Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis.

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 microM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 microM p-nitrophenol and mineralized about 60% of 720 microM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.

A PCR-based method for detection of potato pathogen, Helminthosporium solani, in silver scurf infected tuber tissue and soils.

Silver scurf caused by Helminthosporium solani causes significant economic losses in table stock, seed and processing potatoes. Specific polymerase chain reaction (PCR) primers, Hs1F1/Hs2R1, from H. solani were used for the amplification of a 447-bp product from 20 tissue samples and 54 single spore H. solani isolates, from eastern Canada (27 isolates), western Canada (13 isolates) and North Dakota in USA (14 isolates), but not from other potato fungal pathogens. In addition to PCR analysis, all 54 isolates were studied using conventional detection methods, visual disease symptoms and/or colony morphology and microscopic examination of the morphology of conidiophores and conidia. The PCR assay successfully detected H. solani and the PCR results correlated well with assessments based on conventional techniques. The detection of H. solani by PCR (1 day) is rapid and offers an alternative to the time consuming conventional diagnostic techniques (4-5 weeks). Nested PCR assay was necessary for the detection of H. solani in soils and thus can provide a sensitive technique to study the epidemiology of silver scurf in soils.

Applications of the green fluorescent protein as a molecular marker in environmental microorganisms.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

Electrophoretic Karyotypes of Sclerotinia sclerotiorum.

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.

Bacterial survival and mineralization of p-nitrophenol in soil by green fluorescent protein-marked Moraxella sp. G21 encapsulated cells.

Moraxella sp. G21 cells marked with the green fluorescent protein (gfp) survived in kappa-carrageenan beads and as free cells for a month after inoculation into autoclaved soil and non-sterile soil contaminated with p-nitrophenol (PNP). Similar [U-(14)C]PNP mineralization values were produced by encapsulated Moraxella sp. G21 cells and as free cells (53 and 60% mineralization). There was no significant difference between cell survival and [U-(14)C]PNP mineralization activity in soil by the rifampicin-resistant Moraxella sp. mental strain and Moraxella sp. G21. The ability of encapsulated Moraxella sp. G21 cells to survive, retain their green fluorescence and mineralize [U-(14)C]PNP suggests that the GFP-marked strain encapsulated in kappa-carrageenan may be useful for bioremediation of toxic chemicals in soil.

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis.

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.

Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.