Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

The effect of diffusion and convection on the rate of transfer of solutes across an interface

The transfer of substances across the interface between water and a membrane or between water and a solvent occurs in series with transport up to and away from the interface. These processes have been difficult to resolve. Recently D. M. Miller (Biochim Biophys Acta 856: 27–35, 1986) has used a moving drop technique to measure the rates of transfer of short-chain alcohols and tritiated water between water andn-octanol. This technique produces equivalent unstirred layers which are less than about 10 m thick. Based on the trends in the observed rates of phase transfer, he proposes that the transfer is limited by the actual interfacial step. If so, water-oil interfacial transfer would be sufficiently slow to limit the rate of permeation of lipid membranes by these substances. It is shown here that the observed rates of phase transfer can be explained quantitatively if they are limited by convection or by diffusion across the combination of 5–10 m unstirred layers both inside and outside the moving drops. For water, comparison of the observed rates with the rate of evaporation from a clean surface, suggests that the interfacial step at the water-octanol interface is not rate-limiting.     

Tn1545: a conjugative shuttle transposon

Summary Tn1545, from Streptococcus pneumoniae BM4200, confers resistance to kanamycin (aphA-3), erythromycin (ermAM) and tetracycline (tetM). The 25.3 kb element is self-transferable to various Gram-positive bacterial genera where it transposes. Tn1545 was cloned in its entirety in the recombination deficient Escherichia coli HB101 where it was unstable. The three resistance genes aphA-3, ermAM and tetM were expressed but were not transferable to other E. coli cells. Tn1545 transposed from the hybrid plasmid to multiple sites of the chromosome of its new host. The element re-transposed, at a frequency of 5×10^-9, from the chromosome to various sites of a conjugative plasmid where it could be lost by apparently clean excision. The element transformed and transposed to the chromosome of Bacillus subtilis. The properties of the conjugative shuttle transposon Tn1545 may account for the recent emergence of genes from Gram-positive bacteria in Gramnegative organisms.     

Delayed inducible resistance against a leaf-chewing insect in four deciduous tree species

Summary Both mechanical damage to mountain birch foliage and rearing of moth larvae on the trees reduced the growth of Epirrita autumnata larvae reared on these trees in the following year. The effects of physical damage and some other cues from insects were additive. On bird cherry the performance of Epirrita larvae was equal on untreated trees and on trees artificially defoliated in the previous year, but larval growth was reduced on previously insect-damaged branches. With mountain ash just physical damage per se reduced the performance of Epirrita larvae. On Salix phylicifolia there were no significant differences in the growth or survival of Epirrita on untreated control bushes and on bushes with partial larval damage during the previous year. Among untreated control trees the growth and survivorship of Epirrita were higher on fast-growing willow and bird cherry than on the slow-growing mountain birch. Mountain birch and mountain ash, the two deciduous tree species adapted to nutrient-poor soils, showed delayed inducible resistance triggered by defoliation (artificial or insect-made). This supports the hypothesis that delayed inducible resistance may be a passive response due to nutrient-stress caused by defoliation. On the other hand, the additional increase in the resistance of mountain birch triggered by specific cues from insects suggests that this response may be an evolved defense against leaf-eating insects.     

The effect of simulated acid rain on performance of the aphid Euceraphis betulae (Koch) on silver birch

Summary The effect of artificial acid rain on the reproduction and survival of the aphid Euceraphis betulae on silver birch was studied in Turku, southern Finland. Eight bioassays were done during 1984–1986. In four of the bioassays the aphids produced 40% to over 100% more progeny on birches watered with dilute sulphuric acid (pH 3.5) than on control trees. In four other cases the performance of aphids did not differ between the treatments. An index of aphid reproduction pooled over the whole study was significantly higher on acid-treated than on control birches. The reproduction of aphids on acid-treated birches was enhanced when precipitation was below long term average, suggesting an interaction between the stress caused by acid treatment and dry periods.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

Summary In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H⁺-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu⁺ and Pma⁺ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5 end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.Publication n^o 2456 from the Biology Directorate of the Commission of European Communities     

Plant-water relations in wheat as influenced by root pruning

Summary A greenhouse study in which 24, 54 and 71 per cent roots of wheat (Triticum aestivum L.) were pruned on the 73rd day from the date of planting (anthesis stage) showed that during a 7-day period following root pruning, total transpiration and leaf water potential were significantly lower (P=0.05) and the stomatal resistance was significantly higher (P=0.05) where 54 and 71 per cent roots were pruned, as compared to no root pruning or 24 per cent root pruning. The leaf relative water content, however, showed no significant differences. Thus about one-fourth root sytem could be reduced without adversely affecting the plant-water status.     

Isolation of tomato cell lines with altered response to Fusarium cell wall components

Summary To obtain Tomato cell lines with an altered capacity to respond to heat-released cell wall components (elicitor) of a tomato pathogen (Fusarium oxysporum f. sp. lycopersici), positive and negative selection experiments, using BUdR enrichment techniques, were carried out on suspension cultures of the susceptible, low phytoalexin producer cultivar Red River. Both high and low phytoalexin producing clones were isolated. Further tests demonstrated that not all phytoalexin-producing clones were more susceptible to the elicitor toxic effect, and that they were altered also in the speed of response to fungal cell wall components. Cells selected with Fusarium elicitor showed the same behaviour when challenged by Phytophthora infestans elicitor, thus suggesting in this case lack of specificity. The results are finally discussed with a view to using the technique both as a tool to study the genetics and physiology of hostparasite interactions and as a possible new method for the selection of pathogen resistant genotypes.Paper no. 1224 IPRA-CNR; research supported by an EEC-BAP contract     

Inheritance of resistance to potato y viruses in Phaseolus vulgaris L

Summary Resistance to watermelon mosaic virus-2 in Phaseolus vulgaris L. is conferred by two distinct dominant alleles at independent loci. Based on segregation data one locus is designated Wmv, the other, Hsw. The dominant allele Wmv from cv. Great Northern 1140 prevents systemic spread of the virus but viral replication occurs in inoculated tissue. In contrast, Hsw confers both local and systemic resistance to WMV-2 below 30C. At higher temperatures, plants that carry this allele in the absence of modifying or epistatic factors develop systemic veinal necrosis upon inoculation with the virus that results in rapid death. Patho-type specificity has not been demonstrated for either allele; both factors confer resistance to every isolate tested. A temperature-sensitive shift in epistasis is apparent between dominant alleles at these loci. Because Hsw is very tightly linked if not identical to the following genes for hypersensitivity to potyviruses I, (bean common mosaic virus), Bcm, (blackeye cowpea mosaic virus), Cam, (cowpea aphid-borne mosaic virus) and Hss (soybean mosaic virus), parental, reciprocal dihybrid F₁ populations, and selected F₃ families were inoculated with each of these viruses and held at 35 C. F₁ populations developed vascular necrosis completely or primarily limited to inoculated tissue, while F₃ families from WMV-2-susceptible segregates were uniformly susceptible to these viruses. The relationship between Hsw, Wmv and other genes for potyvirus resistance suggest patterns in the evolution of resistance and viral pathogenicity. Characterization of the resistance spectrum associated with each factor provides an additional criterion to distinguish genes for plant virus resistance.