<Emphasis Type="Italic">Lecophagus vermicola</Emphasis> sp. nov., a nematophagous hyphomycete with an unusual hunting strategy

Lecophagus vermicola sp. nov. is described and illustrated as a predacious (carnivorous) hyphomycete living in bark fissures of living trees of Platanus and other angiosperm and gymnosperm trees, recorded in Hungary, Luxembourg and France. The fungus captures nematodes unlike other Lecophagus species, which are predators of rotifers and tardigrades. The morphology of the sessile, adhesive knobs differ from all previously described species of the genus which form adhesive pegs. Molecular data confirms that the new species belongs to the Lecophagus clade but without matching existing sequences. The fungus captures victims with adhesive knobs and colonizes its prey with a mycelium of rather broad hyphae on which, again, adhesive knobs are formed which penetrate the cuticule of the victim. Clusters of colonized nematodes form a network utilized to capture more prey. The fungus lives in the xeric, ephemerally aquatic habitat of bark fissures of standing, living or dead, corticated trunks and branches. The genus Haptocara is compared, which has similar adhesive knobs capturing nematodes and similar broad hyphae, but for which no molecular data was available.     

Reproductive effort in invasive and non-invasive Rubus

We quantified the physiological costs and the total amount of resources allocated to reproduction in two closely related species of Rubus, one of which is invasive. These two species share several morphological and life-history characteristics and grow together in the Pacific Northwestern United States. Reproductive effort was manipulated in canes of both species by removing flower buds. The non-invasive species, R. ursinus, exhibited significantly greater water stress in the reproductive canes, as indicated by lower leaf water potential (O) and reduced stomatal conductance (g_s). This species also showed a reduction in leaf nitrogen concentration ([N]) associated with reproduction. Combined, these factors led to reduced photosynthesis (A) on a diurnal basis, lower water-use efficiency as inferred from '¹³C, and reduced photosynthetic capacity. All of these effects were more pronounced during the fruiting stage than in the flowering stage. The invasive species, R. discolor, showed no changes in water stress, [N], '¹³C, or A associated with reproduction. A model was used to estimate total gross photosynthesis (A_gross) for reproductive and non-reproductive canes of both species over cane lifetime. Reproduction was associated with a greater decline in A_gross for the non-invasive R. ursinus than for the invasive R. discolor. Although R. discolor allocated more resources directly to flowers and fruit than R. ursinus, the invasive species had significantly lower reproductive effort, or total amount of resources diverted from vegetative activity to reproduction, than the non-invasive species. By minimizing the reduction of photosynthesis associated with reproduction, this invasive species may be able to minimize the trade-offs commonly associated with reproduction.     

Compound nesting of <Emphasis Type="Italic">Strumigenys</Emphasis> sp. (Myrmicinae) and <Emphasis Type="Italic">Diacamma</Emphasis> sp. (Ponerinae), and other nesting symbioses of myrmicine and ponerine ants in Southeast Asia

Summary. A new type of compound nest, inhabited by the small Strumigenys sp.1 (Myrmicinae) and the relatively big Diacamma sp.1 (Ponerinae), was found in Java, Indonesia. Field records as well as results of experimental studies were used to describe this association. The compound nests were relatively frequent in the research area: 75% of the examined Diacamma sp.1 nests were also inhabited by Strumigenys sp.1. Strumigenys sp.1 is specialized on nesting in small, excavated nest chambers, in close vicinity to Diacamma sp.1, while Diacamma sp.1 does not depend on Strumigenys sp.1. The rather small workers of this myrmicine species forage for food inside the Diacamma nests as well as outside, favouring mites and collembolans as prey, but principally not rejecting any other nitrogen source. The refuse pile of Diacamma sp.1 inside the nest houses small living invertebrates and insect parts, preyed upon by Strumigenys sp.1. Given the choice between larvae of Diacamma sp.1 and mites, Strumigenys sp.1 clearly preferred the mites. Thus, the association seems to be beneficial to Strumigenys sp.1, and does not harm Diacamma sp.1. We found two more similar compound nests: In Java, a different Strumigenys species was frequently found nesting within the nest of a Pachycondyla (Ponerinae) species. In Sabah, East Malaysia, we recorded for the first time a Pheidole (Myrmicinae) species nesting in chambers coming out of the walls of Diacamma nest chambers. Existing classifications for compound nest associations (i.e. xenobiosis, cleistobiosis etc.) are too restricted, because they were based on a few cases. Hence, we present a list of non-normative traits describing the various types of nest symbioses by ants.     

