Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.     

Taxane Production in Suspension Culture of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">Media</Emphasis> var. <Emphasis Type="Italic">Hicksii</Emphasis> Carried Out in Flasks and Bioreactor

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus × media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l⁻¹ (on day 14) and 37.5 mg l⁻¹ (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l⁻¹ (on day 4) and 8.8 mg l⁻¹ (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l⁻¹ to 0.6 mg l⁻¹ and to 0.2 mg l⁻¹, respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l⁻¹ to 1.6 mg l⁻¹.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Elicitation of guggulsterone production in cell cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis> by plant gums

Plant gum as an elicitor for guggulsterone production in cell cultures of Commiphora wightii is reported for the first time. Guggulsterone production increased 2.4 fold in the cell cultures by gum Arabic (100 mg l⁻¹), while mesquite gum elicited 2 fold. The cells treated with gum Arabic at 7th and 9th day accumulated enhanced guggulsterones within 24 h, which increased further up to 48 h and then declined. The cells treated at 9th day accumulated higher amount (218 μg l⁻¹) of guggulsterones after 48 h of elicitation as compared to cells treated at 7th day (164 μg l⁻¹). The optimized elicitation conditions were used in vessels of varying capacity where maximum yield of 285 μg l⁻¹ of guggulsterones was recorded in 3 l shake flasks. These experiments enabled highest guggulsterones yield in a short duration of 11 days in cell cultures of C. wightii.     

Xylitol conversion by fermentation using five yeast strains and polyelectrolyte-assisted ultrafiltration

Batch fermentations for xylitol production were conducted using Candida boidinii (BCRC 21432), C. guilliermondii (BCRC 21549), C. tropicalis (BCRC 20520), C. utilis (BCRC 20334), and P. anomala (BCRC 21359) together with a mixture of sugars simulating lignocellulosic hydrolysates as the carbon source. C. tropicalis had the highest bioconversion yield (Y_P/S) of 0.79 g g⁻¹ (g xylitol·g xylose⁻¹) over 48 h. Additional fermentations with C. tropicalis achieved Y_P/S values of 0.6 and 0.39 g g⁻¹ after 96 and 72 h using urea and soybean meal as the nitrogen sources, respectively. Ethanol and arabitol were also produced in all fermentation. Xylitol in the fermentation broth was recovered by cross-flow ultrafiltration. With prior application of 2 mg polydiallyl dimethylammonium chloride l⁻¹ on the membrane surface, protein in the permeate was reduced from 7.1 to 1.5 mg l⁻¹ after 2 h.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l⁻¹) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l⁻¹) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l⁻¹ TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Redirecting Carbon Flux in Torulopsis glabrata from Pyruvate to α-Ketoglutaric Acid by Changing Metabolic Co-factors

The nutrition conditions needed to redirect the carbon flux in Torulopsis glabrata, a pyruvate hyper-production yeast, from pyruvate to α-ketoglutaric acid (KG) were investigated in a stirred fermentor. A minor amount of KG (1.3 gl⁻¹) was produced when NaOH was used to control the pH, while 12 g KG l⁻¹ was produced when CaCO₃ was used instead. When thiamine and biotin were included in the medium, 13 g KG l⁻¹ and 68 g pyruvate l⁻¹ were produced after 48 h when glucose was nearly consumed (approximately 5 gl⁻¹). With fermentation continuing for a further 16 h, the concentration of pyruvate decreased to 31 gl⁻¹, and KG increased to 30 gl⁻¹. KG thus accumulated at the expense of pyruvate consumption. Received 2 June 2005; Revisions requested 30 June 2005 and 1 September 2005; Revisions received 1 September 2005 and 28 October 2005; Accepted 28 October 2005     

Repeated Batch Cultivation of Ralstonia eutropha for Poly (β-hydroxybutyrate) Production

Batch cultivation of Ralstonia eutropha NRRL B14690 attained 21 g biomass l⁻¹ and 9.4 g poly(β-hydroxybutyrate) l⁻¹ (0.45 g PHB g⁻¹ dry wt⁻¹) in 60 h. Repeated batch operation (empty-and-fill protocol) to remove 20% (v/v) of the culture broth and to supplement an equal volume of fresh media resulted in 49 g biomass l⁻¹ and 25 g PHB l⁻¹ (0.51 g PHB g⁻¹ dry wt⁻¹) with an overall productivity of 0.42 g PHB l⁻¹ h⁻¹ in 67 h. In the two cycles of repeated batch fermentation there was a 3-fold increase in productivity as compared to batch.     

Effect of ethrel,chlormequat chloride and paclobutrazol on growth and pyrethrins accumulation in <Emphasis Type="Italic">Chrysanthemum cinerariaefolium</Emphasis> Vis.

Pyrethrins and flower yield of pyrethrum (Chrysanthemum cinerariaefolium Viz.) plants were determined after application of ethrel, chlormequat chloride and paclobutrazol. Ethrel at 50, 100, 250 and 500 mg l^−l produced a significant positive effect on pyrethrins level, decreased plant height, while 50 and 100 mg l^−l significantly increased fresh and dry flower yield. Chlormequat chloride at 1000 and 2000 mg l^−l and paclobutrazol (80 and 160 mg l^−l) increased pyrethrins level, single flower weight and decreased plant height and flower yield. ¹⁴C-acetate incorporation studies further substantiated positive effect of growth retardants on pyrethrins biosynthesis. The effect of growth retardants on pyrethrins seems to be mediated through its effect on biosynthesis.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Heterotrophic production of eicosapentaenoid acid by the diatom Nitzschia laevis: effects of silicate and glucose

The effects of silicate and glucose on growth and eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis were studied. By alternately altering the concentrations of silicate (2.7–64 mg l⁻¹) and glucose (1–40 g l⁻¹) in the medium, the highest cell dry weight (ca. 5.5 g l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest specific growth rate (ca. 0.65 day⁻¹) was obtained at a relatively low glucose concentration (5 g l⁻¹) and high silicate concentrations (32–64 mg l⁻¹). At glucose levels of 5 and 20 g l⁻¹, EPA content was higher with lower silicate concentrations (2.7 and 16 mg l⁻¹ silicate, respectively), while at a silicate level of 16 mg l⁻¹, higher glucose concentrations (20–40 g l⁻¹) facilitated EPA formation. The highest EPA yield (131 mg l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest EPA productivity (15.1 mg l⁻¹ day⁻¹) was obtained at 20 g l⁻¹ glucose and 64 mg l⁻¹ silicate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 218–224. Received 08 May 2000/ Accepted in revised form 21 July 2000     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

Decolorization of 1:2 metal complex dye Acid blue 193 by a newly isolated fungus, Cladosporium cladosporioides

Summary A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l⁻¹ in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l⁻¹) concentration of 564 mg l⁻¹ within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l⁻¹, laccase activity with 1413 U l⁻¹ and lignin peroxidase activity was negligible after day 8 of incubation.