Thidiazuron Promotes <Emphasis Type=Italic>In Vitro</Emphasis> Plant Regeneration of <Emphasis Type=Italic>Arachis correntina</Emphasis> (<Emphasis Type=Italic>Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.     

A new name in <Emphasis Type=Italic>Pavetta</Emphasis> L. (<Emphasis Type=Italic>Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.     

Transposon display supports transpositional activity of <Emphasis Type=Italic>P</Emphasis> elements in species of the <Emphasis Type=Italic>saltans</Emphasis> group of <Emphasis Type=Italic>Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

A new species of <Emphasis Type=Italic>Cortinarius</Emphasis> sect. <Emphasis Type=Italic>Orellani</Emphasis> from Japan

Cortinarius breviradicatus sp. nov., found in deciduous forests, is described and illustrated from Niigata, Japan. It is characterized by its medium-sized to large dark brown basidiocarp, acutely conical pileus, and rooting stipe, and by subglobose to broadly ellipsoid basidiospores. In addition, the extracting solution from its basidiocarps exhibits a strong fluorescence around 400–430 nm in ultraviolet radiation (250 nm), which was observed in a species of Cortinarius sect. Orellani. The new species belongs to the section Orellani. The differences between the new taxon and similar species are briefly discussed.     

The expression of analgesic-antitumor peptide (<Emphasis Type=Italic>AGAP</Emphasis>) from Chinese <Emphasis Type=Italic>Buthus</Emphasis><Emphasis Type=Italic>martensii</Emphasis> Karsch in transgenic tobacco and tomato

The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.     

A high-throughput <Emphasis Type=Italic>Agrobacterium</Emphasis>-mediated transformation system for the grass model species <Emphasis Type=Italic>Brachypodium distachyon</Emphasis> L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA((R)), while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.     

Influence of the<Emphasis Type=Italic>SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type=Italic>Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

A rapid<Emphasis Type=Italic>Agrobacterium</Emphasis>-mediated<Emphasis Type=Italic>Arabidopsis thaliana</Emphasis> transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.     

Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Time Course of <Emphasis Type=Italic>c-fos</Emphasis>, <Emphasis Type=Italic>vasopressin</Emphasis> and <Emphasis Type=Italic>oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.     

Allelopathic effects of the submerged macrophyte <Emphasis Type=Italic>Potamogeton malaianus</Emphasis> on <Emphasis Type=Italic>Scenedesmus obliquus</Emphasis>

Allelopathic effects of the submerged macrophyte Potamogeton malaianus on Scenedesmus obliquus were assessed using a two-phase approach under controlled laboratory conditions. In the co-culture experiment (phase І), the growth of S. obliquus at two different initial cell densities was significantly inhibited by P. malaianus. Moreover, the growth inhibition was dependent on the biomass density of P. malaianus. Antioxidant enzymes (SOD, CAT and POD), MDA, APA, total soluble protein, protein electrophoretic pattern and morphology of S. obliquus were determined after the co-culture experiment was terminated. The activities of SOD, CAT, POD and APA at the low initial cell density were stimulated, the contents of MDA and total soluble protein were increased, and some special protein bands disappeared in P. malaianus treatments. The macrophyte had no effect on the activities of SOD and APA at the high initial cell density, but significantly influenced other physiological parameters of S. obliquus with the increase of biomass density. The morphology of S. obliquus showed no difference in the macrophyte treatments and the controls, and the cultures were dominated by 4-celled coenobia. The results indicated P. malaianus had significant allelopathic effects on the growth and physiological processes of S. obliquus. Moreover, the allelopathic effects depended on initial algal cell density, biomass density of the macrophyte, and their interaction. In the experiment of P. malaianus culture filtrates (phase II), filtrates from combined culture of plant and S. obliquus at the low initial cell density exhibited no apparent growth inhibitory effect on S. obliquus. The result showed that initial addition of growth-inhibiting plant filtrates had no allelopathic effect on S. obliquus. We concluded that the allelopathic effects on S. obliquus were found in the presence of P. malaianus, but not in P. malaianus filtrates. However, the absence of allelopathic effect on S. obliquus might be due to the very low concentrations of allelochemicals in the filtrates. Handling editor: S. M. Thomas     

<Emphasis Type=Italic>Septobasidium parviflorae</Emphasis> sp. nov. on <Emphasis Type=Italic>Pinus parviflora</Emphasis> from Japan

Septobasidium parviflorae sp. nov. on Pinus parviflora is described and illustrated. This species is characterized by its whitish-gray, gray to dark gray-colored fungus body with an indeterminate margin, hyphal strands, and cylindrical basidia with long sterigmata. This is the first report of Septobasidium occurring on a member of the genus Pinus in Japan.     

