Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Direct evidence that cryoprotectant mixtures facilitate individual component permeation into living plant cells.

The fundamental interactions between plant cells and cryoprotectants during vitrification are understudied in the field of plant cryopreservation. Within this area of research, real time cryoprotectant permeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice callus cells when in mixtures with other cryoprotectants. Specifically, we use coherent anti-Stokes Raman scattering (CARS) microscopy to observe the permeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibrational frequencies that correspond to C-D stretching modes of the cryoprotectant molecules. Additionally, we measure cell plasma membrane responses to PVS2 exposure using brightfield microscopy. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell plasma membrane responses observed and therefore permeation and cell plasma membrane response do not appear to be directly correlated. In addition, we observe that cryoprotectant permeation into plant cells occurs more quickly and more uniformly when cryoprotectants are in PVS2 solution than when they are in single component aqueous solutions.

Micrografting: An Old Dog Plays New Tricks in Obligate Plant Pathogens.

Micrografting, which was developed almost 50 years ago, has long been used for virus eradication, micropropagation, regeneration, rejuvenation, and graft compatibility. Recently, micrografting has been used for studies of long-distance trafficking and signaling of molecules between scions and rootstocks. The graft transmissiveness of obligate plant pathogens, such as viruses, viroids, and phytoplasmas, facilitated the use of micrografting to study biological indexing and pathogen transmission, pathogen-induced graft incompatibility, and screening for the pathogen resistance during the past 20 years. The present study provides comprehensive information on the latter subjects. Finally, prospects are proposed to direct further studies.

Cryobiotechnology: A Double-Edged Sword for Obligate Plant Pathogens.

Pathogen-free stock plants are required as propagation materials in nurseries and healthy materials are needed in germplasm exchange between countries or regions through quarantine programs. In addition, plant gene banks also prefer to maintain pathogen-free germplasm collections. Shoot tip cryotherapy is a novel biotechnology method whereby cryopreservation methods are used to eradicate obligate pathogens from vegetatively propagated plants. Long-term preservation of pathogens is necessary in all types of virus-related basic research and applications such as antigen preparation for virus detection by immunology-based methods, production of plant-based vaccines, genetic transformation to produce virus-derived resistant transgenic plants, and bionanotechnology to produce nano drugs. Obligate plant pathogens such as viruses and viroids are intracellular parasites that colonize only living cells of the hosts. Therefore, their long-term preservation is difficult. Cryotreatments cannot completely eradicate the obligate pathogens that do not infect meristematic cells and certain proportions of plants recovered from cryotreatments are still pathogen-infected. Furthermore, cryotreatments often fail to eradicate the obligate pathogens that infect meristematic cells. Cryopreservation can be used for the long-term cryopreservation of the obligate plant pathogens. Thus, cryobiotechnology functions as a double-edged sword for plant pathogen eradication and cryopreservation. This review provides updated a synthesis of advances in cryopreservation techniques for eradication and cryopreservation of obligate plant pathogens.

Cryobiotechnology of apple (Malus spp.): development, progress and future prospects.

KEY MESSAGE: Cryopreservation provides valuable genes for further breeding of elite cultivars, and cryotherapy improves the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. Apple (Malus spp.) is one of the most economically important temperate fruit crops. Wild Malus genetic resources and existing cultivars provide valuable genes for breeding new elite cultivars and rootstocks through traditional and biotechnological breeding programs. These valuable genes include those resistant to abiotic factors such as drought and salinity, and to biotic factors such as fungi, bacteria and aphids. Over the last three decades, great progress has been made in apple cryobiology, making Malus one of the most extensively studied plant genera with respect to cryopreservation. Explants such as pollen, seeds, in vivo dormant buds, and in vitro shoot tips have all been successfully cryopreserved, and large Malus cryobanks have been established. Cryotherapy has been used for virus eradication, to obtain virus-free apple plants. Cryopreservation provided valuable genes for further breeding of elite cultivars, and cryotherapy improved the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. This review provides updated and comprehensive information on the development and progress of apple cryopreservation and cryotherapy. Future research will reveal new applications and uses for apple cryopreservation and cryotherapy.

