Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation

Basidiomycetes present specific problems with regard to their preservation, because most of them do not form resistant propagules in culture but exist only as mycelium. Usually these fungi can only be preserved by serial transfer on agar (labour-intensive procedures that can increase the danger of variation or loss of physiological or morphological features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at −80 °C and lyophilisation could be good alternatives.In this work we set up and tested six protocols of cryopreservation at −80 °C, and 12 protocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The tested protocols were mainly characterized by the use of different growth media, protectants, time and number of perfusion with protectants and finally by the typology and origin of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise (mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisation outcomes were checked, at morphological (macro- and microscopic features), physiological (growth rate and laccase, Mn-independent and Mn-dependent peroxidases activities) and genetic level (Amplified Fragment Length Polymorphisms analysis - AFLP). Vitality of all fungi was successfully preserved by all cryopreservation protocols at −80 °C, and by two lyophilisation methods. Our results showed that cryopreservation at −80 °C did not produce morphological changes in any isolate, while two isolates were affected by lyophilisation. None of the physiological features were lost, even though growth rate and enzyme activities were somehow influenced by all preservation methods. AFLP analysis showed that only the two isolates that varied in their morphology after lyophilisation produced a different DNA fingerprint pattern in comparison with that obtained before lyophilisation. These findings provide evidence that cryopreservation at −80 °C and lyophilisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of some WRF strains.     

Activities of ligninolytic enzymes in some white-rot basidiomycete strains after recovering from cryopreservation in liquid nitrogen

Fourteen strains of white-rot basidiomycetes belonging to eight species of two genera (Inonotus and Pholiota) were tested for their ability to maintain the production of laccase, peroxidase and manganese-dependent peroxidase (enzymes involved in lignin biodegradation) after a short-time preservation in liquid nitrogen with different cryoprotectives (glycerol, dimethyl sulfoxide). No negative effect of cryopreservation or the used cryoprotective on production of the ligninolytic enzymes was found in the fungi tested.     

Basidiomycete cryopreservation on perlite: evaluation of a new method

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.     

Preservation of live cultures of basidiomycetes – Recent methods

Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.     

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.     

CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM

A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) wasdeveloped through the use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to— I96°C by two-step freezing, first to — 76°C and then by plunging in LN. In the case of fertilized eggs, samples were previously frozen to — 40°C, at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time, samples in the double test tubes were thawed in water at 15°C. The post-thaw survival was more than 90% for late embryos, and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete, regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei.     

Survival and ultrastructural features of peach palm (Bactris gasipaes, Kunth) somatic embryos submitted to cryopreservation through vitrification

Bactris gasipaes (Arecaceae), also known as peach palm, was domesticated by Amazonian Indians and is cultivated for its fruit and heart-of-palm, a vegetable grown in the tree’s inner core. Currently, the conservation of this species relies on in situ conditions and field gene banks. Complementary conservation strategies, such as those based on in vitro techniques, are indicated in such cases. To establish an appropriate cryopreservation protocol, this study aimed to evaluate the ultrastructural features of B. gasipaes embryogenic cultures submitted to vitrification and subsequent cryogenic temperatures. Accordingly, somatic embryo clusters were submitted to Plant Vitrification Solution 3 (PVS3). In general, cells submitted to PVS3 had viable cell characteristics associated with apparently many mitochondria, prominent nucleus, and preserved cell walls. Cells not incubated in PVS3 did not survive after the cryogenic process in liquid nitrogen. The best incubation time for the vitrification technique was 240 min, resulting in a survival rate of 37 %. In these cases, several features were indicative of quite active cell metabolism, including intact nuclei and preserved cell walls, an apparently many of mitochondria and lipid bodies, and the presence of many starch granules and condensed chromatin. Moreover, ultrastructure analysis revealed that overall cellular structures had been preserved after cryogenic treatment, thus validating the use of vitrification in conjunction with cryopreservation of peach palm elite genotypes, as well as wild genotypes, which carry a rich pool of genes that must be conserved.     