Structural and molecular analysis of self-incompatibility in almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis>)

. A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S₈) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.     

Plant diversity,herbivory and resistance of a plant community to invasion in Mediterranean annual communities

Several components of the diversity of plant communities, such as species richness, species composition, number of functional groups and functional composition, have been shown to directly affect the performance of exotic species. Exotics can also be affected by herbivores of the native plant community. However, these two possible mechanisms limiting invasion have never been investigated together. The aim of this study was to investigate the relationships between plant diversity, herbivory and performance of two annual exotics, Conyza bonariensis and C. canadensis, in Mediterranean annual communities. We wanted to test whether herbivory of these exotics was influenced either by species richness, functional-group richness or functional-group composition. We also studied the relationship between herbivory on the exotic species and their performance. Herbivory increased with increasing species and functional-group richness for both Conyza species. These patterns are interpreted as reflecting a greater number of available herbivore niches in a richer, more complex, plant community. The identities of functional groups also affected Conyza herbivory, which decreased in the presence of Asteraceae or Fabaceae and increased in the presence of Poaceae. Increasing herbivory had consequences for vegetative and demographic parameters of both invasive species: survival, final biomass and net fecundity decreased with increasing herbivory, leading to a loss of reproductive capacity. We conclude that communities characterised by a high number of grass species instead of Asteraceae or Fabaceae may be more resistant to invasion by the two Conyza species, in part due to predation by native herbivores.     

Influence of a dominant macrophyte, <Emphasis Type="Italic">Juncus effusus</Emphasis>, on wetland plant species richness,diversity, and community composition

Few studies have experimentally investigated the influence of competition for light on structure and composition of wetland vascular plant communities, despite the well-documented high productivity in such systems. Influences of the dominant emergent wetland plant Juncus effusus on the surrounding macrophyte community were evaluated in a shallow freshwater wetland through two consecutive growing seasons. Permanent transects were constructed along diagonals of randomly oriented 1 m² plots centered around isolated, colonizing J. effusus tussocks. Percent areal cover was measured for each species or identifiable taxon group in 400 cm² sub-plots centered 10, 20, 30, and 40 cm from the tussock edge, to evaluate species richness, diversity, and dominance indices. Observational studies during the first growing season indicated that plots having larger, more heavily shading tussocks yielded significantly lower cover and species richness in the surrounding plant community than less shaded plots. Shading by J. effusus was reduced experimentally during the second growing season by holding culms in a vertical position in half of the J. effusus-occupied study plots in order to assess directly the influence of shading by J. effusus. Manipulated plots became more diverse and species-rich and developed higher total percent areal cover. Within shaded reference plots, reductions in cover, richness, and diversity were correlated with intensity of shading; each of these measures was lowest in sub-plots centered 10 cm from the tussock, where measured light reduction was highest. Neighborhood analyses of biomass, species composition, and light reduction indicated that individual species biomass varied with distance from J. effusus tussocks and shading intensity, an indication of the potential for shifts in community composition as populations of this dominant macrophyte expand to fill a wetland area. A mathematical model is presented to illustrate shading effects of J. effusus as a population grows from isolated, colonizing tussocks to an interacting system of tussocks in an established population of this dominant macrophyte.     