Virus infection reduces shoot proliferation of in vitro stock cultures and ability of cryopreserved shoot tips to regenerate into normal shoots in 'Gala' apple (Malus × domestica).

Plant cryopreservation has provide secure back-ups of germplasm collections of vegetatively propagated crops. Often, recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes. The present study investigated the effects of Apple stem grooving virus (ASGV) on shoot proliferation of in vitro stock cultures and recovery of cryopreserved shoot tips of 'Gala' apple. Results showed that virus infection reduced shoot proliferation of in vitro stock cultures and cell ability to regenerate normal shoots in cryopreserved shoot tips. Virus infection increased total soluble protein, total soluble sugar and free proline levels and altered endogenous levels of indoleacetic acid (IAA) and zeatin riboside (ZR), but induced severe cell membrane damage and caused alternation in mitochondria shape of the in vitro stock shoots. The altered levels of IAA and ZR were most likely to be responsible for the reduced shoot proliferation of in vitro stock culture. Cell damage and alternations in mitochondria shape in ASGV-infected shoot tips were most likely responsible for the reduced cell ability to regenerate normal shoots following cryopreservation. To the best of our knowledge, this is the first study on effects of virus infection on recovery of cryopreserved shoot tips. Results reported here emphasize that healthy in vitro stock cultures should be used for cryopreservation.

Combining Thermotherapy with Cryotherapy for Efficient Eradication of Apple stem grooving virus from Infected In-vitro-cultured Apple Shoots.

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips.

KEY MESSAGE: Transcripts related to abiotic stress, oxidation, and wounding were differentially expressed in Arabidopsis shoot tips in response to cryoprotectant and liquid nitrogen treatment. Cryopreservation methods have been implemented in genebanks as a strategy to back-up plant genetic resource collections that are vegetatively propagated. Cryopreservation is frequently performed using vitrification methods, whereby shoot tips are treated with cryoprotectant solutions, such as Plant Vitrification Solution 2 (PVS2) or Plant Vitrification Solution 3 (PVS3); these solutions remove and/or replace freezable water within the meristem cells. We used the model system Arabidopsis thaliana to identify suites of transcripts that are up- or downregulated in response to PVS2 and PVS3 treatment and liquid nitrogen (LN) exposure. Our results suggest that there are many changes in transcript expression in shoot tips as a result of cryoprotection and that these changes exceed the number detected as a result of LN exposure. In total, 180 transcripts showed significant changes in expression level unique to treatment with either the cryoprotectant or cryopreservation followed by recovery. Of these 180 transcripts, 67 were related to stress, defense, wounding, lipid, carbohydrate, abscisic acid, oxidation, temperature (cold/heat), or osmoregulation. The responses of five transcripts were confirmed using qPCR methods. The transcripts responding to PVS2 + LN suggest an oxidative response to this treatment, whereas the PVS3 + LN treatment invoked a more general metabolic response. This work shows that the choice of cryoprotectant can have a major influence on the patterns of transcript expression, presumably due to the level and extent of stress experienced by the shoot tip. As a result, there may be divergent responses of study systems to PVS2 and PVS3 treatments.

Chloroplast heterogeneity and historical admixture within the genus Malus.

UNLABELLED: • PREMISE OF THE STUDY: The genus Malus represents a unique and complex evolutionary context in which to study domestication. Several Malus species have provided novel alleles and traits to the cultivars. The extent of admixture among wild Malus species has not been well described, due in part to limited sampling of individuals within a taxon.• METHODS: Four chloroplast regions (1681 bp total) were sequenced and aligned for 412 Malus individuals from 30 species. Phylogenetic relationships were reconstructed using maximum parsimony. The distribution of chloroplast haplotypes among species was examined using statistical parsimony, phylogenetic trees, and a median-joining network.• KEY RESULTS: Chloroplast haplotypes are shared among species within Malus. Three major haplotype-sharing networks were identified. One includes species native to China, Western North America, as well as Malus domestica Borkh, and its four primary progenitor species: M. sieversii (Ledeb.) M. Roem., M. orientalis Uglitzk., M. sylvestris (L.) Mill., and M. prunifolia (Willd.) Borkh; another includes five Chinese Malus species, and a third includes the three Malus species native to Eastern North America.• CONCLUSIONS: Chloroplast haplotypes found in M. domestica belong to a single, highly admixed network. Haplotypes shared between the domesticated apple and its progenitors may reflect historical introgression or the retention of ancestral polymorphisms. Multiple individuals should be sampled within Malus species to reveal haplotype heterogeneity, if complex maternal contributions to named species are to be recognized.