Typification of <Emphasis Type=Italic>Metrosideros regelii</Emphasis> (<Emphasis Type=Italic>Myrtaceae</Emphasis>) and consideration of its generic position

Summary  The type specimen of Metrosideros regelii is discussed. It contains a mixture of two species, representing different genera, and a lectotype is chosen. The generic position of the species is considered in the light of morphology and recent molecular evidence and the new combination, Mearnsia regelii, made.     

Genetic transformation of <Emphasis Type=Italic>Agave salmiana</Emphasis> by <Emphasis Type=Italic>Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

<Emphasis Type=Italic>Nramp1</Emphasis> deletion does not confer susceptibility to<Emphasis Type=Italic> Rhodococcus equi</Emphasis> infection in mice

Rhodococcus equi is an intracellular bacterium that causes pneumonia in immunocompromised people and foals. The Nramp1 gene influences susceptibility to a variety of intracellular bacteria (including mycobacterial species), but not to Mycobacterium tuberculosis. In this study, we demonstrate that mice functionally deleted of the Nramp1 gene were not more susceptible to infection with virulent R. equi (ATCC 33701) than wild-type mice. Susceptibility of mice to infection with the intracellular bacterium R. equi is more similar to that of M. tuberculosis than to other intracellular bacteria, including other mycobacteria.     

The diagnostic implications of the separation of<Emphasis Type=Italic>Entamoeba histolytica</Emphasis> and<Emphasis Type=Italic>Entamoeba dispar</Emphasis>

For much of the last hundred years most cases of amoebiasis have been diagnosed by light microscopy. Only relatively recently have we become aware that this technique is usually incapable of distinguishing between two species-Entamoeba histolytica andE. dispar-only the first of which is a pathogen. The implications of this for patient management and, even more, for the validity of epidemiological surveys, are only slowly being addressed. What is clear is that methods are urgently required to distinguish between infections with these two species and this review attempts to summarise some of those, which have been developed to meet this need.     

New life cycles of <Emphasis Type=Italic>Porphyra katadae</Emphasis> var. <Emphasis Type=Italic>hemiphylla</Emphasis> in culture

Porphyra katadae Miura var. hemiphylla Tseng et T. J. Chang, a species distributed around the Liaodong and Shandong Peninsulas of China, produces gametophytes from late winter to early spring. These are monoecious with male and female reproductive tissues in distinct halves or sectors. Vegetative tissues from sectors expected to differentiate into sexual tissue were cultured in the laboratory. Male and female reproductive organs, as well as conchocelis and blades, were differentiated from these tissues. The male and female reproductive tissues were in patches and mixed on the cultured tissue pieces. This was quite different from the wild-type sectored individuals. The F₁ conchospore germlings also produced monospores, carposporangia, spermatangia and conchocelis. These carposporangia and spermatangia were in patches and were mixed on the F₁ fronds. The results imply that P. katadae var. hemiphylla is possibly sex-differentiated rather than sex-determined. This is the first report of such a dimorphic life history in the genus Porphyra.     

Leaf spot disease on <Emphasis Type=Italic>Tilia cordata</Emphasis> caused by the fungus <Emphasis Type=Italic>Cercospora microsora</Emphasis>

Increased incidence of leaf spots on many tree species, up to the presence of peripheral importance only, including linden trees was noticed recently. First massive and continuous occurence of the fungus Cercospora microsora Sacc. [teleomorph Mycosphaerella millegrana (Cook.) Schröet., Mycosphaerella microsora Syd.], causal agent of anthracnose on linden trees (Tilia cordata Mill.) grown in urban plantings in Slovakia was reported. Along with this, certain of the important growth characteristics of this fungus were studied under laboratory conditions. To specify Cercospora biology mycelial growth of C. microsora in pure hyphal cultures was observed in relation to medium and locality. One-way ANOVA has confirmed a statistically significant influence of both factors, culture medium and locality on growth rate values of C. microsora. The effect of these factors has not proved unambiguously in all cases. In the case of one locality (Nitra), the significant influence of used media has not been proved (P > 0.05). PDAg showed generally as the most suitable medium, inducing the most intensive growth in three localities (41.06 mm/week on average). Comparing three localities, the effect of this factor is not so unambiguous. Growth rate values from the localities Bratislava and Pribeta indicate unsuitability of medium A for the fast radial growth. A Tukey test separately conducted for the factors medium and the locality revealed the significant combinations of means (P ≤ 0.05).