激光打孔技术在小鼠卵子冷冻保存上的运用

目的比较卵子冷冻复苏后、以及复苏卵子经过激光打孔后,与新鲜精子、冷冻复苏精子体外受精,受精率的变化。方法 （1）通过免疫荧光染色技术,判断卵子冷冻前后透明带糖蛋白-2、微丝以及细胞核的变化;（2）冷冻C57BL/6J小鼠卵子,复苏后一部分卵子打孔,一部分不打孔,然后与9个品系的新鲜精子和冷冻精子体外受精,比较各组受精率的变化。结果 （1）新鲜卵子组透明带糖蛋白-2、微丝及细胞核结构清晰,而冷冻组以及冷冻剂处理组,微丝结构略有变化,透明带糖蛋白-2受到不同程度的损伤,而细胞核在冷冻前后无明显变化;（2）9个品系的新鲜精子与C57BL/6J复苏卵子受精率为17.6%～42.9%,平均为29.6%;新鲜精子与复苏打孔卵子受精率为29.1%～72.3%,平均为49.7%,差异显著（P〈0.05）;9个品系复苏精子与复苏卵子体外受精率为5.4%～23%,平均为15.5%;复苏精子与复苏打孔卵子受精率为16.7%～48.6%,平均为28.8%,差异显著（P〈0.05）。结论 （1）冷冻对卵子的透明带糖蛋白-2有较大的损伤,对微丝有一定的影响,但是对染色体没有影响;（2）冷冻卵子复苏后,体外受精率下降明显,但是复苏卵子经过激光打孔后,可以显著提高体外受精率。     

Maize seed cryo-storage modifies chlorophyll,carotenoid, protein,aldehyde and phenolics levels during early stages of germination

We recorded the crypreservation effects (direct immersion) on various parameters of early germination stages of maize seeds (0, 7 and 14 days). Percentages of germination; fresh mass of different seedling parts; levels of chlorophyll pigments (a, b); carotenoids; malondialdehyde; other aldehydes; phenolics (cell wall-linked, free) and proteins were determined. Various statistically significant effects of seed exposure to liquid nitrogen (LN) were recorded. Maize seeds did not seem to be affected by LN exposure either visually or regarding fresh weight or germination rate. However, delayed growth was observed in seedlings recovered from cryopreserved seeds. This trend indicated an increase in the effect of seed cryopreservation on growing plants. The most significant effects of LN exposure were recorded in the combined fresh weight of stems and leaves at day 7 of germination and in fresh weights of roots, stems and leaves at day 14. At the biochemical level, numerous indicators varied following LN exposure, but the most significant effects were recorded in carotenoids, malondialdehyde and other aldehyde contents. LN exposure modified 50.0% of indicators in cotyledons, 48.1% in stems and leaves, 38.8% in roots and 11.1% in seeds. LN storage modified 11.1% of the variables measured at day 0 of germination, 37.0% at day 7, and 52.7% at day 14. Field performance of cryostored seed-derived plants should be evaluated to measure the durability of the changes observed.     

Effects of overexpression of copper-zinc and manganese superoxide dismutases,catalase, and thioredoxin reductase genes on longevity in Drosophila melanogaster

The overexpression of antioxidative enzymes such as CuZn-superoxide dismutase (SOD), Mn-SOD, and catalase has previously been reported to extend life span in transgenic flies (Drosophila melanogaster). The purpose of this study was to determine whether life-extending effects persist if the recipient control strains of flies are relatively long-lived. Accordingly, the life spans of large numbers of replicate control and overexpressor lines were determined in two long-lived genetic backgrounds involving a combined total of >90,000 flies. Significant increases in the activities of both CuZn-SOD and catalase had no beneficial effect on survivorship in relatively long-lived y w mutant flies and were associated with slightly decreased life spans in wild type flies of the Oregon-R strain. The introduction of additional transgenes encoding Mn-SOD or thioredoxin reductase in the same genetic background also failed to cause life span extension. In conjunction with data from earlier studies, the results show that increasing the activities of these major antioxidative enzymes above wild type levels does not decrease the rate of aging in long-lived strains of Drosophila, although there may be some effect in relatively short-lived strains.     

