomic responses of tobacco (Nicotiana tabacum) plants to salt stress

Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry.     

Anther cultures of Nicotiana tabacum L. mutants

Summary The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana tabacum L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther.The anthers from M₁ plants of a diploidized Nicotiana tabacum haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M₁ sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concentration.     

Jasmonate mediates salt induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.)

Jasmonate (JA), as an important signal, plays a key role in multiple processes of plant growth, deve lopment and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a recognized G box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

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.     

Premature cytokinesis in pollen mother cells of transgenic tobacco plants Nicotiana tabacum L

In majority species of dicotyledonous plants cytokinesis in PMCs occurs once after completion of two caryokinesis cycles, that is a simultaneous type. This paper represents cytological picture and frequency characteristics of abnormality which resulted in cytokinesis triggering after first meiotic division in a part of transgenic tobacco PMCs. It was shown that the main process of cytoskeleton reorganization typical for simultaneous cytokinesis remained without any alterations in such cells. However, in most cases premature cell division occurred with abnormalities such as membrane "tunnel" or "gash" formation. It was ascertained that initiation of additional round ofcytokinesis did not block nuclear cycle and cytokinesis after second meiotic division. Thus, transition of cell division from simultaneous type to successive one occurs under this abnormality.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these second generation callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.Abbreviations SH Schenk and Hildebrandt medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Developmental physiology of floral initiation in Nicotiana tabacum L.

The central process in the making of a multicellular organismis the fating of cells and tissues for their terminal phenotypes.The formation of a flower from a shoot apical meristem completesa sequence of fating processes initiated in embryogenesis. Thefating of a vegetative meristem of Nicotiana tabacum L. to initiatea flower involves at least two signals and two developmentalstates. A signal from the roots maintains vegetative growth,or prevents flowering, in the young seedling. As the plant grows,the vegetative meristem gains greater competence to respondto the floral stimulus from the leaves until it is evoked, byfloral stimulus, into a florally determined state. The florallydetermined state is then expressed. These developmental processesnot only establish the time of floral initiation, but also regulateplant size as measured by the number of nodes produced. Key words: Plant size, floral stimulus, competence, floral determination, induction     

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.