Parasites of the Antarctic rattail <Emphasis Type="Italic">Macrourus whitsoni</Emphasis> (Regan, 1913) (Macrouridae,Gadiformes)

A total of 386 Macrourus whitsoni from Antarctic waters were examined for ecto- and endoparasites. Sixty-five M. whitsoni collected near Halley Bay (Weddell Sea) and 321 specimens from the continental slope off King George Island (South Shetland Islands) were studied for sphyriid copepods directly after being caught. A subsample of 25 specimens from the Weddell Sea and of 9 specimens from King George Island were studied for the presence of other metazoan parasites. Twenty-two species were found, including one myxozoan, six digeneans, one monogenean, three cestodes, seven nematodes, one acanthocephalan and three crustacean species/taxa. While Auerbachia monstrosa and Capillaria sp. are reported for the first time from around the Antarctic, the other parasites have been recorded earlier in the Southern Ocean. Many parasite species found have a wide zoogeographical range and a low host-specificity. The parasite fauna of M. whitsoni revealed several similarities with its congeners M. carinatus and M. holotrachys from Antarctic and sub-Antarctic waters. This can be explained by a wide host range of many macrourid deep-sea parasites, together with an overlap in distribution patterns of their hosts. Other supporting factors are host migration and a close phylogenetic relationship between the hosts, which enable the parasites to infest all three macrourids. Eight new host and 14 new locality records are established.     

In vivo study of microsclere formation in sponges of the genus <Emphasis Type="Italic">Mycale</Emphasis> (Demospongiae,Poecilosclerida)

The process of microsclere secretion was examined in vivo through glass coverslip implants in three species of the genus Mycale from São Sebastião channel, southeastern Brazil: Mycale (Aegogropila) angulosa, Mycale (Arenochalina) laxissima, and Mycale (Carmia) microsigmatosa. All three species adhered well to coverslips and developed normally through at least 2 weeks. Similar experiments with different species (Cinachyrella alloclada, Amphimedon viridis, Haliclona melana, and Aplysina caissara) were also successful with one exception (the cartilaginous Chondrilla nucula), indicating that the method can be applied to most demosponges. Microsclerocyte size varied according to the type of microsclere secreted, but all were elongated to fusiform and had small, anucleolated nuclei. Spicules were transported by microsclerocytes alone, without any other cell type ("helper cells") involved. Secretion of a microsclere was performed by a single sclerocyte. Although some axial filaments were found free in the mesohyl, all microsclere secretion in these animals was fully intracellular. Normal axial filaments were observed in most types of microscleres of the Mycale species (sigmas, toxas, and microxeas). Timed observations of sclerocytes suggest that immature spicules with the aspect of short straight rods with thick ends might be the precursors of the anisochelae. Observed differences in the size versus number of toxa secreted may indicate either the presence of two distinct subpopulations of toxa-producing microsclerocytes or that the initial number of axial filaments at the beginning of silica deposition may determine the final size of the spicules. Although other microscleres such as sigmas and chelae are secreted in a one cell-one spicule basis, several toxas and microxeas can be secreted simultaneously in a single cell.     

Patterns of the <Emphasis Type="Italic">Crematogaster-Macaranga</Emphasis> association: The ant partner makes the difference

Summary. One of the most species-rich ant-plant mutualisms worldwide is the palaeotropical Crematogaster-Macaranga system. Although the biogeography and ecology of both partners have been extensively studied, little is known about the temporal structuring and the dynamics of the association. In this study we compared life-history traits of the specific Crematogaster (Decacrema) partner-ants and followed the development of ant colonies on eight different Macaranga host plant species, from colony founding on saplings to adult trees in a snapshot fashion. We found differences in the onset of alate production, queen number and mode of colony founding in the ant species and examined the consequences of these differences for the mutualism with the host plant. The lifespan of some host plants and their specific ant partners seemed to be well matched whereas on others we found an ontogenetic succession of specific partner ants. The partner ants of saplings or young plants often differed from specific partner ants found on larger trees of the same species. Not all specific Crematogaster species can re-colonize the crown region of adult trees, thus facilitating a change of ant species. Therefore lifespan of the ant colony as well as colony founding behaviour of the different partner ant species are important for these ontogenetic changes. The lifespan of a colony of two species can be prolonged via secondary polygyny. For the first time, also primary polygyny (pleometrosis) is reported from this myrmecophytic system.     

