Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Effect of AgNO3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes

The presence of 1–10 M aminoethoxyvinylglycine (AVG) or 5–30 M AgNO₃ markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 M), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO₃ and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO₃ was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 M.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - IBA indole-3-butyric acid - 2ip 6-{ie195-01}-{ie195-02}-dimethylallylamino purine - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Shoot and root organogenesis of Camellia sasanqua

In vitro-derived shoot tips, (10 mm) taken from primary cultures of Camellia sasanqua L., were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA₃. Maximum shoot proliferation and growth for juvenile and mature tissue was obtained when 0.54 M NAA, 8.8 M BA plus 14.4 to 28.9 M GA₃ was added to the culture media, with a pH between 4.5 and 5.0. In vitro-derived shoots (20 mm) from mature C. sasanqua Day Dream and juvenile C. sasanqua cultures initiated roots in vitro after immersion in 2.5 mM IBA for 30 min. Sixty percent of the mature shoots and 90% of the juvenile shoots initiated roots within 3 weeks of treatment with IBA.Abbreviations MS Murashige and Skoog (1962) - IBA lH-indole-3-butanoic acid - BA N-(phenyl-methyl)-lH-purine-6-amine - GA₃ gibberellic acid - kinetin N-(Z-furanyl-methyl)-lH-purine-6-amine - NAA l-naphthaleneacetic acid - L Linear - Q Quadratic     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

Synthesis of Certain 4-Substituted-1-β-D-Ribofuranosyl-3-Hydroxypyrazoles Structurally Related to the Antibiotic Pyrazofurin

Abstract

Several 4-substituted-1-β-D-ribofuranosyl-3-hydroxypyrazoles were prepared as structural analogs of pyrazofurin. Glycosylation of the TMS derivative of ethyl 3(5)-hydroxypyrazole-4-carboxylate (3) with 1-0-acetyl-2,3,5-tri-0-benzoyl-D-ribofuranose in the presence of TMS-triflate gave predominantly ethyl 3-hydroxy-1-(2,3,5-tri-0-benzoyl-β-D-ribofuranosyl)pyrazole-4-carboxylate (4a), which on subsequent ammonolysis furnished 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamide (5). Benzylation of 4a with benzyl bromide and further ammonolysis gave 3-benzyloxy-1-β-D-ribofuranosylpyrazole-4-carboxamide (8a). Catalytic (Pd/C) hydrogenation of 8a afforded yet another high yield route to 5. Saponification of the ester function of ethyl 3-benzyloxy-1-β-D-ribofuranosylpyrazole-4-carboxylate (7b) gave the corresponding 4-carboxylic acid (6a). Phosphorylation of 8a and subsequent debenzylation of the intermediate 11a gave 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamide 5′-phosphate (11b). Dehydration of 3-benzyloxy-1-(2,3,5-tri-0-acetyl-β-D-ribofuranosyl)pyrazole-4-carboxamide (8b) with POCl₃ provided the corresponding 4-carbonitrile derivative (10a), which on debenzylation with Cl₃SiI gave 3-hydroxy-1-(2,3,5-tri-0-acetyl-β-D-ribofuranosyl)pyrazole-4-carbonitrile (13). Reaction of 13 with H₂S/pyridine and subsequent deacetylation gave 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-thiocarboxamide (12b). Similarly, treatment of 13 with NH₂OH afforded 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamidoxime (14a), which on catalytic (Pd/C) hydrogenation gave the corresponding 4-carboxamidine derivative (14b). The structural assignment of these pyrazole ribonucleosides was made by single-crystal X-ray analysis of 6a. None of these compounds exhibited any significant antitumor or antiviral activity in cell culture.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Transformation of vegetable oils by an oleaginous yeast:Rhodotorula glutinis IIP-30

Summary A locally isolated oleaginous yeastRhodotorula glutinis IIP-30 was grown on vegetable oils obtained from coconut, ground nut and til. The fatty acid composition of yeast oil was quite similar to that of the substrate oil in case of ground nut and til, while it was different with coconut oil. Utilization of C₁₂ and C₁₄ fatty acids of coconut oil to yield higher proportions of C₁₈₁ and C₁₈₂ fatty acids was observed.     

9-(6-Deoxy- β -D-Allofuranosyl)Adenine Cyclic 3′,5′ -Phosphor-Amidate: A New Cyclic AMP Amide Derivative Containing an Equatorial Methyl Group at the 5′-Position

Abstract

R _P and S _P phosphorus diastereoisomers of the title compound (2) are prepared from the corresponding cyclic monophosphate. Solution conformation of the dioxaphosphor-inane ring and hydrolysis of R _p-2 and S _p-2 are studied and compared with those of the phosphorus diastereoisomers of the isomeric compound that contains the 5′-methyl group in the axial position.     

