Pollen irradiation and possible gene transfer in Nicotiana species

Summary Progeny from crosses of Nicotiana langsdorffii with gamma irradiated pollen of Nicotiana alata Crimson Bedder showed skewed segregation in the F₂ favoring the maternal parent. This is probably not gene transfer in a strict sense, rather just an extreme case of reduced transmission of irradiated chromosomes, leading to massive overrepresentation of maternal genes. Gene transfer or mutational loss may explain some anomalous F₁ plants. Segregation in the F₂ progeny showed the presence of several genes from the irradiated pollen. Crosses of Nicotiana sylvestris, N. plumbaginifolia N. paniculata, and Petunia parodii with irradiated pollen from N. alata and Petunia hybrida showed no evidence of gene transfer, nor did experiments with irradiated mentor pollen. This indicates that gene transfer with irradiated pollen between non-crossing species or between species giving sterile hybrids is probably a rare phenomenon.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

Wild species of Nicotiana as a new source of tobacco resistance to the tobacco hornworm Manduca sexta

Sixteen species of Nicotiana were examined for resistance to second instar larvae of the tobacco hornworm, Manduca sexta (L.). The tests were designed so as to discriminate between antibiosis and nonpreference. High levels of antibiosis resistance were observed in several wild species such as N. gossei and species in the Repandae section. Several other species, such as N. excelsior, showed antibiosis that appeared to be due to a different mechanism than the alkaloid-trichome exudate based resistance of the above species. The data indicate that these species of Nicotiana may be used as a new source of resistance to larvae of M. sexta.

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D'espèces sauvages de Nicotiania comme nouvelle source de résistance du tabac à Manduca sexta

Résumé La résistance aux chenilles de second stade de Manduca sexta L. a été examinée chez seize espèces de Nicotiana. Les essais ont été conçus pour distinguer absence d'appétance et antibiose. Plusieurs espèces sauvages comme N. gossei et les espèces de la section Repandae ont présenté un niveau élevé d'antibiose. D'autres espèces, comme N. excelsior ont présenté une antibiose vraisemblablement due à un mécanisme différent de la résistance des espèces précédentes provoquée par un exsudat alcaloïde des trichomes. Les résultats indiquent que ces espèces de Nicotiana peuvent être utilisées comme nouvelle source de résistance aux chenilles de M. sexta.

     

Nucleotide sequences of nuclear tRNACys genes from Nicotiana and Arabidopsis and expression in HeLa cell extract

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA_GCA ^Cys as novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA^Cys genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs^Cys. Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA^Cys genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA^Cys genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs^Cys of plant origin.     

Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.