Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Biotransformation of steriodal saponins in Dioscorea zingiberensis C. H. Wright to diosgenin by Trichoderma harzianum

Diosgenin is an important starting material in the steroidal hormone industry. Traditionally, diosgenin is mainly produced by acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) tubers. This method yields numerous byproducts that can cause serious pollution. In this study, diosgenin was obtained by biotransformation of steroidal saponins in DZW afforded by Trichoderma harzianum CGMCC 2979. The medium was optimized for maximum diosgenin production. The addition of phosphate buffer, surfactant Tween-85, and Fe²⁺ increased the yield of diosgenin by 50.28%, 33.35%, and 22.07%, respectively. The optimum medium obtained by response surface methodology was composed of 60 mmol l⁻¹ phosphate buffer, 0.07% (w/v) Tween-85, and 0.93 mmol l⁻¹ Fe²⁺. Under these conditions, a maximum diosgenin yield of 30.05 ± 0.59 mg g⁻¹ was achieved, which was slightly higher than that obtained from traditional acid hydrolysis. By hydrolyzing the un-transformed steroidal saponins after biotransformation, the total diosgenin yield increased by 35% compared to traditional method. Moreover, chemical oxygen demand and residual reduced sugar in the wastewater produced by this integrated process were only 3.72% and 0.3%, respectively, that of the traditional acid hydrolysis method.     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Chemostat study of xylitol production by Candida guilliermondii

The mechanism of production of xylitol from xylose by Candida guilliermondii was studied using chemostat cultures and enzymatic assays. The maximum dilution rate in aerobic conditions was 0.34 1/h. No xylitol was produced. Under oxygen-limited conditions xylose uptake was impaired and glycerol accumulated but no xylitol was detected. Under transient oxygen limitation, caused by a gradual decrease in the agitation rate, onset of xylitol, acetate and residual xylose accumulation occurred simultaneously when q _O2 dropped below 25 mmol/C-mmol cell dry weight (CDW) per hour. Ethanol and glycerol started to accumulate when q _O2 dropped below 20 mmol/C-mmol CDW per hour. The highest in vitro enzyme activities were found at the lowest dilution rate studied (0.091/h) under aerobic conditions. The amount of active enzymes or cofactor availability did not limit the rate of xylose consumption. Our results confirm that a surplus of NADH during transient oxygen limitation inhibited the activity of xylitol dehydrogenase which resulted in xylitol accumulation. Phosphoglucoisomerase (E.C. 5.3.1.9.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) activities suggest re-shuttling of the metabolites into the pentose phosphate pathway. Received: 7 March 2000 / Received revision: 9 June 2000 / Accepted: 18 June 2000     

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Effects of yeast extract on the production and the quality of the exopolysaccharide, zooglan, produced by Zoogloea ramigera 115SLR

Although many studies have examined the influence of culture conditions on the production and composition of polysaccharides, little is known about the factors influencing the quality of exopolysaccharides (EPS). In this work we studied the effect of yeast extract on the production, composition and molecular weight of the EPS zooglan produced by Zoogloea ramigera 115SLR. This bacterium was grown on a new completely defined synthetic medium and on a medium containing yeast extract. Growth and polysaccharide production performances were comparable on the two media with a glucose to exopolysaccharide conversion yield of 35% (g/g). The polysaccharides produced on these two media have an identical composition but a different molecular weight and molecular weight distribution. The yeast extract medium leads to a more homogeneous polysaccharide solution. Received: 12 June 1998 / Received revision: 19 September 1998 / Accepted: 11 October 1998     

High-efficiency somatic embryogenesis and plant regeneration in California poppy, Eschscholzia californica Cham.

 The development of a rapid protocol for high-efficiency somatic embryogenesis and plant regeneration from seed-derived embryogenic callus cultures of California poppy (Eschscholzia californica Cham.) is reported. The optimized procedure required less than 13 weeks from the initiation of seed cultures to the recovery of plantlets and involved the sequential transfer of cultures onto solid Murashige and Skoog basal medium containing three different combinations of growth regulators. All steps were performed at 25  °C. Friable primary callus was induced from seeds of E. californica cultured on medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The primary callus was transferred to medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid and 0.5 mg l⁻¹ 6-benzylaminopurine to establish embryogenic callus and promote somatic embryogenesis. Regenerated plantlets were recovered after the conversion of somatic embryos on medium containing 0.05 mg l⁻¹ 6-benzylaminopurine and showed normal development. Embryogenic callus was induced at a frequency of 85%, an average of 45 somatic embryos were produced per callus, 90% of the somatic embryos converted, and about 70% of the plantlets were recovered in soil. The growth rate of somatic embryo-derived shoots could be increased by gibberellic acid treatment, but the resulting plantlets were hyperhydritic. Received: 14 February 1999 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO₃. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO₃, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C. Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123

Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v) wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation. Received: 22 January 1999 / Received revision: 23 April 1999 / Accepted: 1 May 1999     

In vitro regeneration of loblolly pine and random amplified polymorphic DNA analyses of regenerated plantlets

 Adventitious buds were induced from organogenic callus derived from mature zygotic embryos of three lines (E-311, E-440, and E-822) of loblolly pine (Pinus taeda L.) within 27 weeks of culture. The influence of cytokinins, silver nitrate, and low-temperature treatment on the differentiation of adventitious buds was analyzed. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 1 mg/l 6-benzyladenine (BA). After adventitious shoots had rooted on TE medium supplemented with 0.5 mg/l IBA, 2 mg/l BA, and 0.5 mg/l gibberellic acid, 498 regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1 :>: 1) soil mixture; 351 of these survived in the field. Total DNA was extracted from 21 regenerated plantlets randomly chosen from the 151 regenerated plantlets of line E-822. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed that 21 primers gave 107 clear reproducible bands, with the amplification products being monomorphic for all of the plantlets of line E-822 tested. A total of 2,247 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested plantlets. These results suggest that somatic organogenesis can be used for clonal micropropagation of some lines of loblolly pine without the fear of the appearance of unwanted somaclonal variants. Received: 5 August 2000 / Revision received: 5 September 2000 / Accepted: 10 October 2000     

Xylitol and riboflavin accumulation in xylose-grown cultures of Pichia guilliermondii

Seven strains of Pichia guilliermondii (Candida guilliermondii, asexual state) from diverse isolation sources were examined for the production of xylitol and riboflavin in xylose-grown cultures. Under the conditions tested, all strains produced xylitol from xylose; conversion efficiencies varied, on a strain-specific basis, from 7% to 36% of the initial substrate. Four of seven strains metabolized xylitol immediately as xylose levels became depleted. The remaining three strains metabolized xylitol slowly and incompletely. Surprisingly, utilization of xylitol showed an apparent relationship with riboflavin production. Strains that readily metabolized xylitol produced at least threefold greater levels of riboflavin than did strains that used xylitol slowly. Moreover, riboflavin accumulation took place during xylitol consumption. P. guilliermondii strains that produced the highest levels of riboflavin on xylose produced significantly less riboflavin when grown on glucose or directly on xylitol. Received: 24 April 1996 / Received revision: 29 July 1996 / Accepted: 24 August 1996     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

 Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis. Received: 28 February 2000 / Revision received: 18 August 2000 / Accepted: 22 August 2000