Nitrogen and phosphorus as the factors affecting ginsenoside production in hairy root cultures of <Emphasis Type="Italic">Panax quinquefolium</Emphasis> cultivated in shake flasks and nutrient sprinkle bioreactor

The study assesses the influence of different concentrations of nitrogen and phosphorus sources on ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The saponin content was determined using HPLC. The maximum yield (12.45 mg g^?1 dw) of the sum of six examined ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1) in hairy roots cultivated in shake flasks was achieved in modified Gamborg B-5 medium containing 0.83 mM l^?1 phosphate, 12.4 mM l^?1 nitrate and 0.5 mM l^?1 ammonium. The yield itself was 1.93 times higher than that achieved in standard Gamborg medium. The modified medium also favourably influenced the biosynthesis of studied saponins in bioreactor cultures. The saponin content (35.11 mg g^?1 d.w.) was 2.75-times higher than that achieved in control medium.     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml^?1) were obtained with media containing soy peptone (3–6 g l^?1) and glutamate (3.6 g l^?1). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml^?1) were also much greater (8–150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Definition of culture conditions for Arxula adeninivorans,a rational basis for studying heterologous gene expression in this dimorphic yeast

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l^?1 was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l^?1. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h^?1 and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.     

Reaction engineering analysis of cellulase production with Trichoderma reesei RUT-C30 with intermittent substrate supply

The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L^?1 microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L^?1 day^?1 intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL^?1 with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL^?1).     

Micropropagation of cocoyam (Xanthosoma sagittifolium L. Schott) in temporary immersion bioreactor

This study aims at establishing a temporary immersion technique for large-scale propagation of cocoyam (Xanthosoma sagittifolium). Sucrose was experimented with as a determinant factor for shoot multiplication in this culture system. The highest proliferation rate (68 ± 7) occurred with 20 g l^?1 sucrose in the culture medium. This concentration appeared to be the optimal amount due to its promoting effect on plantlet development. The acclimatized plantlets showed a continuous effect of sucrose treatment during ex vitro growth, especially in low sucrose concentration. This fact is evidenced by the low survival rate (0.13 ± 0.12) and the poor chlorophyll content (1.180 ± 0.076 mg g^?1) recorded on plantlets derived from 15 g l^?1 of sucrose. The treatment with 60 g l^?1 of sucrose prior to acclimatization was efficient for roots induction and elongation. The analysis of pH revealed a fluctuation from one subculture to another, with an overall pH decrease under all treatments tested and, thus, indicates that plants release proton during growth. This feature had an impact on in vitro plantlets growth. The potentiality of the temporary immersion technique to foster the production of Xanthosoma sagittifolium is discussed.     

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal

Mezcal from Tamaulipas (México) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semi-synthetic media. Medium M1 had a sugar content of 100 g l^?1 and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l^?1 and a G/F of 1:9. Medium M3 had a sugar content of 200 g l^?1 and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l^?1) and acetic acid (1 g l^?1) in M2 than in M1 (1.7 and 0.5 g l^?1, respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l^?1 but left a residual 32 g of glucose l^?1. Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains.     

The synergistic effects of sucrose and plant growth regulators on morphogenesis and evaluation of antioxidant activities in regenerated tissues of <Emphasis Type="Italic">Caralluma tuberculata</Emphasis>

Caralluma tuberculata (C. tuberculata) is a very important medicinal plant with a range of anti-diabetic and weight reduction properties. This high-valued medicinal plant is nowadays considered as endangered due to its unsustainable elimination from wild habitats. There is lack of research efforts on its propagation to overcome escalating demand. In this research study, an effort has been made to optimize protocol for large-scale mass propagation and production of natural antioxidants. Highest callogenic response (87.2 %) was observed from shoot tip explants on Murashige and Skoog (MS) medium containing 30 g l^?1 sucrose and combination of 2, 4-D (2.0 mg l^?1) and BA (1.0 mg l^?1). During shoot morphogenesis, 50 g l^?1 sucrose along with BA (2.0 mg l^?1) and GA₃ (1.0 mg l^?1) enhanced shoot regeneration (91.3 %), mean shoot length (2.6 cm) and shoots per explant (24.5) as compared to control. The combination of IBA and IAA (2.0 mg l^?1) was found optimum for root induction (74.98 %), mean root length (4.1 cm) and roots per shoot (6.9) as compared to control. The plantlets were successfully acclimatized in plastic cups and various tissues were investigated for accumulation of antioxidant secondary metabolites including phenolics, flavonoids, stress enzymes and antioxidant activities. The superoxide dismutase enzyme was higher in shoots; protein content was higher in callus cultures; phenolics, DPPH and protease activity were higher in plantlets, while flavonoids, peroxidase, reducing power and total antioxidant activities were higher in wild plants. This simple protocol is very useful for commercial production of consistent plantlets and metabolites of interest.     

