Hybrid plants preserve unique genetic variation in the St Helena endemic trees <Emphasis Type="Italic">Commidendrum rotundifolium</Emphasis> DC Roxb. and <Emphasis Type="Italic">C. spurium</Emphasis> (G.Forst.) DC

The island of St Helena in the South Atlantic Ocean has a rich endemic flora, with 10 endemic genera and 45 recognised endemic species. However, populations of most endemic species have undergone dramatic reductions or extinction due to over-exploitation, habitat destruction and competition from invasive species. Consequently, endemic species are likely to have lost genetic variation, in some cases to extreme degrees. Here, the entire extant wild populations and all planted trees in seed orchards, of two critically endangered species in the endemic genus Commidendrum (Asteraceae), C. rotundifolium and C. spurium, were sampled to assess levels of genetic variation and inbreeding. Six new microsatellite loci were developed from next-generation sequence data, and a total of 190 samples were genotyped. Some seed orchard trees contained alleles from both wild C. rotundifolium and C. spurium indicating they could be hybrids and that some backcrossing may have occurred. Some of these trees were more similar to C. rotundifolium than C. spurium both genetically and morphologically. Importantly, allelic variation was detected in the putative hybrids that was not present in wild material. C. rotundifolium is represented by just two individuals one wild and one planted and C. spurium by seven, therefore the seed orchard trees comprise an important part of the total remaining genetic diversity in the genus Commidendrum.     

Genetic Diversity in the Worldwide <Emphasis Type="Italic">Botrychium lunaria</Emphasis> (Ophioglossaceae) Complex,with New Species and New Combinations

The Botrychium lunaria (Ophioglossaceae) complex worldwide includes the named species B. lunaria, B. crenulatum, B. tunux, and B. yaaxudakeit. These species have been distinguished from each other morphologically and genetically. This study further investigates the genetic diversity and geographic distribution of this complex, examining a large number of plants worldwide. Enzyme electrophoresis was used to examine allelic variation of 22 loci for 1574 plants of putative B. lunaria, B. crenulatum and B. tunux from North America, Eurasia, and New Zealand, and B. dusenii from the Falkland Islands. Variation in allelic composition assessed by genetic identity and cluster analysis using the programs PopGene and STRUCTURE as well as morphology and geography indicated that the complex is composed of six distinct entities; two of which warrant recognition as new species, B. neolunaria, endemic to North America, and B. nordicum, sister to the B. lunaria complex, from Iceland and Norway; and a new combination, B. lunaria var. melzeri , endemic to Greenland, Iceland, and Norway. The new taxa are described in this paper. Three entities within B. tunux are discussed but not proposed for recognition at this time. Botrychium lanceolatum, included in this study, is composed of three morphologically and genetically distinct entities warranting taxonomic recognition.     

Secondary Metabolites of Micromycetes in Plants of the Family Fabaceae,Genus <Emphasis Type="Italic">Trifolium</Emphasis>

The component composition and content of mycotoxins in perennial grasses of five species of the genus Trifolium, selected from natural biocenoses in different periods of vegetation, were studied. In the terrestrial parts of plants, the permanent presence of alternariol, cyclopiazonic acid, and emodin was shown. It was noted that differences in contamination are most clearly expressed in meadow clover Trifolium pratense L. and white clover Trifolium repens L., while alsike clover Trifolium hybridum L. occupies an intermediate position. It was found that zigzag clover Trifolium medium L. and mountain clover Trifolium montanum L., morphologically close to meadow clover, practically did not differ in the composition of toxic metabolites. The ways of formation of metabolic equilibrium in plants with participation of microscopic fungi are discussed.     