CRYOPRESERVATION OF IN VITRO-GROWN SHOOT TIPS OF CHINESE MEDICINAL PLANT Atractylodes macrocephala KOIDZ. USING A DROPLET-VITRIFICATION METHOD.

BACKGROUND: Atractylodes macrocephala Koidz. is an important medicinal species from China that has been used for thousands of years for its special pharmacological antioxidant, hepatoprotective, anti-inflammatory, anti-allergic, antithrombotic, antiviral, and anticarcinogenic activities. OBJECTIVE: The aim of this research was to develop an efficient droplet-vitrification protocol for A. macrocephala shoot tips which could be used as a strategy for long-term conservation within gene banks. MATERIALS AND METHODS: The duration of preculture, loading, and PVS2 steps, as well as the recovery medium formulation, were optimized to achieve high levels of survival and regrowth for A. macrocephala shoot tips after liquid nitrogen exposure. RESULTS: Survival and regrowth levels after cryopreservation in the cultivar 'Baizhu' were as high as 76% and 62%, respectively. Thermal analysis using differential scanning calorimetry suggested that the PVS2 treatment plays a critical role for successful cryopreservation. CONCLUSION: The droplet-vitrification method established in this study could be used to cryopreserve A. macrocephala.

Genetic diversity in Malus ×domestica (Rosaceae) through time in response to domestication.

UNLABELLED: • PREMISE OF THE STUDY: Patterns of genetic diversity in domesticated plants are affected by geographic region of origin and cultivation, intentional artificial selection, and unintentional genetic bottlenecks. While bottlenecks are mainly associated with the initial domestication process, they can also affect diversity during crop improvement. Here, we investigate the impact of the improvement process on the genetic diversity of domesticated apple in comparison with other perennial and annual fruit crops.• METHODS: Apple cultivars that were developed at various times (ranging from the 13th through the 20th century) and 11 of the 15 apple cultivars that are used for 90% of the apple production in the United States were surveyed for genetic diversity based on either 9 or 19 simple sequence repeats (SSRs). Diversity was compared using standard metrics and model-based approaches based on expected heterozygosity (He) at equilibrium. Improvement bottleneck data for fruit crops were also collected from the literature.• KEY RESULTS: Domesticated apples showed no significant reduction in genetic diversity through time across the last eight centuries. Diversity was generally high, with an average He > 0.7 for apples from all centuries. However, diversity of the apples currently used for the bulk of commercial production was lower.• CONCLUSIONS: The improvement bottleneck in domesticated apples appears to be mild or nonexistent, in contrast to improvement bottlenecks in many annual and perennial fruit crops, as documented from the literature survey. The low diversity of the subset of cultivars used for commercial production, however, indicates that an improvement bottleneck may be in progress for this perennial crop.

Cryopreservation of Populus trichocarpa and Salix dormant buds with recovery by grafting or direct rooting.

BACKGROUND: Methods are needed for the conservation of clonally maintained trees of Populus and Salix. In this work, Populus trichocarpa and Salix genetic resources were cryopreserved using dormant scions as the source explant. OBJECTIVE: We quantified the recovery of cryopreserved materials that originated from diverse field environments by using either direct sprouting or grafting. MATERIALS AND METHODS: Scions (either at their original moisture content of 48 to 60% or dried to 30%) were slowly cooled to -35 degree C, transferred to the vapor phase of liquid nitrogen (LNV, -160 degree C), and warmed before determining survival. RESULTS: Dormant buds from P. trichocarpa clones from Westport and Boardman, OR had regrowth levels between 42 and 100%. Direct rooting of cryopreserved P. trichocarpa was also possible. Ten of 11 cryopreserved Salix accessions, representing 10 different species, exhibited at least 40% bud growth and rooting after 6 weeks when a bottom-heated rooting system was implemented. CONCLUSION: We demonstrate that dormant buds of P. trichocarpa and Salix accessions can be cryopreserved and successfully regenerated without the use of tissue culture.