In vitro propagation of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. by somatic embryogenesis in cell suspension culture

An effective protocol was developed for in vitro propagation of Psoralea corylifolia via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 6 μM naphthaleneacetic acid (NAA) and 30 μM glutamine from transverse TCLs from 10-day-old hypocotyl explants with a 96.4% frequency. Embryogenic callus produced a higher number of somatic embryos (123.7 ± 1.24 per gram fresh weight callus) on MS medium containing 30 g l^?1 sucrose, 1 μM NAA, 4 μM benzyladenine (BA), 15 μM glutamine and 2 μM abscisic acid (ABA) after 4 weeks of culture. Somatic embryos successfully germinated (97.6%) on ½ MS medium containing 20 g l^?1 sucrose, 8 g l^?1 agar and supplemented with 2 μM BA, 1 μM ABA and 2 μM gibberellic acid (GA₃) within 2 weeks of culture. Somatic embryos developed into normal plants, which hardened with 100% efficiency in soil in a growth chamber. Plants were successfully transferred to greenhouse and subsequently established in the field. Plant survival percentage in the field differed with seasonal variations. Average psoralen content of 12.9 μg g^?1 DW was measured in different stages of somatic embryo development by high-performance liquid chromatography (HPLC). This protocol will be helpful for efficient propagation of elite clones on a mass scale, conservation efforts of this species and for secondary metabolites production studies.     

Effects of dynamic changes of nutrients on adventitious roots growth and periplocin accumulation in culture of Periploca sepium Bunge

In this study, the dynamics of biomass production, accumulation of periplocin, medium conditions and consumption of carbon, nitrogen and phosphate were investigated in adventitious roots culture of Periploca sepium in shake flasks over a period of 4 weeks. The biomass reached the maximum peak on day 24 (2.46 and 0.213 g of fresh and dry weight, respectively). Similarly, periplocin production got to a peak of 0.083 mg g^?1 on day 24, simultaneously. PH in medium had a decrease tendency at the beginning and then remained stable around 4.0, however, EC declined during the whole culture with the nutrients consumed. Sucrose was almost used up on the first 12 days which led to the increase in glucose and fructose. In case of nitrogen, consumption of ammonium is faster than nitrate at the beginning. Phosphate was almost consumed during the first 8 days. Based on the nutrients consumption, the adding time of nutrients (1/2 MS medium and 30 g L^?1 sucrose) was investigated and it obtained highest content of periplocin (0.106 mg g^?1) and yield (0.513 mg L^?1) on day 12.     

Withanolide A production from Withania somnifera hairy root cultures with improved growth by altering the concentrations of macro elements and nitrogen source in the medium

Withania somnifera is an important medicinal plant that contains withanolides as bioactive compounds. We have investigated the effects of macroelements and nitrogen source in hairy roots of W. somnifera with the aim of optimizing the production of biomass and withanolide A content. The effects of the macroelements NH₄NO₃, KNO₃, CaCl₂, MgSO₄ and KH₂PO₄ at concentrations of 0, 0.5, 1.0, 1.5 and 2.0× strengths and of nitrogen source [NH₄ ⁺/NO₃ ^? (0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20 and 14.38/37.60 mM)] in Murashige and Skoog medium were evaluated for biomass and withanolide A production. The highest accumulation of biomass (139.42 g l^?1 FW and 13.11 g l^?1 DW) was recorded in the medium with 2.0× concentration of KH₂PO₄, and the highest production of withanolide A was recorded with 2.0× KNO₃ (15.27 mg g^?1 DW). The NH₄ ⁺/NO₃ ^? ratio also influenced root growth and withanolide A production, with both parameters being larger when the NO₃ ^? concentration was higher than that of NH₄ ⁺. Maximum biomass growth (148.17 g l^?1 FW and 14.79 g l^?1 DW) was achieved at NH₄ ⁺/NO₃ ^? ratio of 14.38/37.60 mM, while withanolide A production was greatest (14.68 mg g^?1 DW) when the NH₄ ⁺/NO₃ ^? ratio was 0.00/18.80 mM. The results are useful for the large scale cultivation of Withania hairy root culture for the production of withanolide A.     