Production of virus resistant and insect tolerant transgenic tobacco plants

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r₀ and R₁ tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV cucumber mosaic virus - Bt toxin Bacillus thuringiensis -endotoxin - CaMV cauliflower mosaic virus - NOS nopaline synthase - Kan Kanamycin - Spe spectinomycin - Carb Carbenicillin     

Simultaneous saccharification and fermentation by mixed cultures ofBrettanomyces clausenii andPichia spipitis R of SO2-prehydrolysed wood

Summary Using pilot scale Wenger and Stake II reactors for prehydrolysing aspen and coniferous wood chips in the presence of SO₂ catalyst, highly digestible lignocellulosic substrates were generated from which about 90% yields of hemicellulose mostly in monomeric form could be recovered. Simultaneous saccharification and fermentation (SSF) of these SO₂ feedstocks by a mixed culture ofBrettanomyces clausenii andPichia stipitis R resulted in rapid and efficient fermentation giving a final yield of 369 and 360 L ethanol/tonne of the prehydrolysed woods, respectively. BecauseB. clausenii is an excellent cellobiose fermenter, no -glucosidase was needed during SSF.     

Synthesis and Biological Evaluation of some Acyclic Nucleoside Cyclic Phosphoramidate Derivatives

Abstract

The acyclic nucleosides 2 were treated with 2-chloro-3-methyl-1-oxa-3-aza-2-phosphacyclopentane (3) in the presence of diisopropylethylamine to give the corresponding phosphoramidite derivatives (4). The phosphoramidite intermediates (4) were oxidized with m-chloroperbenzoic acid to the phosphoramidate derivatives (5). Treatment of 5a,b with ZnBr₂ in CH₃NO₂ gave the corresponding acyclic nucleoside cyclic phosphoramidates (6a,b). Attempts to desilylation of 5c by tetrabutylammonium fluoride (TBAF) resulted in opening of the phosphoramidate ring. The newly synthesized compounds were evaluated for antiviral and antitumor cell activity.     

A Reinvestigation of the Synthesis of 4-Thio-Pentofuranosides for Further Nucleosidic Synthesis

Abstract

We demonstrate that the procedure of Huang and Hui⁽⁴⁾, applied to the D-ribose and involving Ph₃P/I₂/Imidazole reagent system do not lead to the desired 4-thio-D-ribofuranoside derivative but gives its diastereoisomer 4-thio-L-lyxofuranoside derivative 4      

Metal Carbonate-Mediated Complete Deacylation of Polyacyl Protected Nucleosides

Abstract

Treatment of poly-acetyl or -benzoyl protected ribonucleosides (1a-i) and 2′-deoxyribonucleosides (3a-d) with metal carbonates such as NaHCO₃ or Na₂CO₃ in MeOH gave the corresponding deacylated free ribomucleosides (2a-d and 4a-b) in excellent high yields.     

Citrate inhibition of glucose uptake in Aspergillus niger

Summary The in vitro uptake of glucose and 2-desoxyglucose by whole mycelia of Aspergillus niger, pregrown under citric acid producing conditions, is inhibited by citrate (I _0.5 15 mM), which affects a high affinity glucose transport system (K_m 0.14 – 0.17 mM).     

Nucleosides,XLVI1 Syntheses and Reactions of 6- and 7-p-Chlorophenyllumazine Nucleosides

Abstract

The syntheses of 6-(4) and 7-p-chlorphenyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-lumazine (6), was well as the debenzoylation to the corresponding free nucleosides 5 and 7, were improved. Thiation of 4 and 6 by P₄S₁₀ led in excellent yields to 4-thiolumazine nucleosides (8, 10) which could be deblocked to 9 and 11 and converted on treatment with ammonia into the isopterin-N-1- ribofuranosides 13 and 14. 2,2′-Anhydro-nucleoside formation worked well with 5 and 7 respectively to give 15 and 16, which formed on acid hydrolysis the 6- and 7-substituted 1-β-D-arabinofuranosyl-lumazines 18 and 19. The new nucleosides have been characterized by UV and ¹H-NMR spectra.     

Chloroplast acclimation to low osmotic potential

Photosynthetic potential of isolated chloroplasts was investigated during in situ water deficits. An eight day stress cycle imposed on spinach plants reduced leaf _w by 0.57MPa, and leaf by 0.50MPa, resulting in partial turgor maintenance during the stress cycle. Pressure/volume curves confirmed the occurrence of osmotic adjustment. Leaf depression was associated with an altered response of chloroplasts to low in vitro. Optimum reaction medium for photosynthesis shifted from –1.04 to –1.57MPa, and low was not as inhibitory to photosynthesis of plastids pre-exposed to stress in situ. These data indicate that chloroplasts acclimate to low external in response to leaf water deficits. This response was still evident four days after a stress cycle ended, but was nearly reversed eight days after stress. Repeated stress cycles in situ did not increase the degree of chloroplast acclimation to low in vitro. Fast dehydration of leaves did not induce this apparent chloroplast acclimation.Abbreviations osmotic potential - _w water potential - PEG polyethylene glycol 8000 - MPa megapascals     

Accumulation of silver by Chromatium vinosum from solutions containing silver thiosulfate

The photosynthetic sulfur bacterium, Chromatium vinosum, was cultured in inorganic photographic processing solutions containing silver thiosulfate complex salt (AgNa₃(S₂O₃)₂) under light. It was found that Chromatium was resistant to Ag and accumulated granular silver in the membrane during growth. The amount of Ag accumulated in the cells depended on the initial concentrations of the Ag salt in the culture solution. When the concentration of Ag was 300 mg/l, the bacteria accumulated Ag as high as 30% of the dry cell weight. The size of the granules was 0.1 to 0.3 m. Results from X-ray microanalysis indicated that these granules consisted mostly of Ag^o with small fractions of Ag₂S and AgCl.