Trichomes and photosynthetic pigment composition changes: responses of <Emphasis Type="Italic">Quercus ilex </Emphasis>subsp. <Emphasis Type="Italic">ballota</Emphasis> (Desf.) Samp. and<Emphasis Type="Italic"> Quercus coccifera</Emphasis> L. to Mediterranean stress conditions

Sun and shade leaves of two Mediterranean Quercus species, Quercus ilex subsp. ballota (Desf.) Samp. and Quercus coccifera L., were compared by measuring leaf optical properties, photosynthetic pigment composition and photosystem II efficiency. The presence of trichomes in the adaxial (upper) leaf surface of Q. ilex subsp. ballota seems to constitute an important morphological mechanism that allows this species to maintain a good photosystem II efficiency during the summer. Q. coccifera has almost no trichomes and seems instead to develop other physiological responses, including a smaller light-harvesting antenna size, higher concentrations of violaxanthin cycle pigments and a higher (zeaxanthin + antheraxanthin)/(violaxanthin + antheraxanthin + zeaxanthin) ratio. Q. coccifera was not able to maintain a good photosystem II efficiency up to the end of the summer. In Q. ilex subsp. ballota leaves, natural loss or mechanical removal of adaxial-face leaf trichomes induced short-term decreases in photosystem II efficiency. These changes were accompanied by de-epoxidation of violaxanthin cycle pigments, suggesting that the absence of trichomes would trigger physiological responses in this species. Our data have revealed different patterns of response of Q. ilex subsp. ballota and Q. coccifera facing the stress conditions prevailing in the Mediterranean area.     

Genotypic variation in the response of two perennial grass species to elevated carbon dioxide

Shoot and reproductive biomass of genotypes of Bromus erectus and Dactylis glomerata grown in competition at ambient and elevated CO₂ were examined for 2 consecutive years in order to test whether genetic variation in those traits exists and whether it is maintained over time. At the species level, a positive CO₂ response of shoot biomass of both species was only found in the first year of treatment. At the genotype level, no significant CO₂2genotype interaction was found at any single harvest either for vegetative or reproductive biomass of either species. Analysis over time, however, indicated that there is a potential for evolutionary adaptation only for D. glomerata: (1) repeated measures ANOVA detected a marginally significant CO₂2genotype2time interaction for shoot biomass, because the range of the genotypes CO₂ response increased over time; (2) genotypes that displayed the highest response during the first year under elevated CO₂ also showed the highest response the second year. Null (B. erectus) or weak (D. glomerata) selective potentials of elevated CO₂ were detected in this experiment, but short time series could underestimate this potential with perennial species.     

Efficient Agrobacterium-mediated transformation of <Emphasis Type="Italic">Lotus japonicus</Emphasis> with reliable antibiotic selection

We have developed a new procedure for transforming a model legume, Lotus japonicus, that yields transformed plants from transverse cotyledon segments without contamination from the presence of non-transformants that survived the antibiotic selection. L. japonicus was transformed with the HPT gene as a selectable marker and the GUS reporter gene, both of which were driven by cauliflower mosaic virus 35S promoter. The efficacy of selection with hygromycin was tested using the assay of GUS activity in putative transformants. The integration of the GUS gene was also confirmed by polymerase chain reaction analysis of the genomic DNA. The integrated T-DNA was stable and inherited as a dominant trait. This procedure may have potential effectiveness and application in large-scale transformation for insertional mutagenesis or gene tagging.     

Nutrient partitioning and storage in arid-zone forests of the mangroves Rhizophora <Emphasis Type="Italic">stylosa</Emphasis> and <Emphasis Type="Italic">Avicennia</Emphasis><Emphasis Type="Italic">marina</Emphasis>