The EU protected taxon <Emphasis Type="Italic">Morimus funereus</Emphasis> Mulsant, 1862 (Coleoptera: Cerambycidae) and its western Palaearctic allies: systematics and conservation outcomes

Morimus funereus is a large longhorn beetle included in the European Habitats Directive and in previous releases of the IUCN red list. It represents a flagship species of old-growth forest saproxylic communities in E and SE Europe. The morphologically based taxonomy of W Palaearctic Morimus is rather unstable due to high phenetic intrapopulational and geographic variability and different authors have attempted to recognise one to five different taxa of specific/subspecific rank. No previous molecular data are available for the genus Morimus. Here, for the first time, a molecular approach based on COI and ITS2 gene sequences was applied in European and Anatolian Morimus specimens. The genetic variability among Euro-Anatolian Morimus populations and the geographical structure suggest that they can not be ascribed to the currently accepted five W Palaearctic Morimus species and may actually represent a single, genetically and morphologically variable biological species (M. asper), highlighting the necessity of an extended taxonomical revision. In light of these results, a phylogeographical hypothesis of postglacial colonisation of the central Mediterranean area has been developed and the consequences of this new taxonomic arrangement regarding conservation strategies for “Morimus funereus” and allied taxa in Europe and Turkey are discussed.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Foraging habitats and floral resource use by colonies of long- and short-tongued bumble bee species in an agricultural landscape with kabocha squash fields

Bumble bees pollinate and forage on flowers of crop and wild plants in agricultural landscapes. These interactions may depend on landscape patterns and bumble bee traits. We studied the abundance, colony density, and foraging range in long-tongued Bombus diversus Smith and short-tongued B. hypocrita Pérez, and evaluated their visits to flowers of wild plants and cultivated kabocha squash (Cucurbita maxima Duchesne). In forests in a farmland, B. hypocrita workers were trapped more frequently in the canopy. Full-sibs determined by nuclear microsatellite genotypes among workers collected in the farmland showed higher colony density and a larger foraging radius in B. hypocrita (30.8 km^?2 and 848 m) than in B. diversus (8.3 km^?2 and 723 m), respectively. Regarding wild plants, workers more frequently visited shallow flowers in B. hypocrita and deep flowers in B. diversus. These results suggest that bumble bees with different traits forage on different wild flowers in different habitats. Squash flowers were visited by both bumble bee species at similar frequency in the latter period of colony growth when males and new queens appeared. Composition of full-sib workers visiting squash and wild flowers did not depend on the number of collected workers of individual colonies, indicating that foraging on squash flowers was not associated with colony growth. Thus, growth and reproduction of bumble bee colonies may be supported by various wild plants and cultivated squash, respectively.     

Oviposition Preferences of <Emphasis Type="Italic">Plutella xylostella</Emphasis> are Influenced by the Type of Plant Induction and Glucosinolate Hydrolysis Profiles

Effects of inducing plants by exposing them to insect herbivory, mechanical damage or damaged neighboring plants were evaluated on the oviposition preferences of Plutella xylostella. The role of plant genotypes differing in their glucosinolate hydrolysis profiles was also evaluated using a wild ecotype (Col-0) and a genetically modified line (tgg1tgg2) of Arabidopsis thaliana. While the Col-0 line has normal production of glucosinolate hydrolysis products, the double myrosinase knockout (tgg1tgg2) is defective in the production of these volatiles. Dual choice oviposition assays were performed using naïve P. xylostella females, and the two A. thaliana lines, which were exposed to the three types of induction treatments. Female oviposition preferences were significantly influenced by both the type of plant induction and the plant genotypes differing in their volatile profiles. Plutella xylostella females significantly preferred to oviposit on herbivore-damaged plants (versus undamaged controls) when Col-0 plants were used, but chose control plants over the double myrosinase knockout tgg1tgg2. However, plant genotype did not influence oviposition choices between plant-plant primed or mechanically damaged plants and paired undamaged controls. Given the prevalent use of genetically modified plants and the potential differences in their responses to different types of induction, these factors may be important to consider in the management of specialist pests such as the diamondback moth P. xylostella.     

