The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

Flower Visitation by Bats in Cloud Forests of Western Ecuador1

The importance of bat pollination has been demonstrated for many plant species. Yet this mutualism has rarely been studied on a community–wide level. In this paper we present results of a yearlong study of a bat–flower community in cloud forests on the western slopes of the Ecuadoran Andes. Of eight plant–visiting bat species caught, only Anoura caudifera and A. geoffroyi were carrying pollen. These species of Anoura supplement their diets with insects. Unlike glossophagines in other environments, however, which switch completely to a frugivorous or insectivorous diet during certain seasons, they are nectarivorous year–round and were never found with seeds or fruit pulp in their feces. Of the 13 morphotypes of pollen carried by the bats, 11 were identified to genus and 7 to species. Floral characteristics of all of these plants fit the traditional chiropterophilous syndrome well. Our study represents the first direct evidence of bat pollination for those plants identified to species, including four species of Burmeistera (Campanulaceae), as well as the first record of bat pollination for a plant of the genus Meriania (Melastomataceae). While overlap in the diets of the two Anoura was high, significant differences in visitation frequencies to particular plant species were detected. The larger bat species (A. geoffroyi) preferred large flowers, whereas the smaller species (A. caudifera) preferred small flowers.     

Contrasting effects of fire frequency on plant traits of three dominant perennial herbs from Chaco Serrano

Fire frequencies are currently increasing in many regions across the world as a result of anthropic activities, affecting ecological processes and plant population dynamics. Fire can generate important changes in soil properties, altering nutrient dynamics and thereby plant growth. Here we analyse fire frequency effects on soil quality and plant traits of three native perennial herbaceous plants (Cologania broussonetii, Desmodium uncinatum and Rhynchosia edulis; Fabaceae) with the capacity for biological N₂ fixation that resprouts and are abundant after fire in Chaco Serrano forests. Based on 22‐year fire history, we assessed plant traits in sites with low and high fire frequencies along with unburned scenarios. We found significantly lower water content, nitrates and electrical conductivity in frequently burned soils. As a result, the three species showed consistently lower leaf area and specific leaf area in both fire frequencies, implying lower growth rates in comparison to unburned sites. However, total leaf biomass was not affected by fire, leaf phosphorus concentration was higher in R. edulis in high fire frequency and leaf N concentration was twice as large in plants growing in sites of high fire frequency in C. broussonetii and R. edulis. Such an increase in N and phosphorus concentrations is likely a result of both their conservative use of resources and their biological N₂ fixation capacity. To our knowledge, this is the first record of such contrasting fire effects observed consistently in three co‐occurring species: while plant growth decreased with fire frequency, leaf nutritional traits remain unchanged or increased in frequently burned sites. Quality‐depleted and drier soils that result from increased fire frequencies may not only affect trait variation at the intraspecific level but can also drive to a homogenization of the plant community, selecting species with particular combinations of morphological and functional traits.     

Mycorrhizal colonization status of plant species established in an exposed area following the 2000 eruption of Mt. Usu, Hokkaido, Japan

We investigated the ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) colonization status of plant seedlings that established in areas devastated by the eruption in March 2000 of Mt. Usu, Hokkaido, Japan. In 2005, we estimated the mycorrhizal colonization ratios and frequencies in seedlings of twelve herbaceous and seven woody plant species. Although arbuscular mycorrhizas were found to colonize Equisetum arvense and Polygonum sachalinense, they were presented at very low frequencies and colonization ratios. Other herbaceous plants exhibited higher frequencies of AM associations and either AM and/or ECM fungal associations were observed in all of the woody plant seedlings investigated. The dominant woody plant species (Populus maximowiczii, Salix sachalinensis and Salix hultenii var. angustifolia) associated mainly with ECM fungi and exhibited variable associations with AM fungi. Mycorrhizal associations were common and significant events for plant establishment, even in the early stages of the revegetation process.     

