Genetically stable regeneration of apple plants from slow growth

Shoot-tips of apple cultivar `Gala' were stored in vitrousing a low temperature slow-growth culture method. All shoot-tips survived 1-year storage, with a significant height increment over that period. Eight `Gala' single-bud sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variation in plants recovered from slow growth culture, the ploidy remained genetically stable relative to the before-storage cultures. An amplified fragment length polymorphism (AFLP) assay was performed to detect DNA sequence variation. No differences in the DNA fragment patterns were observed using 20 primer combinations between the before-storage and the stored samples. In addition, a methylation sensitive amplified polymorphism (MSAP) assay was performed to investigate the DNA methylation status in both the before-storage and stored samples. It was found that the slow-growth storage resulted in a significant DNA methylation change in the stored shoots compared with the before-storage samples.     

Field performance evaluation and genetic integrity assessment of cryopreserved papaya clones

This paper is the first report of field performance and evaluation of morphological traits following cryopreservation in four genotypes of Carica papaya (Z6, 97, TS2 and 35). It also describes the successful establishment of in vitro plantlets following vitrification-based cryopreservation of shoot tips and their acclimatisation through to field establishment. Cloned plants resulting from untreated controls, as well as controls taken at three other stages of the cryopreservation process (dissection, pre-treatment, plant vitrification solution 2 (PVS2) treatment) and cryopreserved plants were established to ensure a rigorous appraisal of any variation. Results indicate no differences between any of the control plants or cryopreserved plants for either growth performance or morphology. In addition, both randomly amplified DNA fingerprinting and amplified DNA methylation polymorphism markers were used to assess any genomic or methylation changes in genotype 97 at four different developmental stages post cryopreservation (in vitro, acclimatisation and field). Only small genomic DNA modifications (0–8.3%) were detected in field stage plants and methylation modifications (0–4.3%) were detected at both the in vitro and field stages for samples treated with PVS2 or cryopreservation.     

Genetic and epigenetic evaluations of citrus calluses recovered from slow-growth culture

The embryogenic callus of "Red Marsh" grapefruit was stored in vitro by slow growth culture method for one year, and survived with a significant weight increment over that period. The survivers regenerated somatic embryos more easily than the controls. Eight callus lines were used for genetic analyses. Although chromosome number variations were verified by cytological examination both in the controls and the stored samples, the ploidy level remained relatively stable during the storage period. Randomly amplified polymorphic DNA (RAPD) analysis was performed to detect DNA sequence variation. No difference in RAPD pattern was found with the 102 primers used. However, a methylation sensitive amplified polymorphism (MSAP) assay showed DNA methylation changes in the stored samples compared with the controls.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

Effect of freezing–thawing process and quercetin on human sperm survival and DNA integrity

We aimed in the first part of our work to study the effect of cryopreservation on the human sperm DNA integrity and the activation of caspase 3, the main apoptosis indicator. In the second part, we were interested in testing the effect of quercetin, as an antioxidant, in preventing sperm damage during the freeze–thawing process. Seventeen semen samples were obtained from 17 men recruited for infertility investigations. Liquefied sperm was cryopreserved using spermfreeze®. Nine of the used samples were divided into two aliquots; the first one was cryopreserved with spermfreeze only (control) and the second one was cryopreserved with spermfreeze supplemented with quercetin to a final concentration of 50 μM. Sperm motility and viability were assessed according to WHO criteria. We used TUNEL assay and the Oxy DNA assay to assess sperm DNA integrity. Activated caspase 3 levels were measured in spermatozoa using fluorescein-labeled inhibitor of caspase (FLICA). Cryopreservation led to a significant increase in sperm DNA fragmentation, DNA oxidation and caspase 3 activation (p < 0.01). Supplementation of the cryopreservation medium with quercetrin induced a significant improvement in post thaw sperm parameters, compared to those of control, regarding sperm motility (p = 0.007), viability (p = 0.008) and DNA integrity (p = 0.02); however, it had no effect on caspase 3 activation (p = 0.3). We conclude that oxidative stress plays a major role in inducing sperm cryodamage but implication of apoptosis in this impairment requires further investigations. Quercetin could have protective effect during cryopreservation but further research is needed to confirm this effect.     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥（Arabidopsis thaliana）幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明,在扩增的662条带中,对照和2个处理及其第2代间完全一致的带型有598条：发生变化的带型有64条,其中能遗传给第2代的有48条,占变异条带的75％。与对照相比,经超低温保存的样品新产生的甲基化位点有14个,而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条,差异条带有38条,占5.7％,而对照与未冷冻处理组的差异率是7．45％,对照与冷冻处理组之间的差异率是6。63％。可见,拟南芥在超低温保存中,无论是经液氮冷冻还是未经冷冻处理,对材料的甲基化状态均有影响,而这种甲基化变化大部分是可以遗传的。     

