The deliberate release of genetically engineered microorganisms requires a thorough characterization of the microbes in question. For the two bioluminescent Rhizobium meliloti strains, L1 and L33 [Selbitschka et al. (1992) Mol Ecol 1:9-19; Selbitschka et al. (1995) FEMS Microbiol Ecol 16:223-232], designated for field release, the sites of genetic modifications in the chromosomes were sequenced from amplified genomic DNA. This indicated no unexpected alterations in the nucleotide sequence. The bioluminescent phenotype was stably inherited over more than 100 generations in liquid cultures. The presence of the luciferase gene in both strains did not have secondary effects on a variety of metabolic pathways as assessed by the Biolog GN system. A specific polymerase chain reaction amplification, based on the chromosomal insertion site of the luc cassette, allowed the discrimination between the two strains and thus simplifies monitoring. The RecA-deficient strain L1 showed a strongly (more than 90%) reduced ability to nodulate alfalfa in competition with its parent strain R. meliloti 2011 and its RecA+ counterpart L33.
The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set-up was designed for investigating the mechanisms of seed coat permeation, which allows steady-state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water-filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.
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