Mangrove partitioning and storage of macronutrients and trace metals were examined in different arid coastal settings of Western Australia. Total living biomass in three Rhizophora stylosa forests, which ranged from 233 to 289 t DW ha^-1, was significantly greater than biomass in three Avicennia marina forests (range: 79-155 t DW ha^-1). Although prop roots and stems were the largest single tree components for R. stylosa and A. marina, respectively, most nutrients were stored in leaves and living roots of both species. However, only a small fraction of the total nutrient pool was vested in tree biomass; the vast bulk was in soils. A large below-ground pool of dead fine roots was identified at all stands, equivalent to 36-88% DW of total living tree biomass. The amount of Ca, S, Cl, Na, Si, Fe, Mn, Zn, B, Mo and Cu vested in dead roots of both species was greater than in the total living tree biomass. The proportion of Fe and S vested in live and dead roots was exceptionally large, consistent with previous evidence of metal plaques on mangrove roots. Sulphur, iron and zinc in dead roots of both species constituted the bulk of these metals. R. stylosa trees preferentially accumulated more Mg, S, Cl, Na, Si, Fe, Mn, B and Mo than A. marina trees. Proportionally greater storage of P, N, Ca, K, Cu and Zn occurred in two of the three A. marina forests. Foliar concentrations of Mg, S, Mn, B and Mo in mangrove leaves were at the high end of the range reported for other tropical trees, but other elemental concentrations were at the low or mid-range. Nitrogen limitation in these forests is implied by a positive correlation between total tree N and net canopy production and by a lower percentage of ecosystem N in tree biomass as compared with other forests. Unlike terrestrial forests where a large proportion of nutrient capital is vested in floor litter, most elements in these mangrove forests are stored in dead roots. A large reservoir of dead roots below the forest floor may serve as a conservation mechanism, particularly in such arid oligotrophic environments.     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species with <Emphasis Type="Italic">Pesotum</Emphasis> anamorphs associated with bark beetles infesting <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Three species of Ophiostoma possessing Pesotum anamorphs isolated from bark beetles and their galleries infesting Abies species in Nikko, Japan, are described as new species. Ophiostoma nikkoense is characterized by brush-shaped synnemata producing long septate clavate conidia, perithecia with neck, and allantoid ascospores. Ophiostoma microcarpum has smaller perithecia with hyphoid ostiolar hyphae on the neck, and the ascospores are cylindrical or ossiform in side and face views. Ophiostoma abieticola has perithecia without ostiolar hyphae on the neck and produces orange-section-shaped or reniform ascospores.Contribution no. 187, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Ganoderma carocalcareus sp. nov., with crumbly-friable context parasite to saprobe on Anthocleista nobilis and its phylogenetic relationship in G. resinaceum group

A new species Ganoderma carocalcareus (Basidiomycota, Ganodermataceae) was collected on living trunk and dead stumps of Anthocleista nobilis (Gentianaceae) in waterlogged swamps in the Mbalmayo Forest Reserve, Cameroon, and identified on the basis of morphology and phylogenetic analyses inferred from mitochondrial small subunit (mtSSU) and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences. Distinct phenotypic characteristics of the new species are dimorphism of basidiomata and variability in context structure and texture over developmental stages. The young basidiomata is ungulate to punk-shaped with context composed of vegetative hyphae attended by scattered, orbicular, smooth, thick-walled chlamydospores, and the mature basidiomata is cushion- to bracket-like with context entirely consisting of chlamydospores masses. This ontogeny intimates the origin of chlamydospores, for which the biogenesis correlates the vanishing of vegetative hyphae throughout the basidiomata maturation. Morphological comparison included Tomophagus colossus (=G. colossus), G. subamboinense and G. weberianum, the known Ganodermataceae species producing chlamydospores and or gasterospores in basidiomata tissues, and G. resinaceum, the closest species with regard to morphology. It followed that G. carocalcareus could not be assigned to these or any other known Ganoderma species. Analyses of mtSSU and ITS rDNA sequence data resolved G. carocalcareus in the G. resinaceum group as a distinct species, but being a close relative of both G. subamboinense and G. weberianum.     

<Emphasis Type="Italic">Campylonotus arntzianus</Emphasis>, a new species of the Campylonotidae (Crustacea: Decapoda: Caridea) from the Scotia Sea (Antarctica)

Two specimens of Campylonotus arntzianus sp. nov. were caught in the Antarctic Scotia Sea off Saunders Island (57°40.31'S, 26°27.81'W) using an Agassiz trawl at one station (depth: 475-589 m). The new species described here is the fifth representative of the monogeneric family Campylonotidae, and the first of the family south of the Antarctic Convergence. Campylonotus arntzianus sp. nov. is a shrimp of about 5 cm in total length. Due to similarities in adult morphology, C. arntzianus sp. nov. seems to be closely related to C. capensis, a deep-sea species from the Southern Atlantic Ocean. A simple key for the species identification of the Campylonotidae is provided.     