Involvement of ethylene in UV-B-induced changes in polyamine content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Components of the ethylene signal perception and transduction pathway (ethylene signaling pathway, ESP) were studied in respect to their involvement in regulation of UV-B-induced changes in levels of polyamines in plants Arabidopsis thaliana (L.) Heynh. Experiments were performed on 15-day old wild type (WT) plants, the mutant etr1-1 with impaired ethylene reception, and the ethylene-insensitive mutant ctr1-1 with constitutively activated ESP. The plants were cultivated aseptically. It was found that exogenous ethylene or an inhibitor of its action 1-methylcyclopropen (1-MCP), which blocks ethylene receptors did not affect the polyamine content in leaf rosettes of plants, which had not been subjected to UV-B stress. A day after UV-B irradiation at intermediate (9 kJ/m²) or high doses (18 kJ/m²), the putrescine levels increased, respectively, 6.4 and 3.0 times in WT, 4.5 and 3.2 times in etr1-1, and 5.5 and 4.7 in ctr1-1. Pretreatment with ethylene (1 μL/L) for 24 h reduced the putrescine accumulation along with the loss in spermidine and spermine pools in WT plants and, to a lesser extent, in etr1-1 mutant. Treatment with 1-MCP (50 nL/L, 3 h before and 24 h after the irradiation) enhanced plant sensitivity to UV-B, putrescine accumulation, as well as spermidine and spermine consumption in WT and, to a lesser degree, in etr1-1. The mutant ctr1-1 was insensitive to both ethylene and 1-MCP. The results show that the activation of ESP by ethylene increases plant resistance to UV-B because the irradiation stimulates accumulation of putrescine, which converts to spermidine and spermine functioning as ROS traps.     

Elucidating the multiple genetic lineages and population genetic structure of the brooding coral <Emphasis Type="Italic">Seriatopora</Emphasis> (Scleractinia: Pocilloporidae) in the Ryukyu Archipelago

The elucidation of species diversity and connectivity is essential for conserving coral reef communities and for understanding the characteristics of coral populations. To assess the species diversity, intraspecific genetic diversity, and genetic differentiation among populations of the brooding coral Seriatopora spp., we conducted phylogenetic and population genetic analyses using a mitochondrial DNA control region and microsatellites at ten sites in the Ryukyu Archipelago, Japan. At least three genetic lineages of Seriatopora (Seriatopora-A, -B, and -C) were detected in our specimens. We collected colonies morphologically similar to Seriatopora hystrix, but these may have included multiple, genetically distinct species. Although sexual reproduction maintains the populations of all the genetic lineages, Seriatopora-A and Seriatopora-C had lower genetic diversity than Seriatopora-B. We detected significant genetic differentiation in Seriatopora-B among the three populations as follows: pairwise F _ST = 0.064–0.116 (all P = 0.001), pairwise G′′_ST = 0.107–0.209 (all P = 0.001). Additionally, only one migrant from an unsampled population was genetically identified within Seriatopora-B. Because the peak of the settlement of Seriatopora larvae is within 1 d and almost all larvae are settled within 5 d of spawning, our observations may be related to low dispersal ability. Populations of Seriatopora in the Ryukyu Archipelago will probably not recover unless there is substantial new recruitment from distant populations.     

Metabolic changes of genetically engineered grapes (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) studied by <Superscript>1</Superscript>H-NMR,metabolite heatmaps and <Emphasis Type="Italic">i</Emphasis>PLS

Introduction

The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch.

Objectives

This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome.

Methods

The effects of the genetic modification upon the grape metabolome were evaluated through ¹H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one.

Results

The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration.

Conclusion

NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.

     

Effects of medium-wave ultraviolet radiation on levels and spectrum of polyamines in leaves and roots of wild-growing plants

The work continues serial studies on short-term effects of medium-wave ultraviolet radiation (UV-B) at 12.5 kJ/m² on plants. Special attention is paid to the rapid response of the antioxidant system. Free and conjugated forms of putrescine polyamines (putrescine, spermine, and spermidine), as well as those of cadaverine, are recognized to be constituents of the antioxidant system. These compounds were analyzed in plants 24 h after UV-B irradiation. Thellungiella salsuginea (Pallas) O.E.Schulz, Salvia officinalis L, Plantago major L., and Geum urbanum L. grown in aquatic culture under phytotron conditions were examined. The results support the hypothesis that putrescine plays the chief role in the plant defense response against medium-wave ultraviolet irradiation. Three of four plants manifested an increase in the content of this polyamine in leaves. It is the change that determines the enhanced total level of free polyamines. We failed to reveal a general tendency in dynamics of levels of conjugated forms of spermine, spermidine, and cadaverine; only conjugates of putrescine demonstrated a distinct increase. This study allows a conclusion that contributions of particular polyamines to the protective response primarily depend on the species to which the investigated plant belongs. It is likely that conjugated polyamines can be reserved as a pool necessary for rapid recovery of free polyamine levels.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Low genetic differentiation among altitudes in wild <Emphasis Type="Italic">Camellia oleifera</Emphasis>, a subtropical evergreen hexaploid plant