Over a century of pear breeding at the USDA.

The U.S. Department of Agriculture (USDA) has performed European pear (Pyrus communis L.) scion breeding for over a century. The breeding program started in the early 1900s by Merton B. Waite in the Washington D.C. area and the program's main goal was to develop host resistance to the devastating disease fire blight, caused by Erwinia amylovora. Most of the historic European pear cultivars being produced in the U.S. were susceptible to fire blight, prompting a need to breed for resistance. More than six generations of USDA breeders have continued this effort to breed disease-resistant European pears. Along with fire blight resistance, the pear breeding programs sought improved fruit quality, cold hardiness, and resistance to psylla (Cacopsylla pyricola Foërster), a significant insect pest of pear. Herein, we discuss the history of the program through each generation of breeder(s). We also present breeding aims, parental selection, and releases. In total, the program has released ten named pear varieties between 1938 and 2022.

Conserving Citrus Diversity: From Vavilov's Early Explorations to Genebanks around the World.

Citrus is among the most economically important fruit crops. Its vast species diversity and global production was observed by N.I. Vavilov during his international plant explorations from the early to mid-1900s. Currently, ex situ citrus collections located around the world conserve and protect citrus genetic resources, as revealed in a survey conducted in 2021. Responses were received from 43 collections in 27 countries, of which 35 provided data regarding collection composition, management practices, and security, as well as other information. The six largest citrus collections have between 1000 and 1735 accessions. The largest accession holdings are mandarins and sweet oranges, although all citrus fruit types are maintained: mandarin, sweet orange, lemon, pummelo, grapefruit, hybrids, lime, sour orange, citron, kumquat, papeda, finger lime, and crop wild relatives. Diseases pose significant threats to collections, though some collections are maintained in a clean-plant state as a result of intensive sanitation efforts. National and regional quarantine regulations often limit the export and import of citrus plants or propagative materials, thus limiting the availability of materials at an international level. Resources, both financial and human, are necessary to ensure the long-term safety and security of citrus collections on a global scale. Future efforts to develop citrus genebanking communities will provide opportunities for improved conservation, as well as collaborations and training.

Tracking Permeation of Dimethyl Sulfoxide (DMSO) in Mentha × piperita Shoot Tips Using Coherent Raman Microscopy.

Cryopreservation has emerged as a low-maintenance, cost-effective solution for the long-term preservation of vegetatively propagated crops. Shoot tip cryopreservation often makes use of vitrification methods that employ highly concentrated mixtures of cryoprotecting agents; however, little is understood as to how these cryoprotecting agents protect cells and tissues from freezing. In this study, we use coherent anti-Stokes Raman scattering microscopy to directly visualize where dimethyl sulfoxide (DMSO) localizes within Mentha × piperita shoot tips. We find that DMSO fully penetrates the shoot tip tissue within 10 min of exposure. Variations in signal intensities across images suggest that DMSO may interact with cellular components, leading to its accumulation in specific regions.

Cryopreservation of citrus shoot tips using micrografting for recovery.

The USDA-ARS National Plant Germplasm System (NPGS) and the University of California Citrus Variety Collection maintain more than 888 unique accessions representing 132 taxa of Citrus, Fortunella, and citrus wild species within field, screenhouse, and greenhouse collections. We have identified a cryopreservation method by which Citrus genetic resources that are not maintained in vitro can be successfully conserved. Shoot tips were excised from actively growing vegetative flushes of protected trees. Surface-disinfected shoot tips were precultured overnight in 0.3 M sucrose, loaded with a loading solution for 20 min and treated with PVS2 for 30 or 60 min at 0 degree C, prior to direct immersion in liquid nitrogen. Rewarmed shoot tips post-cultured overnight on survival medium were then micrografted on 'Carrizo' seedling rootstocks to produce whole plants. Micrografted shoot tips recovered quickly and rooted plants could be transferred to the greenhouse within months. Regrowth of whole plants after micrografting averaged 53 percent for cryopreserved shoot tips of cultivars representing eight Citrus and Fortunella species. This method has several advantages: it uses screenhouse or greenhouse plants as source materials, it is not dependent upon cultivar-specific recovery media, and it avoids seedling juvenility.

