Transient gene expression in electroporated Picea glauca protoplasts

The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm^–1 having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli -glucuronidase gene (-GUS) as a marker gene revealed very high levels of -GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since -GUS-like activity increased when plasmid DNA was present during electroporation.Abbreviations CAT chloramphenicol acetyltransferase - -GUS -glucuronidase - MUG 4-methyl umbelliferyl glucuronide - F microfarads NRCC No. 29150     

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro

To develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. (Seckel and Louise Bonne) and one cultivar of P. bretschneideri Rehd. (Crystal Pear). Half-leaves, taken from shoots proliferating on Lepoivre medium, were plated in petri-dishes on medium supplemented with various combinations of cytokinins and auxins. Cultures of the above cultivars had been established from mature trees. Among the growth regulators tested, thidiazuron (TDZ), combined with naphthalene-acetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 uM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 uM and 2.5 to 13 um, respectively). Among different macronutrient compositions, 1/2 and 1/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L-1) had a linear significant effect on shoot regeneration of Crystal Pear.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - NAA a-naphthaleneacetic acid - NoA 2-naphthoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TDZ thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea) - MS Murashige and Skoog (1962) macroelements - L Lepoivre (Quoirin and Lepoivre, 1977) macroelements     

Micropropagation of Phytolacca dodecandra through shoot-tip and nodal cultures

A procedure for micropropagation of endod (Phytolacca dodecandra) is described. BA at 0.44 M produced 3.1 new shoots per expiant in six weeks using shoot tips. Nodal expiants, however, produced up to 4.7 shoots per explant on medium with 0.44 M BA and 0.27 M GA,. IBA at 0.49 M induced 90% rooting with minimal callus. Plantlets were successfully transferred to the greenhouse and some staminate clones produced flowers after six months.Abbreviations BA 6 benzylaminopurine - kinetin 6-furfurylaminopurine - 2iP N⁶-(2-isopentyl)adenine - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - GA₃ Gibberellic acid - IBA indole-3-butyric acid     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Evidence that sister chromatid exchanges and chromatid breaks are two independent events

The relative frequencies of sister chromatid exchanges (SCE) and chromatid breaks in BrdU (5-bromodeoxyuridine) — sensitive site (lq22 lq23) in Chinese hamster cells after BrdU incorporation were studied. The results show that chromatid breaks do not follow the exchange hypothesis and provide evidence that chromatid breaks and SCEs are two independent events despite some common features.     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10⁴M^–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - ELISA enzyme linked immunosorbent assay - DP degree of polymerization - PVC polyvinyl chloride P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper.     

Sterol conjugates of two phenotypically different calli of Beta vulgaris

Steryl glycosides are the predominant form of sterol at 88% of the total sterol in non-betalain producing calli of Beta vulgaris. The total sterol decreases and sterol form shifts from steryl glycosides to 97% free sterol upon the transition of non-betalain to betalain producing calli. A substantial decrease in stigmasterol (24--ethylcholesta-5,22E-dien-3-ol) and sitosterol (24-ethylcholest-5-en-3-ol) levels is observed during this transition, and alters the ratio of ⁷:⁵ sterols. Spinasterol (24- ethyl-5-cholesta-7,22E-dien-3-ol) is the dominant sterol at 43% and 95% of the total sterol in non-betalain producing and betalain producing calli. The level of 22-dihydrospinasterol (24-ethyl-5-cholest-7-en-3-ol) is reduced in both calli to 3% from 25% in leaves. Lanosterol (4,4,14-trimethyl-cholesta-8(9),24-dien-3-ol) and cycloartenol (9,19-cyclopropyl-4,4,14-trimethyl-cholest-24-en-3-ol) were identified in betalain and nonbetalain producing callus respectively.     

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes

Fumonisins, mycotoxins produced byFusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals.In this study we investigated the effect of fumonisin B₁ on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B₁ a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B₁ on the glycosphingolipid pattern of the chick embryos. In the presence of 72 µg fumonisin B₁ per egg the incorporation of [¹⁴C]galactose and of [¹⁴C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B₁ treated embryos are presented. Fumonisin B₁ caused haemorrhages under the skin as well as in the liver of treated embyros. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.Abbreviations Cer ceramide (N-acylsphingosine) - FB₁ fumonisin B₁ - GM3 NeuAc23Gal14Glc11Cer - GD3 NeuAc28NeuAc23Gal14Glc11Cer - GD1a NeuAc23Gal13GalNAc14(NeuAc23)Gal14Glc11Cer - GT1b NeuAc23Gal13GalNAc14(NeuAc28NeuAc23) Gal14Glc11Cer - HPLC high pressure liquid chromatography - PBS phosphate buffered saline - PDMP 1-phenyl-2-dodecanoylamino-3-morpholino-1-propanol - Sa sphinganine - So sphingosine - Sa/So sphinganine-over-sphingosine - TLC thin layer chromatography - Tris Tris(hydroxymethyl)aminomethan Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Methyl methane-sulphonate (MMS) induced SCEs are reduced by the BrdU used to visualise them

SCE induction in synchronised CHO cells treated with methyl methane sulphonate (MMS) in G₁ was studied over successive pairs of cell cycles by introducing bromodeoxyuridine (BrdU) at consecutive G₁ stages. When individual cell cycle SCE values were calculated from the data, anomalous results were obtained with ratios of 1.01.82.1 for the first three cycles but a negative value for the fourth cycle. Further studies using different BrdU concentrations showed that MMS induced SCEs were reduced by values exceeding 50% in DNA containing high levels of incorporated BrdU. This reduction was dose dependent and accounted for the anomalous results obtained over successive cycles. Lesions leading to chromatid exchanges were also reduced by the same mechanism. SCEs induced by UV irradiation were also decreased but those induced by the cross-linking agent nitrogen mustard (HN₂) remained unaffected. The results indicate that not only are SCE lesions induced by MMS, UV or HN₂ expressed independently of the spontaneous SCEs induced by BrdU but that SCE lesions are multiple in nature. Mechanisms by which SCE lesions could be repaired in BrdU containing DNA are discussed. SCE lesions in MMS treated cells arrested in G₁ with arginine deprived medium (ADM) are repaired without the presence of BrdU in the DNA. An opposite effect is seen however in the control cells, where SCEs are increased with time spent in ADM arrest. These interactions between the effects of MMS, BrdU and ADM arrest are discussed.     

