Effects of ribavirin and adenine arabinoside on tobacco mosaic virus in Nicotiana tabacum L. var Xanthi tissue cultures

Although both ribavirin (1-β-ribofuranosyl-1,2,4-triazole-3carboxamide) and adenine arabinoside inhibited the multiplication of tobacco mosaic virus (TMV) in mechanically inoculated leaf tissues, neither chemical inhibited virus multiplication in unorganized tobacco callus after in vitro inoculation. The adenine deaminase inhibitor, pentostatin, did not increase the activity of adenine arabinoside in cultured cells. Several different developmental conditions and media did not increase the ability of either chemical to eradicate the virus from tobacco tissue cultures. However, the virus was eradicated from TMV-infected callus when grown in the presence of combinations of ribavirin and adenine arabinoside in shoot inducing medium.     

Growth and Endogenous Cytokinins in Tobacco Callus as Affected by N-(2-chloro-4-pyridyl)-N′-phenylurea

The effect of N-(2-chloro-4-pyridyl)-N-phenylurea (4PU-30) on the growth and content of endogenous cytokinins of adenine type in tobacco (Nicotiana tabacum L.) callus was investigated. Biomass accumulation in calli grown on Murashige and Skoog (MS) medium with 4PU-30 was higher than that on MS medium with kinetin. The obvious presence of isopentenyladenine type cytokinins and traces of zeatin type cytokinins supposes modification in the endogenous cytokinin metabolism of the tobacco callus grown on 4PU-30.     

Constitutive Expression of Interferon-Induced Human MxA Protein in Transgenic Tobacco Plants Does not Confer Resistance to a Variety of RNA Viruses

MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses,including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express MxA in plants in an attempt to create resistance to tospoviruses. Here, we report the generation of transgenic tobacco plants that constitutively express MxA under the control of the 35S cauliflower mosaic virus promotor. Northern and western blot analysis confirmed the expression of MxA in several transgenic plant lines. MxA expression had no obvious detrimental effects on plant growth and fertility. However, challenge experiments with tomato spotted wilt virus, tomato chlorotic spot virus, and groundnut ringspot virus revealed no increased resistance of MxA-transgenic tobacco plants to tospovirus infections. Neither was the multiplicationof tobacco mosaic virus, cucumber mosaic virus and potato virus Y inhibited in MxA-transgenic plants. The results indicate that the expression of human MxA alone does not enhance virus resistance in planta.     

Isolation of mutants of Arabidopsis thaliana in which accumulation of tobacco mosaic virus coat protein is reduced to low levels.

Summary We have found that Arahidopsis thaliana is susceptible to infection with a crucifer strain of tobacco mosaic virus (TMV-Cg); the coat protein of TMV-Cg accumulated to a high level in uninoculated rosette leaves several days after inoculation. As a first step in the search for host-coded factors that are involved in virus multiplication, we isolated mutants of A. thaliana in which the accumulation of TMV-Cg coat protein was reduced to low levels. Of 6000 M₂ plants descended from ethyl methanesulfonate-treated seeds, two such lines (PD 114 and PD378) were isolated. Genetic analyses suggested that the PD 114 phenotype was caused by a single nuclear recessive mutation, and that PD114 and PD378 belonged to the same complementation group. The coat protein accumulation of a tomato strain of TMV (TMVL) was also reduced in PD 114 plants compared to that in the wild-type plants. In contrast, PD114 plants infected with turnip crinkle or turnip yellow mosaic viruses, which belong to taxonomic groups other than Tobamovirus, expressed similar levels of these coat proteins as did infected wild-type plants.In this paper, we use the term multiplication (of a virus in a plant) to mean a substantial increase in virus concentration in the uninoculated leaves of the infected plant. Therefore, the efficiency of each process of invasion of the plant by the virus, uncoating, replication and degradation of the virus genome, formation and degradation of the virus particles, and spreading of the virus in the plant will affect the degree of multiplication     

Substituted nitroguanidines provide cytokinin activity during in vitro cultivation of plant tissues

Synthetic nitroguanidine derivatives can be used as alternatives to the traditional adenine-containing cytokinins used in plant tissue culture. First, nitroguanidine derivatives (NG) mimicked the typical activity of two standard cytokinins, 6-benzylaminopurine (BAP) and 2-isopentenyladenine (2iP) in the soybean callus (Glycine max) growth bioassay. NGs caused unanticipated responses as well, as demonstrated in three lines of tobacco (Nicotiana tabacum), when the auxin concentration was reduced from the standard concentration of 2 ug/ml NAA, to much lower concentrations of 0.01 ug/ml NAA or 0.02 ug/ml IAA. At the low auxin concentrations, kinetin lost the ability to promote either growth or differentiation, while the NG cytokinins were fully able to promote both. NGs promoted growth and differentiation in the presence of 0.01 ug/ml NAA in a newly initiated, totipotent line of Coker 319 tobacco. NGs plus 0.02 ug/ml IAA also promoted callus growth in a cytokinin-habituated tobacco line, Havana 425-CH. Lastly, NGs stimulated the outgrowth of healthy callus from aged callus that had been allowed to deteriorate through lack of subculture. Upon transfer of aged NTP callus to fresh media with NGs and 0.02 ug/ml IAA, healthy cell clusters were rapidly produced. In all three cases cited above, kinetin was ineffective at the low auxin concentrations. The NGs are therefore cytokinins, with the additional possibility of reducing the level of auxin required for their activity to be expressed.Abbreviations BAP 6-benzylaminopurine or N⁶- benzyladenine - IAA indoleacetic acid - 2iP N⁶-(²- isopentenyl)adenine - NAA -naphthaleneacetic acid - NG nitroguanidine derivative     

The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus

Summary Four antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 M APM or 10 M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 M), however, at a higher concentration (5 M) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations.     

