Storage and preservation of temperate mushroom cultures, agaricus bisporus and pleurotus Florida

Two temperate mushroom cultures namely Agaricus bisporus (U-3) and Pleurotus florida (PAU-5) were evaluated for their physiological (linear growth and biomass production), biochemical (β-1,4 endoglucanase production) and fruiting behaviour after preservation in 10% (v/v) glycerol and storage at room temperature (25–35°C), −20°C and −196°C for 6 months with the objective of establishing the recovery/changes in these fungi after storage. Studies indicated that the viability and recovery of A. bisporus and P. florida is affected by the storage conditions. Both the fungi could be best stored in liquid nitrogen for longer durations but for regular use, conventional sub-culturing was appropriate.     

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Cryopreservation of wheat suspension culture and regenerable callus

Wheat (Triticum aestivum L. cv. Norstar) suspension cultures and regenerable calli initiated from immature embryos can be cryopreserved in liquid nitrogen temperature (–196°C) by slow freezing (0.5°C/min) in the presence of a mixture of DMSO and sucrose or sorbitol. Cold hardening or ABA treatment before cryopreservation increased the freezing resistance and improved the survival of wheat suspension culture in liquid nitrogen. Callus culture, established from immature embryos, prefrozen in 5% DMSO and 0.5M sorbitol survived liquid nitrogen storage and resumed plant regeneration after thawing. The results confirm the feasibility of long term preservation of wheat embryo callus by cryopreservation and retention of plant regeneration ability.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - LN Liquid nitrogen - TTC 2,3,5-triphenyltetrazolium chloride NRCC No. 23850.     

Aminocentesis cells: Culture,cryogenic storage,isozymes, and finite life in vitro

Summary Forty-six cell cultures established from amniocentesis fluids were preserved in liquid nitrogen and later recovered from the frozen state with little loss of viability as compared to prefreeze viability. Five to 10% glycerol was found to be optimal for preservation in liquid nitrogen, and as few as 5×10⁵ viable cells per frozen ampule could initiate cell growth. Storage in liquid nitrogen did not affect the genetic stability of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, leucine aminopeptidase, acid phosphatase, or 6-phosphogluconic acid dehydrogenase isozymes of the amnion cultures. These studies were supported by Contract NIH-NIGMS-72-2070, Grant CA-04953-13 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3,16, 749 from the National Institutes of Health.     

Conservation of reference strains of Fusarium in pure culture

More than 300 reference strains representing 60 species and varieties ofFusarium cultures named according to different taxonomic systems are currently maintained at the American Type Culture Collection (ATCC). They have been preserved by freeze-drying and by freezing and subsequent storage in liquid nitrogen (–196°C) to insure their viability without contamination, variation, mutation, or deterioration. The materials and procedures used at the ATCC for the acquisition, accessioning, cataloguing, preservation and distribution are described. Longevity storage data for the strains available for distribution are presented and discussed.     

Cryopreservation of Embryogenic Suspension Cultures of Barley (Hordeum vulgare L.)*

Anther-derived suspension cultures of Hordeun vulgare L. were cryopreserved by slow cooling and storage in liquid nitrogen for up to 55 days. Cells were pre-cultivated in L3 suspension medium supplemented with sorbitol. For freeze preservation the cells were treated with different combinations of cryoprotectant agents such as DMSO, proline, glycerol and sucrose. After rapid thawing high viabilities of up to 77% could be achieved. Cell growth commenced 2- 3 weeks later. The frequency of plantlet regeneration was 1%.     

Preservation, mass production and storage ofStagonospora convolvuli, a bioherbicidecandidate for field bindweed (Convolvulusarvensis)

Different solid substrates were investigated as spore production methods for Stagonospora convolvulistrain LA39, a potential bioherbicide for field bindweed (Convolvulusarvensis L.). Up to 4 × 10⁸ spores/g of substratewere yielded on cous-cous (cracked hard wheat). Thespores were as pathogenic as those grown on artificial medium (V-8-juice agar). The air-drying on kaolin and storage at 3 °C kept spores viable and pathogenic for 180 days. Spore germination exceeded70% for the first 140 days and then declined to 50%after 175 days. Less than 5% of spores were still viable after 17 months. The preservation of stock cultures in 10% glycerine at −80 ° C and in liquid nitrogen did not affect viability orpathogenicity of the spores. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria

Pot experiments were carried out to investigate the effect of inoculation with pure and mixed cultures of nitrogen fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7 and the phosphate-solubilizing strain Agrobacterium radiobacter 10 on growth and mineral nutrition of two barley cultivars. A significant positive effect on grain yield both of the studied barley cultivars was obtained after inoculation with mixtures of A. lipoferum 137 + A. radiobacter 10 and A. lipoferum 137 + A. mysorens 7 only. The acetylene reduction activity on roots or in batch culture was significantly higher when A. lipoferum 137 and A. radiobacter 10 were combined. Using ¹⁵N isotope dilution technique it was established that these mixed cultures significantly increased the accumulation of nitrogen fertilizer in the plants. The strain A. radiobacter 10 promoted a better accumulation of phosphorus fertilizer by plants and A. mysorens 7 increased the total phosphorus content in plant tissues. The maximum positive effect of joint inoculation on plant development was observed when the combined nitrogen in soil was in short supply. It was concluded that inoculation with bacterial mixtures provided a more balanced nutrition for the plants and the improvement in root uptake of nitrogen and phosphorus was the major mechanism of interaction between plants and bacteria. The introduced bacteria were able to colonize actively the rhizoplane of barley. No interspecific competition or antagonism were established between components of the bacterial mixtures in the rhizoplane. The strains A. mysorens 7 and A. radiobacter 10 improved viability of A. lipoferum 137 when the plants were grown in acid soil. Field experiments carried out on 3 barley cultivars confirmed the assertion that inoculation with mixed cultures significantly increases the grain yield and nitrogenous nutrition of plants as compared with single cultures.     

Long‐term preservation of a collection of Rhizoctonia solani using cryogenic storage

Rhizoctonia solani is an important plant pathogen for a number of crops and maintaining an extensive collection of reference isolates is important in understanding relationships of this pathogen with multiple hosts. Current long‐term storage methods typically call for frequent transfer increasing the risk of changes in morphological, physiological or virulence characteristics. Cryopreservation using storage in liquid nitrogen (LN) was evaluated to examine the potential for storage of a R. solani culture collection containing 106 isolates (primarily from sugar beet). Cultures were stored on autoclaved barley grains in the vapour phase of LN. After 60 days, 5 years and 10 years in storage, all isolates were tested for viability by calculating the percentage of barley grains from which R. solani mycelia grew. Five years after initial storage, all isolates except one had no change in viability. After 10 years in storage, 67 of 106 isolates had no significant decrease in viability, 39 of 106 isolates had a significant decrease in viability but only 9 isolates had less than 10% growth, with 4 having no growth. A subset of isolates stored for 10 years were tested for pathogenicity on a susceptible (FC901) and resistant (FC703) sugar beet germplasm. All isolates tested maintained approximately the same level of virulence that they had prior to storage on both germplasms. This indicates that cryogenic methods are suitable for the preservation or storage of R. solani culture collections, although efficacy may vary with individual isolates.     

The medicinal <Emphasis Type="Italic">Agaricus</Emphasis> mushroom cultivated in Brazil: biology,cultivation and non-medicinal valorisation

Sun mushroom is a cultivated mushroom extensively studied for its medicinal properties for several years and literature abounds on the topic. Besides, agronomical aspects were investigated in Brazil, the country the mushroom comes from, and some studies focus on the biology of the fungus. This review aimed to present an overview of the non-medicinal knowledge on the mushroom. Areas of commercial production and marketing trends are presented. Its specific fragrance, taste, nutritional value and potential use of extracts as food additives are compared to those of the most cultivated fungi and laboratory models. The interest of the mushroom for lignocellulosic enzyme production and source of biomolecules for the control of plant pathogens are shown. Investigation of genetic variability among cultivars is reported. Growing and storage of mycelium, as well as cultivation conditions (substrate and casing generally based on local products; indoor and outdoor cultivation; diseases and disorders) are described and compared to knowledge on Agaricus bisporus.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

THE USE OF IMMOBILIZATION IN ALGINATE BEADS FOR LONG-TERM STORAGE OF PSEUDANABAENA GALEATA (CYANOBACTERIA) IN THE LABORATORY1

A new method for long-term storage of algal cultures in the laboratory was tested. The procedure is based on the cell immobilization technique. Cells of the filamentous cyanobacterium Pseudanabaena galeata Bocher were immobilized in sodium-alginate beads and stored for 14–18 months. The structure and functional features of the organism were maintained in this immobilized state and no ultrastructural biochemical, or growth rate differences were detected between stock and previously immobilized cell cultures after this period. We discuss the advantages of this method compared to other preservation methods and recommend the use of immobilization in alginate beads for long-term algal storage.     

Preservation of laboratory strains of Toxoplasma gondii in vitro

A procedure to obtain viable stabilates of virulent laboratory strains of Toxoplasma gondii with a prolonged storage life is described. Viable endozoites recovered from the sediment of mouse exudate or tissue cultures (LEP, HeLa) are suspended in Eagle's MEM medium supplemented with 10% calf serum and 10% dimethylsulfoxide and sealed into glass ampoules of 1-2 ml in volume. The ampoules are placed in an apparatus for gradual cell freezing, frozen to --35 degrees C at a rate of --1 degree C/min, and stored in liquid nitrogen. Reinoculation experiments on mice given the suspension intraperitoneally confirmed that such Toxoplasma gondii strains retain viability for at least 4 years. This in vitro preservation technique is compared with the analogous T. gondii preservation procedures described in the literature.     