Maintenance of Critical Properties of Brain Tumor Stem-like Cells After Cryopreservation

It would be very useful to be able to classify brain tumor stem cells (BTSCs) by certain criteria to afford the design of specific or individualized treatment. Here, we studied two BTSC lines with differing biological and molecular features and whose respective features were well preserved after cryopreservation as single cells in SFM or 90% serum with 10% DMSO, a method not previously reported. The resuscitated BTSCs shared properties indistinguishable from their respective parental cells, including tumor sphere forming potentials, growth and differentiation properties, and tumorigenesis in vivo. The two cell lines also had differing molecule profiles, which can be well preserved after cryopreservation, similar to that of their respective primary tumors. Therefore, BTSCs from different patients, that have their own properties, were well retained by the present cryopreservation method, which might be a useful and reliable method for preserving BTSCs for long-term studies, such as classification and specific therapy design.     

Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintains (epi)genetic uniformity of regenerants

The embryogenic cell suspension culture of Gentiana cruciata, cryopreserved by the encapsulation/dehydration method, survived both short- (48 h) and long-term (1.5 years) cryostorage with more than 80% viability. To assess the influence of cryotreatments on the embryogenic potential, a proembryogenic mass was encapsulated and exposed to the following treatments: (1) osmotic dehydration (OD), (2) OD + air desiccation (AD) and (3) OD + AD + cryostorage (LN). The somatic embryogenesis efficiency increased ten times after osmotic dehydration. The AD and LN cryotreatments did not cause any significant alterations in somatic embryo production. We monitored the (epi)genetic stability of 288 regenerants derived from: non-cryotreated, short-term, and long-term cryostored tissue using metAFLP markers and ten primer combinations. Changes in the sequence and DNA methylation levels were studied by subjecting the DNA to digestion with two pairs of isoschisomer restriction enzymes (KpnI/MseI and Acc65I/MseI). Two new AFLP unique DNA fragments at the DNA sequence level, with no differences at the methylation level, were found between regenerants derived from cryopreserved tissue, compared with the non-cryotreated controls. The Acc65I/MseI methylation levels for the three groups of regenerants were not significantly different. Cluster analysis was capable of identifying a number of sub-clusters. Only one of the sub-clusters comprises almost all regenerants derived from non-cryotreated and short-term cryostored tissue. Plantlets derived from long-term cryostored tissue were grouped into separate clusters. The observed AFLP alterations did not appear to be associated with the use of cryopreservation, but were probably related to the process of in vitro culture.     

Microsatellite primers for Texas wild rice (<Emphasis Type="Italic">Zizania texana</Emphasis>), and a preliminary test of the impact of cryogenic storage on allele frequency at these loci

Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive insurance for species that survive in conventional cold storage (–18 °C). Seeds that cannot survive these conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which presents unique technical and methodological challenges. Implicit in this approach is the assumption that these added manipulations do not change the genetic diversity of the preserved collections. We used polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.     

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

Summary Embryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.Abbreviations ABA ± abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - CTAB cetyldimethylethyl-ammonium bromide - DMSO dimethylsulfoxide - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid disodium salt - IBA indole-3-butyric acid - LN liquid nitrogen - PEG polyethylene glycol - SLS N-lauroyl sarcosine - Tris tris[hydroxymethylamino] methane - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effect of cryogenic preservation (−80 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