Nonstructural carbohydrate allocation following different frequencies of simulated browsing in three semi-arid shrubs

Nonstructural carbohydrate allocation patterns in response to different frequencies of simulated browsing (leaf and twig removal) were studied in the following semi-arid shrubs: Osteospermum sinuatum, a dwarf deciduous shrub, Pteronia pallens, a dwarf evergreen shrub, and Ruschia spinosa, a dwarf leaf-succulent shrub. Simulated browsing at all frequencies resulted in the elevation, or had no effect, on total nonstructural carbohydrate (TNC) concentrations of O. sinuatum plant parts, and resulted in the decrease in TNC concentrations of R. spinosa plant parts. The responses of P. pallens were intermediate with elevations as well as declines in TNC concentrations of plant parts measured in response to various clipping frequencies. At the low frequency of simulated browsing (every 26 weeks) elevations in plant TNC content were measured in the two non-succulent shrubs O. sinuatum and P. pallens. It was concluded that the overcompensation with respect to TNC accumulation observed in the two non-succulent species represents one of the ways in which excess photosynthate is utilized by browsed shrubs with a limited regiowth potential. Simulated browsing was the least detrimental with respect to biomass production to the non-succulent O. sinuatum and P. pallens, and most injurious to the leaf-succulent shrub, R. spinosa. The observed TNC allocation patterns could not adequately explain the variation among species in the production of new growth and it was concluded that some factor(s) other than the carbon resource was limiting regrowth.     

Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics

In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.     

Trinucleotide’s quadruplet symmetries and natural symmetry law of DNA creation ensuing Chargaff’s second parity rule

For almost 50 years the conclusive explanation of Chargaff’s second parity rule (CSPR), the equality of frequencies of nucleotides A=T and C=G or the equality of direct and reverse complement trinucleotides in the same DNA strand, has not been determined yet. Here, we relate CSPR to the interstrand mirror symmetry in 20 symbolic quadruplets of trinucleotides (direct, reverse complement, complement, and reverse) mapped to double-stranded genome. The symmetries of Q-box corresponding to quadruplets can be obtained as a consequence of Watson–Crick base pairing and CSPR together. Alternatively, assuming Natural symmetry law for DNA creation that each trinucleotide in one strand of DNA must simultaneously appear also in the opposite strand automatically leads to Q-box direct-reverse mirror symmetry which in conjunction with Watson–Crick base pairing generates CSPR. We demonstrate quadruplet’s symmetries in chromosomes of wide range of organisms, from Escherichia coli to Neanderthal and human genomes, introducing novel quadruplet-frequency histograms and 3D-diagrams with combined interstrand frequencies. These “landscapes” are mutually similar in all mammals, including extinct Neanderthals, and somewhat different in most of older species. In human chromosomes 1–12, and X, Y the “landscapes” are almost identical and slightly different in the remaining smaller and telocentric chromosomes. Quadruplet frequencies could provide a new robust tool for characterization and classification of genomes and their evolutionary trajectories.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Identification of genic moss SSR markers and a comparative analysis of twenty-four algal and plant gene indices reveal species-specific rather than group-specific characteristics of microsatellites

Background  

The moss Physcomitrella patens is an emerging model in comparative plant science. At present, the Physcomitrella genome is sequenced at the Joint Genome Institute (USA). In this study we present our results on the development of expressed sequence tag-derived microsatellite markers for Physcomitrella patens, their classification and applicability as genetic markers on the intra- as well as on the interspecies level. We experienced severe restrictions to compare our results on Physcomitrella with earlier studies for other plant species due to varying microsatellite search criteria and a limited selection of analysed species. As a consequence, we performed a side by side analysis of expressed sequence tag-derived microsatellites among 24 plant species covering a broad phylogenetic range and present our results on the observed frequencies.     