Polysaccharide and protein secretion by grass microhairs

Summary Secretory activities of bicellular microhairs from grasses belonging to the subfamilies Chloridoideae, Arundinoideae, Panicoideae, and Bambusoideae, and including the chloridoid, panicoid and Enneapogon microhair morphological types, have been investigated. Light microscopic histochemistry indicated that all microhairs studied secrete polysaccharide and protein (or glycoprotein), including those which also secrete salt. Localization of polysaccharide at ultrastructural level using periodic acid-thiocarbohydrazidesilver proteinate staining revealed that in panicoid type microhairs dictyosomes are involved in polysaccharide secretion, whereas in the chloridoid and Enneapogon types partitioning membranes seem to be involved instead.Abbreviations Ag silver precipitates representing localization of polysaccharide - BC basal cell - C cuticle - CC cap cell - CH cuticular chamber - CN system of membrane bound channels and vesicles - CP chloroplast - CW cell wall - D dictyosomes - M mitochondria - N nucleus - PTM partitioning membranes - RER rough endoplasmic reticulum - S secretory material - St starch grain - US unstained dictyosome cisternae - V vesicle     

Immobilization of digitoxin 12β-hydroxylase,a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH

Attempts were made to immobilize digitoxin 12-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl₂. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using -methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12-hydroxylase is described.Abbreviations -mdg -methyldigoxin - -mdt -methyldigitoxin     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Syntheses of α-D-glucosyl-D-fructoses by use of a reversed hydrolysis activity of α-glucosidase

Summary -D-Glucosyl-D-fructoses were synthesized by use of a reversed hydrolysis activity of -glucosidase fromSaccharomyces sp. Although -D-glucosyl-(1–1)-D-fructose was synthesized predominantly by the incubation of D-glucose solution in the presence of -glucosidase (batch method reaction), -(1–4)-linked disaccharide was a major product in a procedure by use of an immobilized -glucosidase column and an activated carbon column (column method reaction).     

Polymerization of amino acids containing nucleotide bases

Summary We have prepared the nucleoamino acids 1-(3-amino, 3-carboxypropyl)uracil (3) and 9-(3-amino, 3-carboxypropyl)adenine (4) as (l)-enantiomers and as racemic mixtures. When3 or4 is suspended in water and treated with N,N-carbonyldiimidazole, peptides are formed in good yield. The products formed from the (l)-enantiomers are hydrolyzed to the monomeric amino acids by pronase.Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (l)-enantiomer of3 did not act as templates to facilitate the oligomerization of4.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Effect of organic and inorganic fertigation on yields, δ15N values, and δ13C values of tomato (Lycopersicon esculentum Mill. cv. Saturn)

We examined the effects of fertilizer application, especially the effects of fertigation and types of fertilizer (inorganic and organic) on yields and ¹⁵N and ¹³C values of tomato (Lycopersicon esculentum Mill. cv. Saturn). Fertigation is a method in which an appropriate diluted liquid fertilizer is applied to the plants each time they are drip-irrigated. We developed a method of organic fertigation using corn steep liquor (CSL) as the liquid fertilizer, because it is an industrial byproduct of cornstarch manufacture and can be used very effectively. We compared fruit yield, mineral content, ¹⁵N value, and ¹³C value of tomatoes grown under three different fertilizer treatments, basal dressing: basal dressing with granular chemical fertilizer; inorganic fertigation: fertigation with liquid chemical fertilizer; and organic fertigation: fertigaion with CSL. Mineral contents of tomatoes grown with basal dressing were generally lower than those grown under either fertigation treatment. These results indicated that yields and mineral contents were influenced more by the method of fertilizer application than by whether the fertilizers were inorganic or organic. There were, however, significant differences in the ¹⁵N values of tomato fruits grown under different types of fertilizer applications, especially between inorganic and organic fertilizers. The ¹⁵N value of the chemical fertilizer used for basal dressing was 0.81 ± 0.45{}, that of the chemical fertilizer for fertigation was 0.00 ± 0.04{}, and that of CSL was 8.50 ± 0.71{}. The ¹⁵N values of the soils reflected the ¹⁵N values of the fertilizers. Moreover, the ¹⁵N values of the fruits corresponded to the ¹⁵N values of the applied fertilizers. The ¹⁵N values were 3.18 ± 1.34{} in the fruits grown with a basal dressing of chemical fertilizer, 0.30 ± 0.61 in those grown under inorganic fertigation, and 7.09 ± 0.68 in those grown under organic fertigation. On the other hand, although the ¹³C values in the soil also reflected the ¹³C values of the applied fertilizers, there was no significant difference in the ¹³C values of fruits among the different treatments. In conclusion, because the ¹⁵N values of fertilizers correlated well with those of the fruits, it may be possible to use ¹⁵N values as an indicator of organic products.