Transformation of pollen embryo-derived explants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient -glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, -glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.     

Thidiazuron induced adventitious shoot regeneration in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

A high frequency adventitious shoot regeneration protocol was developed for henbane (Hyoscyamus niger L.) using thidiazuron (TDZ). Hypocotyl, cotyledon and stem explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of N⁶-benzylaminopurine and TDZ. MS medium supplemented with 16 μM TDZ was the most effective for providing 100 % regeneration frequency associated with a 19.53 shoots per hypocotyl explant. Plantlets were rooted on MS medium supplemented with different concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid. High rooting and survival was achieved using half strength MS medium supplemented with 8 μM IBA.This study was supported by The State Planning Commission of Turkey (DPT) and University of Ankara (Project Nos.: 98K120640 and 2001K120240).     

Proteins and polyamines during dormancy breaking of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.     

The germination characteristics of <Emphasis Type="Italic">Scrophularia marilandica</Emphasis> L. (Scrophulariaceae) seeds

Investigations on seeds of Scrophularia marilandica L. were undertaken to determine their germination requirements. Seeds were collected from three naturally occurring sites and one greenhouse-grown population in London, Ontario in September and October of 1997. Some were set to germinate immediately after collection; others were stored in or on soil outside and/or under controlled laboratory conditions before testing. Germination was assessed under two light/temperature regimes (35°C 14 h light, 20°C 10 h dark and 25°C 14 h light, 10°C 10 h dark), in continuous darkness, and in the presence of two germination-promoting chemicals (GA₃ and KNO₃). Fresh seeds germinated best at 35/20°C, while stored seeds germinated best at 25/10°C. No differences in percent germination were found among three seed-maturity stages. All chemical treatments, except 0.01 M KNO_3, increased percent germination. Significant differences were found both among and within sites for most chemical treatments, but exposure to 3 × 10⁻⁴ M GA₃ caused almost every seed to germinate. When compared to the control, both the gibberellic acid and the soil-storage treatments contributed to faster germination. Exposure of seeds to naturally prevailing conditions on the soil surface followed by testing under the 25/10°C regime produced the highest percent germination. No seeds germinated in the dark. In summary, seeds of S. marilandica exhibit physiological dormancy, which can be alleviated by exposure to light, after-ripening and/or cold stratification. It is probable that the differences in germination response among sites can be attributed to differences in environmental conditions during seed production. These experiments indicate that the seeds of S. marilandica must be buried shortly after dispersal in order to form a persistent seed bank.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

Wounding and pathogen infection induce a chloroplast-targeted lipoxygenase in the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Chloroplastic LOXs are implicated in the biosynthesis of oxylipins like jasmonic acid and C₆ volatiles among others. In this study, we isolated the cDNA of a novel chloroplast-targeted Phaseolus vulgaris LOX, (PvLOX6). This gene is highly induced after wounding, non-host pathogen infection, and by signaling molecules as H₂O₂, SA, ethylene and MeJA. The phylogenetic analysis of PvLOX6 showed that it is closely related to chloroplast-targeted LOX from potato (H1) and tomato (TomLOXC); both of them are implicated in the biosynthesis of C₆ volatiles. Induction of PvLOX6 mRNA by wounding ethylene and jasmonic acid on the one side, and non-host pathogen, salicylic acid on the other indicates that common bean uses the same LOX to synthesize oxylipins in response to different stresses. PvLOX6 accession number: EF196866.     

Adenine nucleotides and energy charge during dormancy breaking in embryo axes of <Emphasis Type="Italic">Acer platanoides</Emphasis> and <Emphasis Type="Italic">Fagus sylvatica</Emphasis> seeds

We analysed changes in AMP, ADP, and ATP concentrations and adenylate energy charge in Norway maple (Acer platanoides L.) and European beech (Fagus sylvatica L.) seeds during dormancy breaking (at 3 °C) and in the control variant at 15 °C. Values of the studied indicators in stratified beech seeds were generally higher at 15 °C, at least until germination (+3 °C). By contrast, in maple seeds, the values recorded during dormancy breaking by cold stratification were much higher than at 15 °C. Three peaks (usually in weeks 3, 6, and 8) were observed in maple seeds at 3 °C, but not at 15 °C. Among adenine nucleotides, AMP reached the highest levels in both species in both variants of the experiment.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

Expression and functional analysis of <Emphasis Type="Italic">ZmDWF4</Emphasis>, an ortholog of <Emphasis Type="Italic">Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.