Cryopreservation of virus: a novel biotechnology for long-term preservation of virus in shoot tips.
ABSTRACT: Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications. Development of efficient, reliable strategies for long-term preservation of plant virus would largely assist these studies.The present study reported a novel biotechnology allowing cryopreservation of Apple stem grooving virus (ASGV) in living shoot tips. Following cryopreservation by droplet-vitrification or encapsulation-dehydration, about 62-67% of shoot regrowth and 100% of ASGV cryopreservation were obtained. Although shoot proliferation and virus concentration were reduced in cryopreserved diseased shoots after 8 weeks of shoot regeneration, continuous subculture for 4 times (16 weeks) increased shoot proliferation and virus concentration to comparative levels as those produced by shoot tip culture (as a control to shoot tip cryopreservation). Cryopreserved ASGV was efficiently transmitted to a woody plant by micrografting and to a herbaceous indicator by mechanical inoculation. Gene sequencing in three fragments of ASGV genome including coat protein and movement protein showed that cryopreserved ASGV shared 99.87% nucleotide identities with shoot tip culture-preserved virus, indicating cryopreserved virus is genetically stable.The present study demonstrates ASGV, a representative virus that can infect meristematic cells of shoot tips, can be efficiently cryopreserved in shoot tips. To the best of our knowledge, this is the first report on plant virus cryopreservation in living tissues, and has great potential applications to long-term preservation of plant viruses.
Project description:Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy.At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C.Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.
Project description:The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.
Project description:This study develops protocols for the micropropagation and cryopreservation of Dracocephalum austriacum (Lamiaceae). It is a perennial herbaceous plant that overwinters with ground-level sprouts and is classified as critically endangered in Europe. In vitro cultures were initiated from seeds on growth-regulator-free Murashige & Skoog (MS) medium after nicking the seed coat. Propagation via shoot culture was achieved on ½ MS medium with 1 µM benzyl adenine (BAP). Rooting on various indole-3-butyric acid (IBA)-media was not reliable, but the rooting success was 80% after 10 weeks on medium with 1 µM BAP. Two starting materials underwent cryopreservation: (1) shoot tips from cold-acclimated in vitro plantlets and (2) axillary buds from winter shoots from field plants. For the cryopreservation of in vitro shoots, plant vitrification solution (PVS)3 and incubation over ice yielded the best results (~?34% regeneration success). However, regeneration using winter acclimated buds were 100, 76 and 30% for collections in December, February and March, respectively, using the same protocol. Moreover, the ploidy levels of cryopreserved plantlets were estimated using flow cytometry. The use of winter-acclimated field material of temperate herbaceous plants or subshrubs has high potential as explant source for cryopreservation and calls for exploring this technique for other species.
Project description:The aim of this study is to optimize and evaluate the effectiveness of vitrification, droplet-vitrification, and encapsulation-vitrification techniques in the cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara 'Gold Heart', a popular medicinal and ornamental plant species. In vitro-derived shoot tips were used in the experiments. All three techniques were based on explant dehydration with plant vitrification solution 3 (PVS3; 50% glycerol and 50% sucrose) for 0, 30, 60, 90, 120, 150, or 180 min. The recovered microshoots were subjected to morphometric, biochemical, and molecular analyses (RAPD, ISSR, SCoT). The highest recovery level was reported with the encapsulation-vitrification protocol based on 150 min dehydration (73.1%), while the vitrification technique was the least effective (maximum 25.8% recovery). Explants cryopreserved with the encapsulation-vitrification technique produced the highest mean number of shoots (4.9); moreover, this technique was optimal in terms of rooting efficiency. The highest fresh weight of shoots, on the other hand, was found with the vitrification protocol based on a 30-min PVS3 treatment. The concentrations of chlorophyll a and b were lower in all cryopreservation-derived plants, compared to the untreated control. On the other hand, short dehydration and cryopreservation of non-encapsulated explants stimulated the synthesis of anthocyanins. A small genetic variation in 5% of all samples analyzed was detected by RAPD and ISSR marker systems. Only plants recovered from the encapsulation-vitrification protocol had no DNA sequence alternations.
Project description:BACKGROUND:Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. METHODS AND FINDINGS:To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. CONCLUSIONS:Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.
Project description:To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question of whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoot cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Thus, global host gene expression changes do not necessarily lead to virus disease symptoms. Our data suggest that the general approaches to correlate host gene expression changes under viral infection conditions to specific disease symptom, based on the interpretation of transcription profiling data and altered individual gene functions, may have limitations depending on particular experimental systems.
Project description:The medaka (Oryzias latipes) is a teleost model distinguished from other model organisms by the presence of inbred strains, wild stocks, and related species. Cryopreservation guarantees preservation of these unique biological resources. However, because of their large size, cryopreservation techniques for their eggs and embryos have not been established. In the present study, we established a methodology to produce functional gametes from cryopreserved testicular cells (TCs). Whole testes taken from medaka were cryopreserved by vitrification. After thawing, the cells dissociated from cryopreserved testicular tissues were intraperitoneally transplanted into sterile triploid hatchlings. Some cells, presumably spermatogonial stem cells, migrated into the genital ridges of recipients and resulted in the production of eggs or sperm, based on sex of the recipient. Mating of recipients resulted in successful production of cryopreserved TC-derived offspring. We successfully produced individuals from the Kaga inbred line, an endangered wild population in Tokyo, and a sub-fertile mutant (wnt4b-/-) from cryopreserved their TCs. This methodology facilitates semi-permanent preservation of various medaka strains.
Project description:Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.
Project description:Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.
Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies. Overall design: Examination of DMSO cryopreservation in rat liver immune cells of four animals. Eight samples were analyzed represented by fresh cells and cells cryopreserved for 3 weeks per animal.