Sucrose and light effects on in vitro cultures of potato,chokecherry and saskatoon berry during low temperature storage

Cultures of potato (Solanum tuberosum) cv. Atlantic, chokecherry (Prunus virginiana L.) cv. Garrington and saskatoon berry (Amelancher alnifolia Nutt.) cv. Northline grown in vitro for 3 weeks at 24/22 °C, 16-h photoperiod, 150 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) mixed fluorescent/incandescent light were stored for 6, 9 and 12 weeks at 4 °C under 0 (darkness) and 3 μmol m⁻² s⁻¹ PPFD (690 nm red light continuous illumination). Growth regulators free MSMO medium either with or without 30 g l⁻¹ sucrose was used to store the cultures. All cultures retained capacity to re-grow after storage. Tested factors, sucrose, light and the length of the storage period had an impact on shoot quality and re-growth capacity of the cultures. For either light treatment sucrose was essential for the low temperature maintenance of vigorous stock plants of potato, if stored for over 6 weeks. Chokecherry and saskatoon cultures stored well without sucrose; although chokecherry benefited from sucrose in the storage medium when the stock cultures were kept at the low temperature for 12 weeks. Low light significantly improved quality of the stored potato cultures, but had very little effect on both chokecherry and saskatoon berry cultures. The woody plant cultures grew during storage, and the longer the stock plants were stored, the more vigorous cultures they generated. The results indicate that growers can successfully use their existing facilities, small refrigerators and coolers with low light intensity, set at 4 °C, for short term storage of potato, chokecherry and saskatoon berry cultures. The potato cultures, which are known to be sensitive to prolonged low temperature storage, should be frequently monitored and subcultured as required. On the other hand, the woody plant stock cultures do not require any special attention when kept at 4 °C and re-grow the most vigorous shoots if stored for at least 12 weeks. This revised version was published online in August 2006 with corrections to the Cover Date.     

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     

Temperature and genotype affect asparagus somatic embryogenesis

Summary Calluses from five asparagus genotypes G14, G32, G171, G203, and G447 and hybrid Jersey Giant (JG) were incubated at three temperature regimes (24, 27, and 30°C) on embryo induction medium to assess somatic embryo development and conversion to plantlets. The calluses from three genotypes (G14, G32, and G171) were not responsive, failing to produce somatic embryos at any temperature regime. For three responsive genotypes (G203, G447, and JG), both incubation temperature and genotype significantly affected the numbers of somatic embryos produced. The calluses produced the most and the least numbers of total, bipolar, and globular embryos when incubated at 27°C and 24°C, respectively. When incubated at 27°C, G203 produced the highest numbers of total and globular embryos, 178 g⁻¹ callus and 142 g⁻¹ callus, respectively while G447 produced the highest number of bipolar embryos, 77 g⁻¹ callus. Incubation temperature but not genotype significantly affected the conversion of somatic embryos to plantlets. The somatic embryos recovered from the three responsive genotypes incubated at 27°C also converted to plantlets at the highest frequencies, 60–63% of the bipolar embryos and 42–43% of the globular embryos converted to plantlets, while the somatic embryos recovered from the calluses incubated at 24°C converted to plantlets at the lowest frequencies.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Triacylglycerol phase and ‘intermediate’ seed storage physiology: a study of Cuphea carthagenensis

Seeds with ‘intermediate’ storage physiology store poorly under cold and dry conditions. We tested whether the poor shelf life can be attributed to triacylglycerol phase changes using Cuphea carthagenensis (Jacq.) seeds. Viability remained high when seeds were stored at 25°C, but was lost quickly when seeds were stored at 5°C. Deterioration was fastest in seeds with high (≥0.10 g g⁻¹) and low (0.01 g g⁻¹) water contents (g H₂O g dry mass⁻¹), and slowest in seeds containing 0.04 g g⁻¹. A 45°C treatment before imbibition restored germination of dry seeds by melting crystallized triacylglycerols. Here, we show that the rate of deterioration in C. carthagenensis seeds stored at 5°C correlated with the rate that triacylglycerols crystallized within the seeds. Lipid crystallization, measured using differential scanning calorimetry, occurred at 6°C for this species and was fastest for seeds stored at 5°C that had high and very low water contents, and slowest for seeds containing 0.04 g g⁻¹. Germination decreased to 50% (P50) when between 16 and 38% of the triacylglycerols crystallized; complete crystallization took from 10 to over 200 days depending on water content. Our results demonstrate interactions between water and triacylglycerols in seeds: (1) water content affects the propensity of triacylglycerols to crystallize and (2) hydration of seed containing crystallized triacylglycerols is lethal. We suggest that these interactions form the basis of the syndrome of damage experienced when seeds with intermediate storage physiologies are placed in long-term storage.     

