Production of saikosaponins by tissue culture of Bupleurum falcatum L.

The studies for production of saikosaponins by tissue culture of Bupleurum falcatum L. were carried out to produce saikosaponins with several kinds of media and plant hormones. Among the media and plant hormones studied, Gamborg's B-5 [23] medium containing 0.5 ppm kinetin (k) and 1.0 ppm 3-indolebutyric acid (IBA) was the most effective medium and hormone for production of saikosaponins. The highest content of saikosaponin-d in the dried cells was 0.26%, which was similar to a concentration of Bupleuri Radix.Abbreviations MS medium used by Murashige and Skoog [22] - G medium used by Gamborg (B 5) [23] - W medium used by White [24] - NN medium used by Nitsch and Nitsch [25] - k Kinetin - BAP 6-Benzylaminopurine - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA -Naphtylacetic acid - IBA 3-Indolebutyric acid - IAA 3-Indoleacetic acid - ssd saikosaponin-d - PM Production medium - dw Dry weight     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

An effective method for axillary bud culture and RAPD analysis of cloned plants in tetraploid black locust

An efficient micropropagation technique by axillary bud multiplication was established for cloning tetraploid black locust tree (Robinia pseudoacacia L.). The result showed that the optimal medium for shoot multiplication and elongation was Murashige and Skoog (MS) medium supplemented with 0.5 mg/l 6-benzylaminopurine in combination with 0.5 mg/l kinetin and 0.1 mg/l 1-naphthaleneacetic acid. The best medium for rooting was half-strength MS medium with 0.25 mg/l indole-3-butyric acid. In the present report, we examined the genetic fidelity of the micropropagated plants by the randomly amplified polymorphic DNA (RAPD) method with 25 primers. The cloned plants of tetraploid black locust showed complete stability.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - KIN Kinetin - MS Murashige and Skoog medium - NAA 1-Naphthaleneacetic acid - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNACommunicated by M. Beckert     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Extracellular Formation of O-Butylhomoserine by Anaerobes

Clostridium BB–264, Cl. acetobutyricum 314–48, Cl. kaneboi, Cl. saccharoperbutylacetonicum and other two strains of Cl isolated recently produced an unidentified ninhydrin-positive compound in medium containing 5 % glucose, 1 % ammonium acetate, 0.1 % potassium dihydrogen phosphate, 0.04 % magnesium sulfate, 0.001 % ferrous sulfate, 0.1 % yeast extract, 10 μg/liter of biotin and 1 % calcium carbonate.

This ninhydrin-positive compound was eluted with solvent composed of butanol: acetic acid: water (4: 1: 2) by chromatography on cellulose powder column. It was crystallized from ethanol and then identified as an amino acid, O-butylhomoserine (Abbrev. as O-BHSer). Yield of this amino acid increased by adding homoserine or butanol to the medium. The increase was also recognized with addition of glycine, lysine, serine, threonine or valine. The formation of this amino acid was repressed by adding methionine to the medium.

Gas pressure to the culture is one of the important factors that make the amino acid formation by anaerobes possible.     

Volatile acid production from threonine,valine, leucine and isoleucine by clostridia

The amounts of the volatile acids produced from thereonine, valine, leucine and isoleucine by growing cultures of clostridia have been measured. The species used were Clostridium sporogenes; C. caloritolerans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. botulinum proteolytic type F; C. botulinum proteolytic type G; C. putrificum; C. difficile; C. ghoni; C. bifermentans; C. sordellii; C. mangenoti; C. cadaveris; C. lituseburense; C. propionicum; C. sticklandii; C. scatologenes; C. subterminale; C. putrefaciens; C. histolyticum; C. tetanomorphum; C. limosum; C. lentoputrescens; C. tetani; C. melanomenatum; C. cochlearium; C. sporospheroides. Most of the species tested gave increased yields of propionic acid when grown in the threonine medium; in addition, some species resembled C. propionicum and produced n-butyric acid when grown in this medium. C. histolyticum produced only acetic acid in the basal medium; all seven strains of this species produced more acetic acid when grown in the threonine medium than in the basal medium. Species which oxidize valine to iso-butyric acid also oxidize leucine to 3-methyl butyric acid and isoleucine to 2-methylbutyric acid. The iso-caproic fraction produced by some species is shown to be derived from leucine. The identitity of the branched-chain acids produced by C. sporogenes has been confirmed by gas liquid chromatography/mass spectrometry.Abbreviations GLC gas liquid chromatography - RCM reinforced clostridial medium - VFA volatile fatty acid     