Winter diet and foraging range of gentoo penguins (<Emphasis Type="Italic">Pygoscelis papua</Emphasis>) from Kidney Cove,Falkland Islands

The winter diet and foraging range of gentoo penguins, Pygoscelis papua, were studied at Kidney Cove, Falkland Islands. The mean wet mass of the 56 stomach-content samples collected from May to October was 32.7ᇃ.4 g. The diet consisted generally of cephalopods, crustaceans and fish, as well as two other molluscs. Among the six cephalopod species identified, the commercially fished Patagonian squid, Loligo gahi, had the highest abundance and was also the main prey by reconstituted mass (53% of the total reconstituted mass). Lobster krill, Munida gregaria, one of five species of crustaceans, was the most abundant prey species by frequency of occurrence and by number (68% and 60%, respectively). Rock cod, Patagonotothen ramsayi, accounted for the majority of the fish diet with 34% of the total reconstituted mass. Most prey species identified in the winter diet were also abundant diet components during the breeding season. However, the known biology of the prey species and their rate of digestion indicated that, in winter, adults may forage further offshore than during the breeding season. This assumption was supported by the results obtained from two birds satellite-tracked during the study period. Both birds remained mainly in inshore waters and returned frequently ashore, but one penguin foraged up to 276 km from the coast. The differences in the foraging behaviour of the two birds were reflected in significant differences with regard to time spent underwater, distance travelled per day and calculated travelling speed. Furthermore, the progress of cohorts of L. gahi over the winter is consistent with results from life-cycle studies in this region and suggests that birds have been foraging in the feeding grounds of L. gahi.     

Pruning of host plant neighbours as defence against enemy ant invasions: <Emphasis Type="Italic">Crematogaster </Emphasis>ant partners of <Emphasis Type="Italic">Macaranga</Emphasis> protected by "wax barriers" prune less than their congeners

Many ant partners of tropical ant-plants prune the leaves and shoot tips of other plants growing around their hosts. According to the hypothesis proposed by Davidson et al. (Ecology 69:801-808), this specialized behaviour not only protects the host plants against overgrowth, but it also conveys a direct benefit to the ant colony as it removes contact points to the neighbouring vegetation where invasions of enemy ants could occur. Here we test this hypothesis by comparing pruning intensity in five closely related Crematogaster (subgenus Decacrema) plant-ant species (and one species of Technomyrmex) that differ in their exposure to competition by other ants. Pruning intensity was quantified by measuring the area loss of paper tape pieces wrapped around the stems of Macaranga host plants. All Crematogaster (Decacrema) ants tested but not Technomyrmex sp. pruned, but the intensity of the behaviour varied strongly between and within species. Pruning was significantly weaker in the three tested Crematogaster species inhabiting Macaranga host plants with a slippery, waxy stem surface, which functions as a mechanical barrier protecting the specific ant partners against generalist competitors. Pruning was generally stronger on more densely ant-populated trees. Even though the number of ants per twig length was lower in associations of ants with glaucous Macaranga hosts, only part of the variation of pruning activity could be explained by "ant density". When corrected for ant density, "wax-running" Crematogaster (Decacrema) ants still pruned more weakly than their congeners inhabiting non-glaucous Macaranga hosts. Pruning is obviously most important when an ant-plant is potentially accessible to intruders, but less necessary when the ant colony is isolated by a protective wax barrier. Our results support the hypothesis that "selfish" defence against invasions is the major selective pressure that has led to the development and maintenance of pruning behaviour in weakly competitive plant-ants.     

Tissue-culture enhanced transposition of the maize transposable element <Emphasis Type="Italic">Dissociation</Emphasis> in <Emphasis Type="Italic">Brassica oleracea</Emphasis> var. '<Emphasis Type="Italic">Italica</Emphasis>'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli (Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct (Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.