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO₂ g dry cell weight (DCW)^?1 h^?1, the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO₂ g DCW^?1 h^?1 allowed maximization of the ethanol yield over xylose (0.327 g g^?1), the average productivity (2.2 g l^?1 h^?1) and the ethanol final titer (48.81 g l^?1). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g^?1) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO₂ g DCW^?1 h^?1, whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l^?1 h^?1 and 54.19 g l^?1 when the specific OUR was the highest.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l^?1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l^?1 α-naphthalene acetic acid and 1 g l^?1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l^?1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.     

Evaluation of ethanol production by a new isolate of yeast during fermentation in synthetic medium and sugarcane bagasse hemicellulosic hydrolysate

A new xylose fermenting yeast was isolated from over-ripe banana by enrichment in xylose-containing medium. The phylogenetic analysis of ITS1-5.8S-ITS2 region sequences of ribosomal RNA of isolate BY2 revealed that it shows affiliation to genus Pichia and clades with Pichia caribbica. In batch fermentation, Pichia strain BY2 fermented xylose, producing 15 g l^?1 ethanol from 30 g l^?1 xylose under shaking conditions at 28°C, with ethanol yield of 0.5 g g^?1 and volumetric productivity of 0.31 g l^?1 h^?1. The optimum pH range for ethanol production from xylose by Pichia strain BY2 was 5–7. Pichia strain BY2 also produced 6.08 g l^?1 ethanol from 30 g l^?1 arabinose. Pichia strain BY2 can utilize sugarcane bagasse hemicellulose acid hydrolysate for alcohol production, efficiency of fermentation was improved by neutralization, and sequential use of activated charcoal adsorption method. Percent total sugar utilized and ethanol yield for the untreated hydrolysate was 17.14% w/v and 0.33 g g^?1, respectively, compared with 66.79% w/v and 0.45 g g^?1, respectively, for treated hemicellulose acid hydrolysate. This new yeast isolate showed ethanol yield of 0.45 g g^?1 and volumetric productivity of 0.33 g l^?1 h^?1 from sugarcane bagasse hemicellulose hydrolysate detoxified by neutralization and activated charcoal treatment, and has potential application in practical process of ethanol production from lignocellulosic hydrolysate.     

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l^?1 glutamine, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l^?1 activated charcoal, and 500 mg l^?1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization

Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l^?1), yeast extract (25.93 g l^?1), and corn steep liquor (10.45 g l^?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW^?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet^?1 h^?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.     

Enhanced production of hairy root metabolites using microbubble generator

Previously, increased partitioning of the natural product nicotine from tobacco hairy roots into the culture media was achieved by altering the expression of the nicotine uptake permease gene. The present study demonstrated that further increases in nicotine yield in the media were attained by using surfactant-stabilized microbubbles. Compared to other non-ionic surfactants (Tween 20 and Tween 80) and the ionic surfactant SDS, Triton X-100 (TX100) both increased total nicotine production and exudation into the hairy root culture media. In comparison to surfactant-free medium, TX100 at 10, 25, and 50 mg l^?1 did not show strong inhibition of hairy root growth. At 4,000 rpm shear speed, microbubbles stabilized by 10, 25, and 50 mg l^?1 TX100 had k _L a of 22.3, 36.2, and 44.1 h^?1 in Gamborg’s B5 medium, respectively, in comparison to 16.4 h^?1 with conventional air sparging. In a 1-l bioreactor, microbubbles stabilized by TX100 were applied to hairy roots after the inoculated root tips were self-immobilized by branching. With microbubble dispersion, dissolved oxygen rapidly increased from 60 to 85 %, and hairy root growth rate increased. Nicotine accumulation in culture medium with microbubbles reached 146 mg l^?1 after 30 days cultivation. These results show that combining genetic modification with surfactant-stabilized microbubble dispersion can substantially increase levels of nicotine in the media of hairy root cultures.