Regulation of phenylpropanoid metabolism by exogenous precursors in axenic cultures of Sphagnum fallax

Sphagnum plantlets, cultivated in continuous-feed bioreactors, are characterised by high levels of free endogenous phenolics and a pronounced excretion of some phenolics into the effluent culture medium. The transfer of Sphagnum fallax, precultivated in continuous-feed bioreactors, to batch cultures resulted in an increased flux through phenylpropanoid metabolism and an accumulation of p-coumaric acid to 0.1 μM and of trans-sphagnum acid up to 0.5 μM in the external medium [³H]-labelled L-phenylalanine (7.7 GBq mol^?1) was rapidly taken up, resulting in an enhanced synthesis and excretion of p-coumaric and trans-sphagnum acid. Specific activities were 6.9 and 5.4 GBq mol^?1, respectively, for these cinnamic acids excreted into the external medium. Endogenous pools of trans-cinnamic and p-coumaric acid did not increase and no labelling could be detected in these compounds. Cell wall-bound activity amounted to ca 14% of the applied activity after 48 h of incubation, 59% of which was recovered in dioxane/2 M HCl extracts of the cell wall. Exogenously applied trans-cinnamic acid (0.1 mM) was taken up to 46% and resulted in a transient endogenous accumulation of trans-cinnamic acid, the level of free endogenous p-coumaric and trans-sphagnum acid was found to have decreased. The concentrations of p-coumaric and trans-sphagnum acid in the culture medium rose to 17 and 2.4 μM, respectively, after 48 h of incubation in 0.1 mMtrans-cinnamic acid. Exogenously applied p-coumaric acid (0.1 mM) was taken up to 79% from the incubation solution but not stored endogenously, as metabolic products trans-sphagnum acid and an unknown p-coumaric acid-conjugate accumulated in the external medium and endogenously. These results give evidence for the biosynthetical route from phenylalanine to sphagnum acid and a channelling of pathway intermediates by the enzymes L-phenylalanine ammonia-lyase (EC 4.3.1.5) and cinnamic acid 4-hydroxylase (EC 1.14.13.11).     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

Beneficial Effect of Corncob Acid Hydrolysate on the Lipid Production by Oleaginous Yeast Trichosporon dermatis

In this work, corncob acid hydrolysate and its simulated medium whose sugar composition was the same as the corncob acid hydrolysate were used as fermentation substrate for lipid production by oleaginous yeast Trichosporon dermatis. On the corncob acid hydrolysate, after 7 days of fermentation, the biomass, lipid content, lipid yield, and lipid coefficient of T. dermatis were 17.3 g/L, 40.2%, 7.0 g/L, and 16.5%, respectively. Interestingly, during the lipid fermentation on the corncob acid hydrolysate, glucose, xylose, arabinose, and even acetic acid could be well utilized as carbon sources by T. dermatis. Surprisingly, the lipid yield (7.0 g/L) of T. dermatis on the corncob acid hydrolysate was much higher than that (3.8 g/L) on the simulated medium, in spite of the fact that the lipid coefficient (17.4%) on the simulated medium was a little higher. This phenomenon further showed that lignocellulosic acid hydrolysate was a suitable substrate for lipid fermentation by T. dermatis. This work would help the comprehensive utilization of lignocellulosic biomass for lipid production.     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

Callus initiation and plant regeneration from inflorescence primordia of the intergeneric hybrid Agropyron repens (L.) Beauv.xBromus inermis Leyss. cv. nanus on a modified nutritive medium