Expressed resistance to black shank among tobacco callus cultures

Summary Quantitatively inherited resistance to the black shank pathogen (Phytophthora parasitica var. nicotianae) was expressed among callus tissue cultures of tobacco (Nicotiana). Tissue cultures of genotypes known to posses polygenic mechanisms for black shank resistance expressed that resistance in vitro when challenged by the viable pathogen. Callus of a susceptible cultivar was readily parasitized in culture. Furthermore, single gene resistance to the common pathogen race was also shown to operate in vitro. Nongenetic factors examined did not contribute significantly to the observed differences. Disease expression in vitro appeared to be highly correlated with its expression at the whole plant level.Screening for quantitative disease resistance can be complicated at the whole plant level by variable hostpathogen reactions and by significant genotype × environment interactions. Since quantitatively inherited mechanisms of black shank resistance are expressed in tobacco callus cultures, an in vitro host-pathogen system may be useful in screening tobacco lines for black shank resistance.The research reported in this paper (No. 82-3-6) is in connection with a project of the Kentucky Agr. Exp. Stn., and the paper is published with the approval of the director     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

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     

The Mode of Inhibition of TMV- and PVX-Induced RNA-Dependent RNA Polymerases by some Antiphytoviral Drugs

Six nucleobase and nucleoside analogues (6-aminouracil [6-AU], 6-aminothymin [6-AT], 6-fluorothymin [6-FT], 2,4-dioxohexahydro-1,3,5-triazine [DHT], 9-(2,3-dihydroxypropyl) adenine [DHPA] and 1-β-D-ribofuranosyl-1,2,4-triazol-3-carboxamid [ribavirin]) with potential antiphytoviral activity were tested in an in vitro replication system for their effect on tobacco mosaic virus (TMV)-and potato virus X(PVX)-induced RNA dependent RNA polymerase. 6-AU, 6-AT, 6-FT, and DHPA inhibited both polymerases which may be a target of these compounds. A Putative noncompetitive mode of inhibition is discussed. Whereas ribavirin did not affect the in vitr RNA synthesis in the established systems, the uracil-derivative DHT inhibited only the PVX-specific enzyme.     

Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Growth of and quassin accumulation by cultures of Quassia amara

Callus and suspension cultures were established from Quassia amara, a member of the Simaroubaceae. Analysis of the tissue culture showed that quassin was present in both callus and suspension cultures. The effect of variation in auxin and cytokinins on both callus growth and the presence of quassin was examined. The suspension culture was grown in a 7 liter bioreactor when good yields of quassin were achieved.Abbreviations IAA indole-3-acetic acid - IBA indolebutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6BA 6-benzyladenine - IpA N⁶ (-isopentenyl) adenine - IpAR N⁶ ( isopentenyl) adenine riboside - td doubling time     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

Dependence of the kinetics of secondary active transports in yeast on H+-ATPase acidification

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H⁺-ATPase, was inhibited to different degrees by D₂O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mm KC1 had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and pH were unchanged.Other types of acidification (spontaneous upon suspension; K⁺ stimulated) did not affect the secondary uptake systems.     

Plant regeneration through somatic embryogenesis in the forage grass Caucasian bluestem (Bothriochloa caucasica)

Plantlets were regenerated from cultured seed explants of the forage grass Caucasian bluestem [Bothriochloa caucasica (Trin.) C.E. Hubbard] via somatic embryogenesis. Embryogenic callus was produced in four weeks when surface sterilized seeds were cultured on a medium containing MS-salts, B-5 vitamins, 12 mM L-proline, 2% sucrose, 0.8% agar and 5M 2,4-D. Plantlets were regenerated in 6–8 weeks after culture initiation. Healthy root and shoot systems were produced within three weeks after the plantlets were transferred to a medium lacking 2,4-D. Approximately 95% of the plantlets survived greenhouse acclimation and produced healthy plants and viable seeds. Caucasian bluestem callus cultures exhibit natural resistance to kanamycin. High levels of kanamycin (up to 800 mg/l) did not completely inhibit callus growth. However, the regeneration of healthy-plantlets was completely inhibited by kanamycin even at low levels (50 mg/l).     

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. Crandon) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 10⁷ colony-forming units ml^-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 250 mg l^-1 cefotaxime. Levels of cefotaxime greater than 250 mg l^-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 M thidiazuron. In contrast to thidiazuron, N⁶-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 M, and growth was inhibited when concentrations of 0.11 or 0.22 M were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.Abbreviations BA N⁶-benzyladenine - CIM callus initiation medium - CaMV cauliflower mosaic virus - cfu's colony-forming units - HPT hygromycin phosphotransferase - MS Murashige and Skoog medium (Murashige & Skoog 1962) - NPT-II neomycin phosphotransferase-II - PAR photosynthetically active radiation - PCR polymerase-chain-reaction - TDZ thidiazuron - WPM Woody Plant Medium (Lloyd & McCown 1980) - 2,4-d 2,4-dichlorophenoxyacetic acid     

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3molg^–1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.