Effects of cryopreservation on developmental competency, cytological and molecular stability of citrus callus

Cell suspensions of twelve citrus genotypes were successfully cryopreserved by vitrification. All genotypes survived cryopreservation with >90% viability and the surviving cells of some genotypes regenerated somatic embryos better than the controls. Single-cell sibling lines of cultivar Newhall were used for cytological and molecular examination. It was found that the ploidy constitution remained genetically stable and that no DNA sequence variation was detected by randomly amplified polymorphic DNA (RAPD) assay after cryopreservation. In addition, the methylation sensitive amplified polymorphism (MSAP) assay indicated that cryopreservation caused a significant change in DNA methylation status.     

Influence of cryopreservation on the cytosine methylation state of potato genomic NDA

Shoot tips of Solanum tuberosum (Désirée) were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9 % of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6 % (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.     

Detection of polymorphism in the methlyenetetrahydrofolate reductase gene by Raman spectroscopy

A novel approach using micro‐Raman spectroscopy for the detection of alteration of global DNA methylation levels in human individuals with polymorphism in the methlyenetetrahydrofolate reductase (MTHFR) gene was tested. Comparison of measured Raman spectra from samples with wild type genotype to those with polymorphism (homozygous and heterozygous ) in the MTHFR gene revealed that the intensity of the ~ 2945 cm^–1 peak, representing C–H stretches in the DNA constituents and in the methyl group is sample dependent. The intensity ratios between the area of these peaks in the different samples and the peaks at 1667 cm^–1 were used to assess the extent of methylation, allowing classification of the DNA samples for the common MTHFR polymorphism, MTHFR 677 C > T. These results suggest that improvement of the identification capabilities of Raman spectroscopy for DNA methylation can contribute to disease diagnosis and to a better understanding of the physical and biomolecular processes related to epigenetic regulation of the genome. Copyright © 2012 John Wiley & Sons, Ltd.     

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique

Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.     

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD,biochemical and morphological analysis

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.     

Cryopreservation of human skeletal muscle impairs mitochondrial function

Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity of oxidative phosphorylation was significantly (P < 0.05) reduced in cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P < 0.05). Addition of cytochrome c revealed an increase in respiration indicating cytochrome c loss from the mitochondria. The results from this study demonstrate that normal mitochondrial functionality is not maintained in cryopreserved human skeletal muscle samples.     

Geneticallly enginereed trehalose accumulation improves cryopreservation tolerance of chrysanthemum (Dendranthema grandiflorum kitam.) shoot-tips

Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48 percent and 67 percent for transgenic lines and between 33 percent and 36 percent for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci.     

Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.     

Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.     

Cryopreservation of garlic (allium sativum L.) using plant vitrification solution 2

Most cryopreservation procedures are optimized using a small number of germplasm accessions. We classified the garlic (Allium sativum L.) accessions that were selected for our studies based on genotype as identified using amplified fragment length polymorphism markers. Although recovery was variable, shoots regenerated from a broad range of the accessions after cryo-exposure. Garlic shoot tips were excised from cloves, surface sterilized, and placed on media at 5 degree C for 2 days prior to cryopreservation. Shoot tips were then treated with sucrose-glycerol for 20 minutes, plant vitrification solution 2 (PVS2; 15 percent w/v ethylene glycol, 15 percent w/v DMSO, 30 percent w/v glycerol, 13.7 percent w/v sucrose) at 0 degree C, and then plunged on foils into liquid nitrogen slush. Explants were recovered in 1.2 M sucrose for 20 minutes and then plated onto Gamborgs B5 medium containing alpha-naphthaleneacetic acid (NAA) and 6-(gammagamma-dimethylallylamino purine) (2-iP). Our results demonstrate that genotypically diverse accessions of garlic can be successfully cryopreserved.     

Cryopreservation of Dioscorea rotundata poir.: a comparative study with two cryogenic procedures and assessment of true-to-type of regenerants by RAPD analysis

The aim of this study was to develop a cryopreservation protocol for Dioscorea rotundata with maintenance of genetic stability of regenerated plants after cryopreservation. In vitro shoot tips were cryopreserved using vitrification and encapsulation-dehydration to compare the efficacy of the two methods. Both methods produced high levels of plant regeneration from cryopreserved shoot tips. The regeneration level obtained using vitrification (71%) was not significantly different from that obtained using encapsulation-dehydration (67%). Genetic stability of plants derived from cryopreserved shoot tips was evaluated using RAPD markers. Analysis of 50 cryopreserved-derived and 20 in vitro- maintained (control) plantlets showed that 10 primers produced 77 clear, reproducible bands, with the amplification products being monomorphic for all the plantlets tested. A total of 5,390 bands obtained from this study exhibited no aberration in RAPD banding. Thus, the present study showed that both vitrification and encapsulation-dehydration methods are equally applicable to D. rotundata for cryopreservation. The in vitro plantlets derived from cryopreservation were genetically stable at the molecular level tested.     