Plant regeneration from callus of intergeneric hybrid Agropyron repens (L.) Beauv. x Bromus inermis Leyss cv. nanus (AGROMUS) was carried out on a new culture medium designated medium-F. Within 21 days of the plating of inflorescence primordia the initiated callus showed globular structures. From the 21st day of culture, one step plant regeneration occurred on the callus without subculture. The new basal medium reported in this work was effective in callus initiation and plant regeneration of the hybrid AGROMUS by (i) the reduction of the total ion strength (2.6 g/l, 22.5 mM) of macroelements compared to MS (4.5 g/l,45.2 mM), (ii) the use of NH₄NO₃ as the sole N-source, and (iii) the application of KH₂PO₄ at an 8 times higher concentration (1160 mg/l,8.5 mM) when compared to the Murashige and Skoog medium composition. This medium provided a 2 to 10 fold reduction in the 2,4-dichlorophenoxyacetic acid supplement needed for the callus initiation and one step plant regeneration after a gibberellic acid (2 mg/l, for 5 days) pretreatment of tillers. The regenerated plantlets were subcultured in multi-shoot culture and potted in soil to grow for further analysis.Abbreviations AA amino acid medium (Müller and Grafe 1978, Toriyama and Hinata 1985) - B5 Gamborg et al. (1968) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - N6 Chu et al. (1975) medium - SH Schenk and Hildebrandt (1972) medium - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Co-production of caffeic acid and p-hydroxybenzoic acid from p-coumaric acid by Streptomyces caeruleus MTCC 6638

In a culture medium of Streptomyces caeruleus MTCC 6638 grown with p-coumaric acid (5 mM) as the sole source of carbon, co-production of caffeic acid and p-hydroxybenzoic acid was observed. Both caffeic acid and p-hydroxybenzoic acid are important phenolic compounds with pharmaceutical importance. These biotransformed products were identified by high-performance liquid chromatography and electrospray ionization mass spectrometry. Obtained data suggest that p-coumaric acid was possibly utilized by two different routes, resulting in the formation of a hydroxycinnamate and a hydroxybenzoate compound. However, higher concentration of p-coumaric acid (10 mM) favoured caffeic acid formation. Addition of 5 mM p-coumaric acid into S. caeruleus cultures pre-grown on minimal medium with 1.0 g/l glucose resulted in the production of 65 mg/l caffeic acid. Furthermore, S. caeruleus cells were able to produce the maximum amount of caffeic acid when pre-grown on nutrient broth for 16 h. Under this condition, the addition of 5 mM p-coumaric acid was sufficient for the S. caeruleus culture to produce 150 mg/l caffeic acid, with a molar yield of 16.6% after 96 h of incubation.     

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

Summary Thirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.     

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

Summary Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×10³ ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants

Summary Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.Abbreviations NAA -napthaleneacetic acid - KT Kinetin - 2,4-D 2, 4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - BAP N⁶ benzylaminopurine - NFM NaCl-free medium     

Effects of plant growth regulators on high frequency shoot multiplication and callus regeneration of an important Indian medicinal plant, nirgundi (Vitex negundo L.)

Summary A rapid shoot multiplication protocol was established for an important medicinal plant, Vitex negundo L., belonging to the family Verbenaceae, using Murashige and Skoog medium, achieved by shoot multiplication as well as callus regeneration. Shoot multiplication was induced by different concentrations of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ), Benzyladenine and 6-furfuryl amino purine separately along with 10% (v/v) coconut water. Green organogenetic callus was obtained by the combined effect of 0.5–2.15 μM TDZ and 1.7 μM indole-3-acetic acid (IAA) along with 1% polyvinylpyrrolidone (PVP), and produced the maximum number of shoots when subcultured onto medium containing 2.7 μM TDZ alone. Elongation of in vitro shoots was observed in MS medium containing 2.4 μM gibberellic acid and rooting was induced by the combined effect of 1.71 μM IAA and 1.62 μM α-naphthalene acetic acid.     

Inhibitory action of toxic compounds present in lignocellulosic hydrolysates on xylose to xylitol bioconversion by <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8–2.6 g/l), ferulic acid (0.2–0.6 g/l), and/or syringaldehyde (0.3–0.8 g/l), according to a 2³ full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds, mainly by acetic acid.     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Sugar-cane pressmud as a novel and inexpensive substrate for production of lactic acid in a solid-state fermentation system

Lactobacillus casei subsp. casei CFTRI 2022 produced a higher concentration of lactic acid (5.27 g/100 g dry sugar-cane pressmud) in a solid-state fermentation (SSF) system as compared to L. helveticus CFTRI 2026 and Streptococcus thermophilus CFTRI 2034. The lactic acid production by L. casei subsp. casei CFTRI 2022 was found to be significantly influenced by the initial moisture content, initial pH and initial sugar concentration of the medium. Studies on four inert materials to reduce the initial sugar concentration in the medium showed the high potential of microcrystalline cellulose whereas the use of diatomaceous earth, acid-washed river sand and washed pith bagasse posed problems. The data indicate the potential of lactic acid production from sugar-cane pressmud in an SSF system.