Lichens toGunnera — with emphasis onAzolla

Summary N₂-fixing cyanobacteria occur in symbiotic associations with fungi (ascomycetes) as lichens and with a few green plants. The associated cyanobacterium is always a species ofNostoc orAnabaena. Only a small number of plant genera are involved but there is a remarkable range of host diversity. Associations occur with several bryophytes (e.g.Anthoceros, Blasia, Cavicularia), a pteridophyte (Azolla), cycads (nine genera includingMacrozamia andEncephalartos) and an angiosperm (Gunnera). Except forGunnera, where the cyanobacterium penetrates the plant cells, the cyanobacteria are extracellular with specialized morphological modifications and/or structures of the host plant organs providing an environment which facilitates interaction with the prokaryote.Salient aspects of current knowledge pertaining to the establishment, perpetuation, and functioning of the individual symbioses are summarized. Where possible this includes information concerning recognition and specificity, mode(s) of infection, morphological modifications/adaptations of the host plant and a synopsis of morphological, physiological and biochemical changes common to the symbiotic cyanobacteria. The latter encompasses heterocyst frequencies, enzymes involved in ammonia assimilation, photosynthetic capability and metabolic interaction with the host.TheAzolla-Anabaena symbioses, which have potential agronomic significance as an alternative nitrogen source and maintain continuity with the endophyte through the sexual cycle, are emphasized.     

Conservation of medicinal plants of Western Ghats,India and its sustainable utilization through <Emphasis Type="Italic">in vitro</Emphasis> technology

Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25 years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well.     

The role of Acanthospermum hispidum in the phenology of Euschistus heros and of Nezara viridula

Laboratory, greenhouse, and field studies with Euschistus heros (F.) and Nezara viridula (L.) (Heteroptera: Pentatomidae) in northern Paraná, Brazil, demonstrated greater survival and longevity with E. heros than N. viridula when fed the weed Acanthospermum hispidum DC. (Compositae). E. heros was better adapted to A. hispidum than N. viridula but neither species reproduced on this plant. Stink bugs moved from soybean to A. hispidum mainly during mid-March to mid-April when soybean plants matured. The infestation reached 60 and 100% with a maximum of ca. 3 and 10 stink bugs/plant in 1988 and 1989, respectively. Surprisingly, E. heros and N. viridula, which are normally seed-feeders, strongly preferred the high moisture stems of A. hispidum. Both species demonstrated similar feeding frequencies. These results suggest that in northern Paraná, the common weed, A. hispidum functions as a temporary host providing water and nutrients to those two pentatomid pests of soybean. Finally, both species do not seem to recognize A. hispidum as an unsuitable or perhaps toxic plant.     

Importance of <Emphasis Type="Italic">in vitro</Emphasis> technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.     

In situ observation of the <Emphasis Type="Italic">Cardinium</Emphasis> symbionts of <Emphasis Type="Italic">Brevipalpus</Emphasis> (Acari: Tenuipalpidae) by electron microscopy

Brevipalpus (Acari: Tenuipalpidae) mites are important pests on a variety of host plant species. The mites damage their hosts directly by feeding and some species also serve as vectors of plant viruses. Among more than 200 described Brevipalpus species, three are recognized as vectors of plant viruses: B. phoenicis, B. californicus and B. obovatus. These species occur worldwide in subtropical and tropical regions. Brevipalpus mites reproduce mostly by thelytokous parthenogenesis and this condition was attributed to a bacterial endosymbiont, recently characterized as a member of the genus Cardinium. The same symbiont infects many other arthropods and is capable of manipulating their host reproduction in various ways. Generally the presence of Cardinium is determined by molecular, PCR based, techniques. In the current work we present visual evidence for the presence of these bacteria by transmission electron microscopy as a complement of previous detection by PCR. Cardinium is easily identified by the presence of a unique array of microtubule-like structures (ML) in the cell. Symbionts have been observed in several organs and eggs from different populations of all three Brevipalpus species known as vector of plant viruses. Cardinium cells were always immersed directly within the cytoplasm of infected cells. Bacteria were observed in all females of all instars, but were absent from all males examined. Females from some Brevipalpus populations were observed to be uninfected by Cardinium. This observation confirmed previous PCR-based results that these populations were aposymbiotic. The observed distribution of the bacteria suggests that these bacteria could have other functions in the mite biology beside feminization.     