The effect of cryopreservation on plasma membrane and granule associated enzymes of polymorphonuclear neutrophils (PMNs) was studied. The activity of PMNs to generate superoxide anions during phagocytosis was very sensitive to cryopreservation and exhibited approximately 60% inhibition in 24 hr. The total enzyme activity was not as affected during 1-month cryopreservation as that observed with the extracellular release of enzymes. Acid p-nitrophenyl phosphatase and peroxidase were released slightly from frozen and thawed PMNs. However, the extracellular release of LDH, a cytosol marker, and β-glucuronidase and lysozyme, granuleassociated enzymes, increased with cryopreservation time. The degree of release of these enzymes was LDH > β-glucuronidase > lysozyme. A considerable amount of LDH was extracellularly released after 1-month storage. Frozen and thawed PMNs became sensitive to hypotonic solutions, although fresh, nonfrozen PMNs were very resistant to hypotonic lysis. The hypotonic fragility increased even after 1 hr of cryopreservation.Addition of ATP to the preservation medium did not improve enzyme activity, enzyme release, or stimulated superoxide anion generation but increased the hypotonic fragility of PMNs. However, albumin showed protective effects against cryopreservation injury to the O₂^?-generating system, the extracellular enzyme release, and osmotic fragility.     

Cryopreservation of immature Parkia speciosa Hassk. zygotic embryonic axes following desiccation or exposure to vitrification solution

This work reports on the cryopreservation of immature zygotic embryonic axes (EA) of petai (Parkia speciosa Hassk.) for the first time. Two cryopreservation protocols, namely desiccation and vitrification method were tested individually using excised EA. Desiccation of EA to lower moisture content (MC) reduced the survival percentage but a drastic decline in survival percentage (~20 %) was recorded at 16 % MC prior to exposure to LN, rendering the EA to be sensitive to desiccation. Cryopreservation of EA after desiccation, irrespective of the MC, did not result in any survival. On the other hand, post-cryopreservation survival was obtained when the EA were exposed to plant vitrification solution-2 (PVS2) for 75–105 min. The best results were obtained when the EA were exposed to PVS2 for 90 min with an average recovery of 55.5 %. EA recovery into whole plantlets was obtained when the EA were cultured on MS medium supplemented with 2 gl^?1 activated charcoal and 0.1 mgl^?1 of the plant growth regulators α-naphthalene acetic acid, 6-benzylaminopurine and gibberellin A₃, each. EA, exposed for less than 75 min and more than 105 min to PVS2, did not show any survival after cryopreservation. The optimization of exposure time is necessary to increase survival. This study has shown that the employment of suitable method is important for conservation using cryopreservation.     

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

Survival and genetic stability of Picea abies embryogenic cultures after cryopreservation using a pregrowth-dehydration method

A vitrification method enabled efficient cryopreservation of embryogenic tissue (ETs) of Norway spruce (Picea abies L.) at ?196 °C in liquid nitrogen (LN). Correctly formed, normal somatic embryos were generated from ETs that had been thawed after removal from LN. The pregrowth-dehydration method involved preculture of ETs with sucrose (0.25–1.00 M) in the presence or absence of 10 μM abscisic acid (ABA), followed by air-drying for 2 h and rapid freezing in LN. Pretreatment of ETs with both sucrose and ABA promoted ET growth after preculture and thawing more effectively than treatment with sucrose alone. Survival of ETs after thawing from LN using both sucrose and ABA was 54.4 % compared to pretreatment with sucrose alone which was 20 %. Addition of ABA in the preculture medium also improved the ability of ETs to form cotyledonary stage somatic embryos. The somatic embryos, which had normal shoot and root apices and the correct number of cotyledons, were indistinguishable from regenerants obtained from control cultures. Genetic analysis of control and cryopreserved ETs, as well as somatic embryos derived from cryopreserved ETs, indicated that the cryopreservation method had no effect on any of the five microsatellite loci (SpAGC1, SpAGC2, SpAGG3, SpAC1H8, and SpAC1F7) tested. The cryopreservation protocol outlined should enable the long-term storage of valuable clones of Norway spruce in LN, potentially for hundreds of years.     

杜仲,秤锤树花粉的超低温贮藏研究

本文研究了影响杜仲(Eucommia ulmoides Oliv.)和秤锤树(Sinojackia xylocarpa Hu.)花粉超低温贮藏的一些因素,包括花粉含水量、贮藏时间、冻解方法等。利用差热分析(DTA)技术预测花粉在液氮(LN)中安全贮藏含水量的范围。结果表明,含水量的影响最显著。DTA 有望用于预测花粉在 LN 中安全贮藏含水量的范围。