Camellia oleifera is a subtropical evergreen plant. Cultivated C. oleifera is the most important woody oil crop in China. Wild C. oleifera is an essential genetic resource for breeding. The patterns of genetic differentiation among altitudes/latitudes in wild C. oleifera are still unknown. Camellia oleifera may be predominantly hexaploid. The characteristics of polyploidy may lead to considerable biases in estimates of genetic diversity and differentiation. Our study used C. oleifera as a case study for analysing genetic diversity, structure and differentiation in polyploid plants using simple sequence repeats (SSRs). Wild C. oleifera samples were collected at different altitudes on the Jinggang and Lu mountains of China. The ploidy levels were determined with flow cytometry analysis. Eight highly polymorphic SSRs were used to genotype the samples. Genetic diversity and structure were analysed. Various estimates of genetic differentiation were compared. The flow cytometry results indicated that wild C. oleifera samples were all hexaploid at various altitudes of the Jinggang and Lu mountains. High levels of genetic diversity were found on both the Jinggang and Lu mountains. Genetic structure analyses indicated clear genetic differentiation between the Jinggang and Lu mountains and lower genetic differentiation among altitudes within each mountain. Classical genetic differentiation estimates of F_st failed to discriminate genetic differentiation between and within mountains. The Rho statistic showed a moderate level of genetic differentiation between mountains and lower levels of genetic differentiation within each mountain. Our study demonstrates that Rho is the statistic of choice for estimating genetic differentiation in polyploids.     

<Emphasis Type="Italic">In vivo</Emphasis> Role of the UV-Endonuclease from <Emphasis Type="Italic">Micrococcus luteus</Emphasis> in the Repair of DNA

THE UV-endonuclease enzyme of Micrococcus luteus causes single strand breaks in vitro adjacent to or one nucleotide removed from pyrimidine dimers produced in DNA by ultraviolet irradiation^1–4. Excision of the dimers is catalysed by a second enzyme, the UV-exonuclease^1,5. We now report the isolation of strains of M. luteus which possess altered levels of UV-endonuclease. Mutants lacking this enzyme are incapable of excising thymine-containing dimers while strains with decreased UV-endonuclease activity repair more slowly than wild type cells.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

Radiation induced crinkled leaves in jute

Crinkled leaf chimeras appeared regularly, as phenocopies, in the X₁ and P₁ generations of three varieties ofCorchorus olitorius L. The S₁ populations were devoid of this abnormality. These plants were also characterized by late flowering and reduction in the number of flowers per plant. The frequency of crinkled leaf chimeras increased with increase in dosages of X-rays and³²P beta rays. Inheritance studies revealed no immediate genetic background of these abnormal plants. However, mutations showing crinkled leaves were isolated in some X₃ and P₂ lines and their segregation in later generations was found to be disturbed. Chromosomal abnormalities during meiosis were considered as one of the factors responsible for the disturbed segregation. Crinkled leaf mutations also showed variable manifestation of the mutant characteristic in different years.     

Ultraviolet Mutagenesis in Strains of <Emphasis Type="Italic">E. coli</Emphasis> deficient in DNA Polymerase

CERTAIN mutations in Escherichia coli which cause increased sensitivity to ultraviolet light (UV) drastically change the UV mutability of the sensitive strain. Strains lacking the ability to excise pyrimidine dimers, for example, exhibit greatly increased UV mutability, producing induced mutations at doses of UV far smaller than those required to induce mutations in wild type strains^{1, 2}. Mutants owing their UV sensitivity to reduced ability to perform genetic recombination, on the other hand, show reduced mutability in response to UV compared with the wild type and some (recA or exrA strains, for example) are stable to UV, producing no detectable induced mutations at any dose^3–5. Analysis of UV mutagenesis in such strains has led to the hypothesis that most UV-induced mutations in E. coli are errors in the recombinational repair of gaps in the daughter-strand which are located opposite unexcised pyrimidine dimers^{6, 7}.