Diet of the Iberian hare (Lepus granatensis) in a mountain ecosystem

The diet of the Iberian hare (Lepus granatensis) was studied through microhistological pellet analysis in two areas from a mountain ecosystem in Central Portugal. Fecal pellets were collected monthly in 24 plots spatially distributed throughout the two study areas. For each period, a sample of 15 to 20 pellets was milled and 400 epidermal fragments were identified, by comparison with a reference collection. A wide range of plant species was observed in hare’s diet. Grasses represent the basis of the Iberian hare diet, with frequencies always higher than 50% in both study areas (annual average = 69.98%). Most of the 35 species of grasses assembled for the reference collection (91.43%) were identified in the pellets. Nevertheless, only six of these were consumed in proportions greater than 5%, being Anthoxanthum odoratum, Secale cereale and Agrostis spp. the species ingested in higher frequencies. The rate of grasses consumption reached 80.69% in winter but decreased in summer to around 55%. In this season, a concurrent rise in the ingestion of other plant groups, like herbs and shrubs, and of plant inflorescences was observed. This work provides the first results on the Iberian hare’s diet on mountain ecosystems, and suggests that the Iberian hare diet in a mountain ecosystem is similar to the observed in L. europaeus and L. timidus.     

Drosophila simulans' response to laboratory selection for tolerance to a toxic food source used by its sister species D. sechellia

Drosophila sechellia is a specialist species which feeds and breeds on a toxic plant, Morinda citrifolia. All other Drosophila species are killed by ripe fruits of Morinda. D. simulans was subjected to laboratory selection for survival in presence of octanoic acid, the toxic compound of M. citrifolia. After 20 generations of selection, selected lines showed an increased tolerance to octanoic acid, although the response was small compared to the interspecific differences. The genetic response to selection was assessed by studying the changes in allele frequencies at 28 microsatellite loci. Three loci, located in three distinct genomic regions, showed changes in allele frequencies significantly different from what is expected under drift alone in the selected lines. An oligogenic determination for tolerance to octanoic acid is in agreement with published results based on interspecific crosses.     

Biotechnology for saving rare and threatened flora in a biodiversity hotspot

The Southwest Australian Floristic Region (SWAFR) is a plant biodiversity hotspot with a geographically isolated and predominantly endemic flora. Known threatening processes (i.e. excessive clearing of native vegetation, soil salinity, soil erosion and chronic weed infestation) combined with uncertain but potentially deleterious environmental (climate) changes pose great challenges for conservation and restoration efforts. With a paucity of nature reserves, in situ protection of species can be problematic. For many species, ex situ conservation becomes the only viable strategy for saving species from extinction via seed banking or living collections established through vegetative propagation, or tissue (in vitro) culture methods. Development of specific in vitro protocols is necessary to successfully initiate culture lines, with considerable development of nutrient media, plant growth regulator regimes and incubation conditions required to optimise shoot regeneration and multiplication, especially with woody species of the SWAFR. In addition, integration of root induction and acclimatization stages has allowed significant improvements in speed and success of plant production of both endangered and difficult-to-propagate woody species. We contend that there is also considerable potential for expansion of alternative in vitro technologies such as somatic embryogenesis for difficult taxa to complement existing ex situ conservation and restoration strategies in biodiversity hotspots such as SWAFR.     

Impact of grazing regime on a Mongolian forest steppe

Question: What is the impact of grazing regime on plant species abundance, plant growth form, plant productivity and plant nutrient concentrations in a forest steppe? Location: Hustai National Park in the forest steppe region of Mongolia. Methods: On the Stipa steppe we applied three different grazing regimes by using; (1) one type of exclosure which excluded grazing by large mammalian herbivores, mainly takh (Przewalski horse), (2) another type of exclosure that excluded both large and small (Siberian marmots) mammalian herbivores, and (3) control plots which were freely grazed. We measured species frequencies, tiller densities, plant biomass and nitrogen concentrations of the vegetation. Results: Exclusion from grazing by takh and marmots significantly increased plant standing crop, but marmot grazing and full grazing did not show significant differences. Protection from grazing decreased forage quality, shown by a lower N-concentration of the standing crop. However, this was solely the result of the lower live-dead ratio of the vegetation. The frequency of the rhizomatous Leymus chinensis decreased under reduced grazing, as did the frequency of the total of rhizomatous species. The frequency of Stipa krylovii increased under reduced grazing, as did its basal areas, tiller density and tussock height. Conclusion: Reduced grazing leads to a lower abundance of rhizomatous species and an increase in tussock species.     

A defined amino acid exchange close to he putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein

Summary To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A₂ promoter CHI A₂. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5 regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.