Induction and Isolation of Auxotrophic Mutants in Somatic Cell Cultures of Nicotiana tabacum.

A method for the selection of auxotrophic mutants from somatic cell cultures of Nicotiana tabacum depends upon the incorporation of 5-bromodeoxyuridine into the DNA of wild-type cells and upon its lack of incorporation into the DNA of auxotrophic cells. There may be more than one functional copy of essential genes in the haploid genome of Nicotiana tabacum.

Methionine sulfoximine--resistant mutants of tobacco.

Selecting mutants from populations of haploid plant cells cultured in vitro may provide a rapid method for recovering agriculturally useful variants. Mutants of Nicotiana tabacum were recovered which were resistant to methionine sulfoximine, an analog structurally similar to methionine. Induction of chlorosis was prevented or less evident in mutant plants that were inoculated withPseudomonas tabaci, a bacterial pathogen which produces a toxin that is a structural analog of methioning. Several mutants show a specific increase in the level of free methionine.

A morphogenetically competent soybean suspension culture.

A morphogenetically competent suspension culture was derived from embryonic axes of Glycine max cv. Mitchell. The cultural history included visual selection for nonfriable, embryo-like structures, recurrent selection in a regime of 2,4-dichlorophenoxyacetic acid exposure and withdrawal, and the replacement of the nitrogen in a Murashige and Skoog salts-based medium with 20 millimolar ammonium citrate. The embryoids produced by this suspension are capable of completing plantlet development. The suspension can be maintained by serial subculture.

Cloning of cauliflower mosaic virus (CLMV) DNA in Escherichia coli.

A plasmid containing cauliflower mosaic virus DNA can be faithfully cloned in Escherichia coli, but proved to be noninfective in test plants.

Polypeptide composition of fraction 1 protein from parasexual hybrid plants in the genus Nicotiana.

Analysis of the subunit polypeptide composition of Fraction 1 proteins gives information on the expression of both nuclear and chloroplast genomes; the large subunits of the protein are coded by chloroplast DNA, whereas the small subunits are coded by nuclear DNA. Fraction 1 protein isolated from the leaves of parasexual hybrid plants derived from the fusion of protoplasts of Nicotiana glauca and N. langsdorffii contains the small subunit polypeptides of both parent species and the large subunit polypeptides of only N. glauca. Fraction 1 protein isolated from the leaves of a hybrid plant obtained after the uptake of chloroplasts of N. suaveolens by protoplasts of white tissue of a variegating mutant of N. tabacum contains the large subunit polypeptides of both N. suaveolens and N. tabacum, as well as the small subunit polypeptides of both these species.

In vitro selection of Nicotiana sylvestris variants with limited resistance to TMV.

Haploid tobacco (Nicotiana sylvestris) plants were inoculated with a yellow strain of tobacco mosaic virus (TMV-Flavum) and then exposed to 500 rads of acute gamma radiation. Leaf strips cultured on callus-inducing medium yielded two types of colonies: 1) yellow, virus-infected and 2) green, apparently healthy. Of the 3210 calli scored, approximately 5% were virus-free, and after regeneration, 0.2% were resistant at the plant stage. Later, adult plants, both TMV-resistant and TMV-susceptible, produced self-fertile, diploid flowers. Both seedling progeny and rooted cuttings from resistant stock plants showed resistance to TMV infection. This resistance was characterized by restricted virus multiplication and movement within the infected plant resulting in a 3-8 week delay in symptom expression.

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild-type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 +/- 11.1 versus 48). The enzyme from R2 had a lower V(max) for CO(2), although the K(m) values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit.Total polyphenol oxidase activity was the same in leaves from the heterozygous mutant (Su/su) and wild-type (su/su) plants when correlated with developmental age as represented by morphology rather than by the chronological age of the plants. There was a marked increase in the soluble activity of this enzyme with increasing age of both plant types and also as a result of varying environmental conditions. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity correlated inversely with increases in the soluble activity of polyphenol oxidase in crude homogenates from which the carboxylase/oxygenase was crystallized over a generation of Su/su and su/su plants. Criteria are outlined for determining if differences in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase are caused by an effect of polyphenol oxidase activity and/or by some other extrinsic parameter.

Characterization of the thylakoid membranes of the tobacco aurea mutant Su/su and of three green revertant plants.

Phenotypic characterizations of the semidominant aurea tobacco (Nicotiana tabacum L.) mutant Su/su, the homozygous mutant Su/Su and three green revertants (R1, R2, and R3) are presented. The leaf color of Su/su plants varies from yellow to light-green when grown under high and low energy fluence rates (33.0 and 3.3 W m(-2)), respectively. The change in visual phenotype under high-light conditions is correlated with decreased content of chlorophyll per leaf area, agranal chloroplast ultrastructure, changes in the number of chlorophyll-protein complexes, and absence of two or more of the light harvesting chlorophyll-polypeptides of 25,000-29,000 dalton. The homozygous mutant grown under low light was shown to be completely lacking in grana stacks and to be deficient in chlorophyll-protein complexes. Revertant R1 was found to be identical to wild-type plants in all parameters examined (leaf color, chloroplast ultrastructure, chlorophyll-protein complexes, chlorophyll-protein complex polypeptides) except in chlorophyll content. It did not show an increased chlorophyll and carotenoid content as did the wild-type plants when exposed to high light. Revertants R2 and R3 were similar to the heterozygous mutant Su/su in most of the parameters examined. They yellowed because of a loss of chlorophyll and an increase in the amount of carotenoids, had agranal chloroplasts, and had variant chlorophyll-protein complexes when grown under high light intensities. However, each appeared to contain some of the light-harvesting pigment-protein complex polypeptides found to be absent in Su/su when grown under high-light conditions.

Parasexual interspecific plant hybridization.

Interspecific plant hybrids have been produced by parasexual procedures. Protoplasts of Nicotiana glauca and N. langsdorffii were isolated, fused, and induced to regenerate into plants. The somatic hybrids were recovered from a mixed population of parental and fused protoplasts by a selective screening method that relies on differential growth of the hybrid on defined culture media. The biochemical and morphological characteristics of the somatically produced hybrid were identical to those of the sexually produced amphiploid.