Minimizing the deleterious effects of endophytes in plant shoot tip cryopreservation.

Plant cryopreservation technologies are used within gene banks for the long-term preservation of vegetatively propagated collections. Surface-sterilized plant tissues grown in the field, greenhouse/screenhouse, growth chamber, or in vitro are the source of shoot tips subjected to vitrification-based cryopreservation methods. Here, we describe the methods used to minimize microbial contamination during the tissue culture initiation process. We also discuss the occurrence and possible elimination of endophytes after extended in vitro culture and during recovery after liquid nitrogen exposure. We describe two case studies in which bacterial endophytes were observed in Citrus gene bank accessions during recovery after cryopreservation. These were identified using the MinION Oxford Nanopore system and Kirby-Bauer disc diffusion assays to examine the bacterial responses to antibiotic exposure. The methods used in this case study could be applied to identify endophytes to better target antimicrobial treatments of plant tissue collections.

DNA profiling with the 20K apple SNP array reveals Malus domestica hybridization and admixture in M. sieversii, M. orientalis, and M. sylvestris genebank accessions.

The USDA-ARS National Plant Germplasm System (NPGS) apple collection in Geneva, NY, USA maintains accessions of the primary Malus domestica (Suckow) Borkh. progenitor species M. sieversii (Ledeb.) M. Roem., M. orientalis Uglitzk., and M. sylvestris (L.) Mill. Many of these accessions originated from seeds that were collected from wild populations in the species' centers of diversity. Some of these accessions have fruit phenotypes that suggest recent M. domestica hybridization, which if true would represent crop contamination of wild species populations and mislabeled species status of NPGS accessions. Pedigree connections and admixture between M. domestica and its progenitor species can be readily identified with apple SNP array data, despite such arrays not being designed for these purposes. To investigate species purity, most (463 accessions) of the NPGS accessions labeled as these three progenitor species were genotyped using the 20K apple SNP array. DNA profiles obtained were compared with a dataset of more than 5000 unique M. domestica apple cultivars. Only 212 accessions (151 M. sieversii, 26 M. orientalis, and 35 M. sylvestris) were identified as "pure" species representatives because their DNA profiles did not exhibit genotypic signatures of recent hybridization with M. domestica. Twenty-one accessions (17 M. sieversii, 1 M. orientalis, and 3 M. sylvestris) previously labeled as wild species were instead fully M. domestica. Previously unrealized hybridization and admixture between wild species and M. domestica was identified in 230 accessions (215 M. sieversii, 9 M. orientalis, and 6 M. sylvestris). Among these species-mislabeled accessions, 'Alexander', 'Gold Reinette', 'Charlamoff', 'Rosmarina Bianca', and 'King of the Pippins' were the most frequently detected M. domestica parents or grandparents. These results have implications for collection management, including germplasm distribution, and might affect conclusions of previous research focused on these three progenitor species in the NPGS apple collection. Specifically, accessions received from the NPGS for breeding and genomics, genetics, and evolutionary biology research might not be truly representative of their previously assigned species.

Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks.

Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.

Non-Uniform Distribution of Cryoprotecting Agents in Rice Culture Cells Measured by CARS Microscopy.

Cryoprotectants allow cells to be frozen in liquid nitrogen and cryopreserved for years by minimizing the damage that occurs in cooling and warming processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not entirely evident. This contributes to the arduous process of optimizing cryoprotectant formulations for each new cell line or species that is conserved. Coherent anti-Stokes Raman scattering microscopy facilitates the visualization of deuterated cryoprotectants within living cells. Using this technique, we directly imaged the location of fully deuterated dimethyl sulfoxide (d6-DMSO), the deuterated form of a commonly used cryoprotectant, DMSO, within rice suspension cells. This work showed that d6-DMSO does not uniformly distribute throughout the cells, rather it enters the cell and sequesters within organelles, changing our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration within different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distributions of these molecules change when different cryoprotectants are used.