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We report the first linkage map of tobacco (Nicotiana tabacum L.) generated through microsatellite markers. The microsatellite markers were predominantly derived from genomic sequences of the Tobacco Genome Initiative (TGI) through bioinformatics screening for microsatellite motives. A total of 684 primer pairs were screened for functionality in a panel of 16 tobacco lines. Of those, 637 primer pairs were functional. Potential parents for mapping populations were evaluated for their polymorphism level through genetic similarity analysis. The similarity analysis revealed that the known groups of tobacco varieties (Burley, Flue-cured, Oriental and Dark) form distinct clusters. A mapping population, based on a cross between varieties Hicks Broad Leaf and Red Russian, and consisting of 186 F2 individuals, was selected for mapping. A total of 282 functional microsatellite markers were polymorphic in this population and 293 loci could be mapped together with the morphological trait flower color. Twenty-four tentative linkage groups spanning 1,920 cM could be identified. This map will provide the basis for the genetic mapping of traits in tobacco and for further analyses of the tobacco genome.

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BaciUlus thuringiensis was isolated from dried tobacco residues and dead tobacco beetles (Lasioderma serricorne (F.); Coleoptera: Anobiidae) collected in a large number of locations worldwide. Eighty-eight samples of stored tobacco were analyzed and yielded 78 B. thuringiensis strains which were characterized on the basis of parasporal crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, and the results of an immunoblot analysis of the insecticidal crystal proteins. Flagellar antigen identification was used to differentiate selected isolates. Strains that produced rhomboidal crystals associated with the Coleoptera-specific pathotype (Cry Ill group) were the most abundant strains (59%o of the isolates). Preliminary toxicity assays were performed with L. serricorne larvae, and the results suggested that activity is not restricted to isolates related to the Coleoptera-specific group. The results of our survey indicate that B. thuringiensis is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of B. thuringiensis isolates that may be used to control stored-product pests.

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Historically, control of plant virus disease has involved numerous strategies which have often been combined to provide effective durable resistance in the field. In recent years, the dramatic advances obtained in plant molecular virology have enhanced our understanding of viral genome organizations and gene functions. Moreover, genetic engineering of plants for virus resistance has recently provided promising additional strategies for control of virus disease. At present, the most promising of these has been the expression of coat-protein coding sequences in plants transformed with a coat protein gene. Other potential methods include the expression of anti-sense viral transcripts in transgenic plants, the application of artificial anti-sense mediated gene regulation to viral systems, and the expression of viral satellite RNAs, RNAs with endoribonuclease activity, antiviral antibody genes, or human interferon genes in plants.

Experiments were conducted in tobacco (Nicotiana tabacum L.) to investigate the effect of sodium bicarbonate (NaHCO(3)) on the conversion of nicotine to nornicotine, a secondary alkaloid that can form the tobacco-specific nitrosamine N-nitrosonornicotine (NNN). The results showed that, under optimum conditions, NaHCO(3) stimulated nicotine conversion in converter plants to the maximum level predetermined by the genetic background. The conversion level in NaHCO(3)-treated leaves was 2-3 times higher than that in control leaves. For young seedlings the optimum concentration of sodium bicarbonate was 0.8% aqueous solution, and for adult plants the optimum concentration was 1%. Lower concentrations resulted in partial stimulation, whereas higher concentration damaged leaf tissue and resulted in a lower conversion level. Studies with different temperatures (from 22 to 43 degrees C) showed that 37 degrees C was optimal. This temperature allowed the least amount of time, 2-3 days for mature leaves and 4-6 days for green leaves, for the major converters to reach >95% of nicotine conversion. An examination of leaves from different growth stages and stalk positions showed that the amount of time needed for conversion was longer for young leaves and shorter for mature leaves. Treatment of leaves with NaHCO(3) affords a rapid and convenient means of identifying and removing nornicotine converter plants during growth in the greenhouse or field.

A worldwide survey was conducted to evaluate the frequency and distribution of Bacillus thuringiensis populations on cured tobacco leaves during post-harvest storage. In total, 133 tobacco samples of different types and origins were analyzed. Nine percent of the samples showed the presence of B. thuringiensis, and 24 B. thuringiensis strains were isolated and characterized. The majority of the isolates produced bipyramidal crystals, and three fourths of them showed a second type of crystal protein (cuboidal or heterogeneous crystals). Only three isolates showed the rhomboidal crystal morphology characteristic of the anti-coleopteran B. thuringiensis subsp. tenebrionis. PCR analysis with primers specific for cry1 and cry3 genes revealed eight distinct cry gene profiles. The results of this study indicate that B. thuringiensis is naturally present at low frequency on the phylloplane of cured tobacco leaves and that its distribution is worldwide.