拟南芥幼苗超低温保存后DNA甲基化的遗传变异

运用MSAP技术分析了拟南芥(Arabidopsis thaliana)幼苗超低温保存后DNA甲基化的遗传变异情况。结果表明, 在扩增的662条带中, 对照和2个处理及其第2代间完全一致的带型有598条; 发生变化的带型有64条, 其中能遗传给第2代的有48条, 占变异条带的75%。与对照相比, 经超低温保存的样品新产生的甲基化位点有14个, 而去甲基化的位点有22个。经过处理但未冷冻的与冷冻处理组之间带型一致的有624条, 差异条带有38条, 占5.7%, 而对照与未冷冻处理组的差异率是7.45%, 对照与冷冻处理组之间的差异率是6.63%。可见, 拟南芥在超低温保存中, 无论是经液氮冷冻还是未经冷冻处理, 对材料的甲基化状态均有影响, 而这种甲基化变化大部分是可以遗传的。     

Evaluation of genetic stability in cryopreserved Prunus

Summary The evaluation of the genetic stability of Prunus Ferlenain plants regenerated from cryopreserved apices was investigated. The analysis of plants recovered from frozen material was performed at the phenotypic (developmental competence), cytological (chromosomal preparations) and molecular level [random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)]. No genetic change was detected among the plantlets regenerated from frozen apices in comparison to the non-frozen material, including leaves of the mother tree kept in an orchard and vitroplantlets regenerated from non-frozen apices. This result suggested that the procedure used for Prunus cryopreservation could be used for long-term conservation. The relevance of each strategy for the genetic stability evaluation of the cryopreserved material is discussed.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

The effect of egg yolk, low density lipoproteins, methylxanthines and fertilization diluent on cryopreservation efficiency of northern pike (Esox lucius) spermatozoa

The effect of egg yolk, low density lipoproteins (LDL) as well as methylxanthines (caffeine and theophylline) and fertilization diluent on cryopreservation efficiency of northern pike, Esox lucius, spermatozoa was tested. Milt was cryopreserved in pellets on dry ice then stored in liquid nitrogen. The extender consisted of 0.6 M sucrose + 15% DMSO supplemented with egg yolk or LDL fractions. The most effective results (77.3% hatched larvae vs 74.1% in the control group) were obtained from extender that contained only 0.6 M sucrose + 15% DMSO and was used for freezing, while the fertilization diluent was used for thawing. Addition of egg yolk or LDL to the extender did not improve the results. The presence of caffeine in the thawing solution significantly lowered fertilization rate of cryopreserved spermatozoa, whereas theophylline did not significantly affect the results. The addition of fertilization diluent to the eggs prior to insemination was superior to the other treatments. The proposed procedure constitutes a complete method for the efficient cryopreservation of northern pike semen.     

Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Effects of sample collection and storage conditions on DNA damage in buccal cells from agricultural workers

Buccal cells are becoming a widely used tissue source for monitoring human exposure to occupational and environmental genotoxicants. A variety of methods exist for collecting buccal cells from the oral cavity, including rinsing with saline, mouthwash, or scraping the oral cavity. Buccal cells are also routinely cryopreserved with dimethyl sulfoxide (DMSO), then examined later for DNA damage by the comet assay. The effects of these different sampling procedures on the integrity of buccal cells for measuring DNA damage are unknown. This study examined the influence of the collection and cryopreservation of buccal cells on cell survival and DNA integrity. In individuals who rinsed with Hank's balanced salt solution (HBSS), the viability of leukocytes (90%) was significantly (p<0.01) greater than that of epithelial cells (12%). Similar survival rates were found for leukocytes (88%) and epithelial cells (10%) after rinsing with Listerine(?) mouthwash. However, the viability of leukocytes after cryopreservation varied significantly (p<0.01) with DMSO concentration. Cell survival was greatest at 5% DMSO. Cryopreservation also influenced the integrity of DNA in the comet assay. Although tail length and tail moment were comparable in fresh or cryopreserved samples, the average head intensity for cryopreserved samples was ～6 units lower (95% CI: 0.8-12 units lower) than for fresh samples (t(25)=-2.36, p=0.026). These studies suggest that the collection and storage of buccal samples are critical factors for the assessment of DNA damage. Moreover, leukocytes appear to be a more reliable source of human tissue for assessing DNA damage and possibly other biochemical changes.     

Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP)

Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.