Recovery and characterisation of hybrid firs (Abies alba x A. cephalonica, Abies albax A. numidica) embryogenic tissues after cryopreservation

Embryogenic tissues of hybrid firs were cryopreserved using a slow freezing protocol. The procedure involved preculture of tissues for 24, 48 or 72 h in media with different sorbitol concentrations (0.4 or 0.8 M) and addition of 5% (v/v) DMSO as cryoprotectant. The cell lines tested withstood cryopreservation, even though tissue regrowth after thawing was dependent on treatment and cell line. For cell line AN72, regrowth was 100% for all experimental conditions tested. With cell line AC78, regrowth was 100% except after shorter pretreatment durations, which produced 83% and 86% regrowth for 0.4 M and 0.8 M sorbitol pretreatment, respectively. Cell lines AC1 and AC4 were more sensitive to cryopreservation with 37.5 to 100% regrowth, respectively. Growth parameters evaluated 3 months after cryopreservation showed cell line and treatments effects. In most cases, cryopreservation had no negative effect on growth of tissues. Statistically significant differences in fresh mass accumulation were found for four samples out of 24 investigated, although growth increase of these tissues still reached 79.4-84.6%, compared with non-cryopreserved ones (100% increase). Maturation capacity and genetic fidelity were studied in tissues whose growth was not negatively influenced by cryopreservation. Maturation capacity of embryogenic tissues cryopreserved using the optimal protocol was comparable to that of non-frozen controls. RAPD analysis of 88 genomic regions per cell line did not reveal any changes in genetic fidelity of cryopreserved tissues compared to non-cryopreserved controls.     

Developing cryopreservation for Picea sitchensis (sitka spruce) somatic embryos: a comparison of vitrification protocols

Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, approximately 10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P less than 0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation-dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.     

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.     

Genetic stability assessments of plantlets regenerated from cryopreserved in vitro cultured grape and kiwi shoot-tips using RAPD

In vitro cultured shoot-tips of four grape (Vitis vinifera) cultivars and one kiwi (Actinidia deliciosa) cultivar were cryopreserved using the encapsulation-dehydration method. Genomic DNA of plantlets regenerated directly from cryopreserved shoot-tips was extracted and analyzed using the RAPD (Random Amplified Polymorphic DNA) technique. The RAPD profiles obtained were highly reproducible and no differences were found between the DNA patterns obtained with plantlets regenerated from control and cryopreserved plantlets. The RAPD technique therefore appears to be a fast, simple and efficient method for evaluating genetic stability of cryopreserved material, which can be used rapidly after the completion of a freezing experiment and will efficiently complement other genetic stability evaluation methods.     

Cryopreservation of black iris (Iris nigricans) somatic embryos by encapsulation-dehydration

Somatic embryos of black iris (Iris nigricans) were cryopreserved using encapsulation-dehydration. Embryos size of 2-4 mm gave the highest survival after cryopreservation. Preculturing embryos on medium containing 0.75 M sucrose for 3 d at 22 degree C, then at 30 degree C for 1 d ensured maximum survival (60%). Viable embryos were brown in color after cryopreservation and during early recovery while dead ones where light brown or creamy. The first sign of regrowth was noted after 15 d. The final regrowth percentage of living embryo was 90% after 35 d. Only 10% of the embryos in all treatments showed secondary embryogenesis. Direct sowing in vivo of cryopreserved embryos was not successful in achieving germination.     

Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane (Saccharum sp.) embryogenic calluses

In this paper, we investigated if the differences consistently noted in survival and plantlet production between cryopreserved and non-cryopreserved, control sugarcane embryogenic calluses were related to modifications induced during cryopreservation in the structural and functional integrity of cell membranes. For this, the evolution of electrolyte leakage, lipid peroxidation products and cell membrane protein contents was measured during 5 d after cryopreservation. Differences between control and frozen calluses were observed only during the first 2 (electrolyte leakage) or 3 d (lipid peroxidation products and membrane protein content) after freezing. It was not possible to link these differences with the differences noted in survival and plant production between control and cryopreserved calluses. Additional studies are thus needed to elucidate which biochemical factors, linked to survival and plantlet regeneration, are affected by cryopreservation.     

Pollen from Glycine species survive cryogenic exposure

Pollen of 12 genotypes of the annual soybean and its wild perennial relatives were stored without pre-desiccation at low temperatures (-20 C and -196 C) and tested for their viability in vitro. The influence of cryopreserved pollen on pod set and seed production was also investigated. Cryopreserved pollen of all the genotypes showed germination in vitro. Pollen of annual soybean stored at -20 C retained their viability for 4 months, however, pollen of its wild perennial relatives at same storage conditions failed to germinate in vitro. Flowers pollinated with cryopreserved pollen had similar pod set and number of seeds/pod as those pollinated with fresh pollen. Results of this study suggest that cryopreservation of pollen can be used successfully for soybean breeding, and also offers the possibility of conserving the haploid gene pool of soybean and wild perennial species in a cryobank facility.     

GUS gene remains stable in transgenic citrus callus recovered from cryopreservation

The conservation of transgenic materials is very important, paticularly for their potential future use in crop development. In this study, transgenic callus cultures of 'Newhall' navel orange (Citrus sinensis Osbeck) were cryopreserved by a vitrification method. Transgenic calluses survived cryopreservation and recovered under normal culture conditions. The results of PCR (Polymerase Chain Reaction) amplification, Southern blotting and SSCP (Single-Strand Conformation Polymorphism) assay showed that the GUS gene was still maintained in the genome of callus cultures recovered from cryopreservation. X-Gluc staining further indicated GUS gene expression in callus cultures recovered from cryopreservation.