Survival selection on escape performance and its underlying phenotypic traits: a case of many-to-one mapping.

Selection often operates not directly on phenotypic traits but on performance which is important as several traits may contribute to a single performance measure (many-to-one mapping). Although largely ignored in the context of selection, this asks for studies that link all relevant phenotypes with performance and fitness. In an enclosure experiment, we studied links between phenotypic traits, swimming performance and survival in two Enallagma damselflies. Predatory dragonflies imposed survival selection for increased swimming propensity and speed only in E. annexum; probably E. aspersum was buffered by the former species' presence. Accordingly, more circular caudal lamellae, structures involved in generating thrust while swimming, were selected for only in E. annexum. Other phenotypic traits that contributed to swimming speed were apparently not under selection, probably because of many-to-one mapping (functional redundancy). Our results indicate that not only the phenotypic distributions of syntopic prey organisms but also many-to-one mapping should be considered when documenting phenotype-performance-fitness relationships.

Adaptive sex-specific life history plasticity to temperature and photoperiod in a damselfly.

We investigated four predictions about how temperature, photoperiod and sex affect the life history plasticity and foraging activity of a damselfly. (i) As predicted, increased temperatures increased foraging activity and growth rates, but in contrast with the prediction, late photoperiod (high time stress) did not affect foraging activity and growth rate. (ii) Unexpectedly, the increase in growth rate at increasing temperatures was not larger under high time stress. (iii) As predicted, age and size at emergence decreased at higher temperatures and at the late photoperiod. Temperature-induced life history shifts were direct or the result of behavioural growth mediation depending on the temperature range. Photoperiod-induced life history shifts were direct. (iv) As predicted, males emerged before females but at a smaller size. The degree of sexual size dimorphism was influenced by the joint effects of temperature and photoperiod. We could only detect genetic variation in size plasticity to photoperiod. The match between the sex-specific life history responses to temperature and photoperiod and predictions by relevant optimality models suggests adaptive life history plasticity to these variables.

An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems.

In Arabidopsis, mutation at PFL causes pointed first leaves, reduced fresh weight and growth retardation. We have cloned the wild-type PFL gene by T-DNA tagging, and demonstrate that it complements the mutant phenotype. PFL codes for ribosomal protein S18, based on the high homology with rat S18 and on purification of S18-equivalent peptides from plant ribosomes. pfl represents the first mutation in eukaryotic S18 proteins or their S13 prokaryotic counterparts, involved in translation initiation. Arabidopsis contains three S18 gene copies dispersed in the genetic map; they are all transcribed and code for completely identical proteins. No transcript is detected from the mutated gene, S18A. The activity of the S18A promoter is restricted to meristems, with a markedly high expression at the embryonic heart stage, and to wounding sites. This means that plants activate an extra copy of this ribosomal protein gene in tissues with cell division activity. We postulate that in meristematic tissues plants use transcriptional control to synthesize extra ribosomes to increase translational efficiency. In analogy with this, an additional, developmentally regulated gene copy might be expected for all ribosomal proteins.

RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction.

A nonradioactive RNA-RNA in situ hybridization method using digoxigenin-labeled probes is described. The visualization of hybrids is done using the indoxyl-nitro blue tetrazolium alkaline phosphatase reaction. The addition of polyvinyl alcohols of high molecular weight (40-100 kDa) to the reaction medium enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a 20-fold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.

Two T-DNA's co-transformed intoBrassica napus by a doubleAgrobacterium tumefaciens infection are mainly integrated at the same locus.

Hypocotyl explants of threeBrassica napus varieties were infected with two nopaline typeAgrobacterium strains each carrying a distinct disarmed T-DNA containing different selectable markers. Selection was done for only one of the markers, after which the regenerated plants were screened for the presence of the second marker. High co-transformation frequencies of both T-DNA's were obtained (39%-85% of the transformants). Where the two T-DNA's were integrated linked, they were usually present in an inverted orientation relative to each other; in all of the cases observed the two right borders were adjacent. Tandem orientations occurred less frequently. The T-DNA's were mainly integrated as intact copies and deletions did not often occur. The co-transformation system described favors a genetically linked integration of the two T-DNA's (78%), although in a single transformed plant both linked and unlinked copies of both T-DNA's may be present.

Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones.

Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25 degrees C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO(3) (-)/NH(4) (+) ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants.

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO(3) was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens.

Leaves of the in vitro grown potato cultivars 'Bintje', 'Berolina', 'Desiree', and 'Russet Burbank' were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For 'Russet Burbank', it was necessary to include AgNO3 in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants.

Chloroplast transformation by Agrobacterium tumefaciens.

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 mug/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the Cm phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti-plasmid vectors can be used to introduce genes in chloroplasts.

Expression of foreign genes in regenerated plants and in their progeny.

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km and Cm). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.

IS-like element IS8 in RP4 plasmid and its involvement in cointegration.

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi::RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.