液氮冻结法保藏毛霉目菌种的效果

本文报告了用液氮冻结法保藏毛霉目菌种的效果。对14属26种32株进行了液氮保藏试验,其结果慢速冻结孢子成活率高于快速冻结。用10%的甘油、10%的二甲基亚砜和蒸馏水作保护剂制成的孢子悬液,经慢速冻结后储存在液氮罐气态中二年,以甘油作保护剂的孢子成活率为90%,二甲基亚砜的为85%,蒸馏水的为79%。并检测了某些菌株的反丁烯二酸、蛋白酶、α-半乳糖苷酶、脂肪酶、果胶酶、葡萄糖苷酶等生理活性,结果冻结后的活性与冻结前相比,无明显差异。     

The relationship between fungal preservation method and secondary metabolite production in Metarhizium anisopliae and Fusarium oxysporum

The effects of preservation regime on secondary metabolite production in two genera of economically important fungi, Metarhizium anisopliae and Fusarium oxysporum, was assessed using thin layer chromatography and high performance liquid chromatography over a 2-year testing period. Five preservation regimes, commonly used in microbial culture collections throughout the world were investigated: continual sub-culture, lyophilization, storage of mycelial plugs in water, storage at –20 °C and cryopreservation with liquid nitrogen. Preservation regime influenced secondary metabolite production in the test fungi. Changes in secondary metabolite profiles occurred after relatively short storage periods in most strains, irrespective of the preservation treatment used. Although no preservation treatment can be guaranteed to provide total stability of secondary metabolite production, cryopreservation was the best of the methods tested. Response to preservation and storage also differed between strains of the same species. Therefore, there is a need to develop new and existing preservation criteria with an emphasis on strain-specific criteria in order to reduce the prospects of instability in secondary metabolite production.     

Maintenance of cytochrome P-450 levels in hepatocytes preserved on gelatin gels

In culture, cytochrome P-450 levels fall rapidly with the result that hepatocytes are either used quickly or maintained in modified systems which prejudice their subsequent behaviour. In this study the effect of hypothermic preservation of hepatocytes on gelatin gels on levels of cytochrome P-450 was investigated. In marked contrast to conventional cultures, hypothermic preservation (10°) maintained, over a 6-day period, cytochrome P-450 at levels similar to those of the more stable cytochrome b₅. Cell storage on gelatin at 25° was associated with a conversion of cytochrome P-450 to cytochrome P-420. The procedure at 10° provides a valuable tool for toxicity testing, hepatocyte conservation and distribution.     

Preservation of Thiobacillus ferroxidans and Thiobacillus thiooxidans with activity check

Cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, used in biohydrometallurgical processes of economic importance, are very difficult to preserve by conventional methods. Hence, to preserve the cultures with their activity intact, various techniques were tried, after determining their respective activity in terms of Iron Oxidation Rate (IOR) and Sulfur Oxidation Rate (SOR). Among the methods tested, along with the recommended method of serial transfer in a liquid medium, were methods such as lyophilization, storage in a liquid nitrogen and mixing with sterile, inert carriers like lignite or chalcopyrite ores. After a period check-up at 4 months and 8 months storage, it was found that out of these methods, mixing with sterile ore followed by storage at 8°C, kept both types of activities intact. The temperature of storage was observed to have a definite effect on activity, in that when the preserved cultures were stored at 8°C, the activity was retained, whereas at 28–30°C (RT) storage, the activity of all the cultures preserved by various techniques, dropped significantly.     

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain)

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions is presented in this paper. Shoot-tip cultures of Musa cultivars (both banana and plantain) are induced by culturing small excised shoot apices on modified MS semisolid medium supplemented with various concentrations and combinations of auxins and cytokinins. The effects of cytokinin concentration in the medium as well as the genotypic configuration of the cultivars on the rate of shoot-bud proliferation have been tested. The established shoot-tip cultures grown on modified MS semisolid medium supplemented with IAA (0.18 mg/l) and BA (2.30 mg/l) have been successfully stored at 15°C with 1000 lux light intensity up to 13–17 months depending on the cultivar. The cultivars tested in the present investigation seem to vary in their ability to withstand minimal growth temperature.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog     

Influence of wheat cultivars on straw quality and Pleurotus ostreatus cultivation

The main raw material for Pleurotus ostreatus (oyster mushroom) cultivation is wheat straw. Estimation of straw biodegradability from 15 different spring wheat cultivars under irrigation in South Africa was determined using linear discriminant analysis to discriminate or group the 15 cultivars by combining chemical analysis and in vitro enzymatic hydrolysis. Significant differences (P < 0.01) were found between ash, nitrogen, reducing sugars, anthrone reactive-carbohydrates, water-soluble dry matter, and oyster mushroom yields. The significance of these measurements was investigated and discussed.