Changes in fungi and mycotoxins in pearl millet under controlled storage conditions

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25°C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25°C in either air (aerobic) or N₂ (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25°C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), “Helminthosporium”-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25°C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20–22%. Aflatoxin contamination averaged 174 ng g⁻¹ over all treatments, and increased up to 798 ng g⁻¹ in high-moisture grain at stored at 25°C.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana?×?C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Induced tillering ofLolium multiflorum in vitro

Four growth regulators incorporated into Murashige and Skoog's basal medium were tested for their effect on the rate of tillering ofLolium multiforum plantlets in culture. Tillering was stimulated in the presence of 1–2 mg l⁻¹ 6-benzylaminopurine, but 1 mg l⁻¹ gave less plantlet distortion and the tillers could be separated easily. Small shoot tips were considered to be best for establishing aseptic cultures for propagation, but individual tillers were best for transferring to the tiller inducing medium, both initially and at subculturing. Tillering rate was affected by culture vessel, temperature and light intensity. Of the variations tested, the optimum conditions were to culture whole tillers onto a medium containing 1 mg l⁻¹ BAP in 30-ml universal tubes at 20°C with continuous white fluorescent light at 12,000 lx for 4–5 weeks. Rates in excess of 40 tillers per month were obtained in culture compared with 5–12 in the glasshouse.     

Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo

Ovaries from squash plants (cv. Eskandarani) were picked one day before anthesis, and exposed to cold temperature (4 °C) for 0, 2, 4 and 8 days. The ovules were cultured on MS medium with 30 g l⁻¹sucrose, 8 g l⁻¹agar and supplemented with four concentrations of 2,4-D, i.e., 0.1, 1.0, 5 and 10 mg l⁻¹. Then the dishes were incubated at 25 ± 1 °C under 16-h photoperiod for 4 weeks. After that ovules were transferred to growth regulator free MS medium for 4 weeks. Data indicated that the most plantlets per 100 cultured ovules resulted from the ovule of ovaries without cold treatment, when cultured in MS medium supplemented with 1 or 5 mg l⁻¹2,4-D. The cytological study revealed that one third of examined plants were haploid (2n = x = 20) and the others were double haploid (2n = 2x = 40). This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency plant regeneration of garlic (Allium sativum L.) calli immobilized in calcium alginate gel

Calli obtained from a shoot-tip of garlic,Allium sativum L., were encapsulated using a calcium alginate gel. Some of the encapsulated calli were cultured on a 1/2 MS medium supplemented with 3% sucrose, 10⁻⁵ M kinetin, and 5×10⁻⁶ M NAA, whereas the remainder was stored for 40 days at 4°C. All the naked calli regenerated on the solid medium, while 95% of the encapsulated calli regenerated, and 88% of the encapsulated calli regenerated after 40 days of storage at 4°C. The capsule matrix delayed the germination time of the encapsulated calli, yet activated the shoot formation of the artificial garlic seeds. The shoot length of the encapsulated garlic calli was much longer than that of the naked garlic calli. The encapsulated garlic calli were dried in a laminar airflow cabinet and the conversion frequency of the dried artificial garlic seeds on a 1/2 MS medium remained at 93% with a water loss of less than 50%.     

Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N⁶-benzyladenine (BAP) each (20×10⁻⁶ M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10⁻⁶ M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10⁻⁶ M) and 2,4-D (5×10⁻⁶ M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10⁻⁶ M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability. Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal mass in vitro.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Production,formulation and antagonistic activity of the biocontrol like-yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> against <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Aureobasidium pullulans (de Bary) Arnaud (Ach 1-1) was grown in a glucose fed-batch fermentor to 106 g dry wt l⁻¹ in 48 h. The cells were dried in a fluidized bed dryer with a final viability of 62%. After 7 months at 4°C, the viability was 28% of the initial value (= 2.3 × 10¹⁰ c.f.u. g⁻¹ dry matter). A protection level of 89% was achieved with the biomass preparation at 1 × 10⁸ c.f.u. ml⁻¹ after 28 and 7 days for apples stored respectively at 5 and 25°C against Penicillium expansum. Our process is suitable to produce large quantities of the strain Ach 1-1 as biological control agent for apple preservation.     

Early development of germlings of <Emphasis Type="Italic">Sargassum thunbergii</Emphasis> (Fucales,Phaeophyta) under laboratory conditions

Morphology and culture studies on germlings of Sargassum thunbergii (Mertens et Roth) Kuntze were carried out under controlled laboratory conditions. Growth characteristics of these germlings grown under different temperatures (from 10 to 25°C), irradiances (from 9 to 88 μmol photons m⁻² s⁻¹), and under blue and white light conditions are described. The development of embryonic germlings follows the classic “8 nuclei 1 egg” type described for Sargassaceae. Fertilized eggs spent 5–6 h developing into multicellular germlings with abundant rhizoids after fertilization. Under conditions of 20°C, 44 μmol photons m⁻² s⁻¹ and photoperiod of 12 h, young germlings with one or two leaflets reached 2–3 mm in length after 8 weeks. Temperature variations (10, 15, 20, 25°C) under 88 μmol photons m⁻² s⁻¹ significantly influenced the growth rate within the first week, although this effect became less obvious after 8 weeks, especially at 15 and 20°C. Variation in germling growth was highly significant under different irradiances (9, 18, 44, 88 μmol photons m⁻² s⁻¹) at 25°C. Low temperature (10°C) reduced germling growth. Growth of germlings cultured under blue light was lower than in white light. Optimal growth of these germlings occurred at 25°C and 44 μmol photons m⁻² s⁻¹.     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor β chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20°C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10⁹ cfu ml⁻¹) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10⁴ cfu ml⁻¹ at 4°C, and no viable cells were detected at 20°C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l⁻¹ for superantigens and 2 mg l⁻¹ for T-cell receptor β chain. These results contribute to the development of methods for microbial cell preservation under field conditions. Martín F. Desimone and Mauricio C. De Marzi contributed equally to this work