Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Sarcocystis inghami n. sp. (Sporozoa: Sarcocystidae) from the skeletal muscles of the Virginia opossum Didelphis virginiana in Michigan

This report describes the newly identified Sarcocystis inghami n. sp. from the skeletal muscles of opossums (Mammalia: Didelphidae) that were collected from south central Michigan (42° 43-42° 79N, 84° 18-84°mathtype="display">6'W), USA. The new species is distinguished from all species described from North and South American opossums by the distinctive morphology of the villar protrusions on the cyst wall. Sarcocysts of S. inghami are microscopic, up to 700 m long and 110 m wide. The sarcocyst wall is up to 7 m thick, with long, stalked protrusions which average 5.5 × 1.2 m. These are constricted at the base, expanded laterally, rounded off distally and occasionally bifid. The villar protrusions have numerous microtubules without electron–dense bodies that extend from the tips into the granular layer. Bradyzoites are 10.7 × 4.3 (8––12 × 4––5) m. This is the second species of Sarcocystis sarcocyst described from the Virginia opossum in North America.     

Increasing glutaminase activity in shoyu koji using a mixed culture of two koji moulds

For improved fermentation of shoyu (soy sauce), a useful koji-making system has been developed using a mixed tane-koji of two shoyu koji moulds, namely Aspergillus oryzae K2 (length of conidiophores about 350 m) and the late-conidiation strain, A. oryzae HG (length of conidiophores about 2500 m). The mixed culture of strains K2 and HG had about twice the glutaminase activity of the single-strain cultures. In addition, the number of conidia in the mixed culture was about 10% of that in a culture of strain K2 alone.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

Effect of phytotoxic metabolites ofFusarium culmorum on barley root and root-hair development

Summary A mixture of gluconic acid and two unidentified organic acids, isolated from the culture filtrate ofFusarium culmorum, grown in glucose-limited continuous culture, were found to be highly toxic to growth of barley seedlings. A concentration of 900 g ml^–1 of the acid totally inhibited germination of barley seeds, with lower concentrations greatly inhibiting growth and development of germinated seeds. Root extension and root-hair production were inhibited at a concentration of 450 g ml^–1 of the acids, but the number of roots per seedling was increased. Toxicity of the acids was promoted at low pH.     

Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook

Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm^-1 (1000 s), a protoplast viability of 57%, and 47% conversion of ¹⁴C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 g ml^-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - CPW 13M CPW salts medium with 13% (w/v) mannitol - DC direct current - FDA fluorescein diacetate - f. wt fresh weight - GUS -glucuronidase - K Kao (1977) - MES 2-N-morpholinoethane sulfonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000) - TLC thin layer chromatography     

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 M (0.5 mgl^–1) -naphthaleneacetic acid and 0.33 M (0.05 mgl^–1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 M (1.0 mgl^–1) 6-benzylaminopurine and 0.53 M (0.1 mgl^–1) -naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 M (1.5 mgl^–1) -naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Electrotransformation ofStreptococcus sanguis Challis

Plasmid DNA was introduced into noncompetent cells ofStreptococcus sanguis Challis by an electrotransformation technique. The procedure was simple and rapid, did not require elaborate pretreatment of cells, and yielded transformant colonies in 24 h. The maximum transformation efficiency attained was 2.1×10⁴ transformants per g of pVA736. Molecular rearrangements and deletions were not detected in plasmid DNA isolated from transformants.     

Micropropagation of Tamarix gallica from nodal explants of mature trees

Tamarix gallica L. was micropropagated from four-to six-node explants taken from mature trees. Shoot proliferation was induced on Linsmaier and Skoog medium containing 30 g l^-1 sucrose, 7 g l^-1 agar, 200 mg l^-1 reduced glutathione (basal medium) and supplemented with 3.3 M benzyladenine. Adding 0.5 or 1.0 M indole-3-butyric acid (IBA) to the basal medium increased lateral shoot formation and ease of rooting. Microcuttings repeatedly subcultured on 1.0 M IBA produced well-developed roots, a high number of axillary shoots and could be acclimatized in the greenhouse.