Plant Cryopreservation Research Articles

A promising strategy for conservation of endemic plant Euonymus koopmannii

Abstract. Gubaidullin N, Kali B, Tussipkan D, Manabayeva SA. 2024. A promising strategy for conservation of Central Asian endemic plant Euonymus koopmannii. Biodiversitas 25: 3114-3120. Cryopreservation of endemic plants is necessary to safeguard the biodiversity and genetic resources of unique species that may face extinction due to climate change or human activities. This process enables the long-term preservation of their genetic information and ensures the possibility of restoring these species. Euonymus koopmannii Lauche is, known as E. nanus var. turkestanicus, is a native to Kazakhstan and other Central Asian countries. The E. koopmannii is listed in the Red Book of Kazakhstan and has ecological importance in soil stabilization and its decorative value. This study aims to contribute to the conservation efforts of E. koopmannii by establishing an effective cryopreservation protocol for its meristems. This study has identified for the first time an optimized cryopreservation protocol for E. koopmannii meristems involving specific treatment durations and osmotic solutions. The protocol resulted in a significantly higher survival rate (60%) when meristems were precultured with 0.3 M sucrose, treated with Plant Vitrification Solution 2 for 30 min., and regenerated on a medium with 20 g/L sucrose. Secondly, a comparative analysis of cryopreserved regenerates with a control group has revealed that cryopreservation negatively impacted the plant height but significantly increased the number of meristem shoots and roots. This finding highlights the trade-off between growth and root development in cryopreserved plants. Thirdly, the presented optimization of the cryopreservation protocol provides valuable insights for the preservation and regeneration of E. koopmannii meristems. It emphasizes the critical importance of precise treatments and the composition of the nutrient medium to achieve successful cryopreservation and subsequent recovery of plants in tissue culture. This research provides essential information for conserving E. koopmannii, shedding light on the intricate balance required to apply cryopreservation techniques effectively.

Potential of fructans as natural cryoprotectant agents in plant cryopreservation: concept validation on Arabidopsis thaliana L.

BACKGROUND: Today, synthetic chemicals are used in vitrification solutions for cryopreservation studies to mimic natural cryoprotectants that supply tolerance to organisms in nature against freezing stress. In the case of plants, PVS2, containing glycerol, dimethyl sulfoxide (Me2SO), ethylene glycol and sucrose, is considered as the golden standard for successful cryopreservation. However, Me2SO can generally cause toxicity to certain plant cells, adversely affecting viability after freezing and/or thawing. Hence, the replacement (or substantial reduction) of Me2SO by cheap, non-toxic and natural cryoprotectants became a matter of high priority to vitrification solutions or reducing their content gained escalating importance for the cryopreservation of plants. Fructans, sucrose derivatives mainly consisting of fructose residues, are candidate cryoprotectants. OBJECTIVE: Inspired by their protective role in nature, we here explored, for the first time, the potential of an array of 8 structurally different fructans as cryoprotectants in plant cryopreservation. MATERIALS AND METHODS: Arabidopsis thaliana L. seedlings were used as a model system with a one-step vitrification method. PVS2 solutions with different Me2SO and fructan contents were evaluated. RESULTS: It was found that branched low DP graminan, extracted from milky stage wheat kernels, led to the highest recovery (85%) among tested fructans with 12.5% Me2SO after cryopreservation, which was remarkably close to the viability (90%) observed with the original PVS2 containing 15% Me2SO. Moreover, its protective efficacy could be further optimized by addition of vitamin C acting as an antioxidant. CONCLUSION: Such novel formulations offer great perspectives for cryopreservation of various crop species.

Plant Cryopreservation: Principles, Applications, and Challenges of Banking Plant Diversity at Ultralow Temperatures.

Progressive loss of plant diversity requires the protection of wild and agri-/horticultural species. For species whose seeds are extremely short-lived, or rarely or never produce seeds, or whose genetic makeup must be preserved, cryopreservation offers the only possibility for long-term conservation. At temperatures below freezing, most vegetative plant tissues suffer severe damage from ice crystal formation and require protection. In this review, we describe how increasing the concentration of cellular solutes by air drying or adding cryoprotectants, together with rapid cooling, results in a vitrified, highly viscous state in which cells can remain viable and be stored. On this basis, a range of dormant bud-freezing, slow-cooling, and (droplet-)vitrification protocols have been developed, but few are used to cryobank important agricultural/horticultural/timber and threatened species. To improve cryopreservation efficiency, the effects of cryoprotectants and molecular processes need to be understood and the costs for cryobanking reduced. However, overall, the long-term costs of cryopreservation are low, while the benefits are huge.

Nanoparticle-mediated enhancement of plant cryopreservation: Cultivar-specific insights into morphogenesis and biochemical responses in Lamprocapnos spectabilis (L.) Fukuhara 'Gold Heart' and 'Valentine'.

The integration of nanoparticles (NPs) holds promising potential to bring substantial advancements to plant cryopreservation, a crucial technique in biodiversity conservation. To date, little attention has been focused on using nanoparticles in cryobiology research. This study aimed to assess the effectiveness of NPs in enhancing the efficiency of plant cryopreservation. In-vitro-derived shoot tips of bleeding heart (Lamprocapnos spectabilis (L.) Fukuhara) 'Gold Heart' and 'Valentine' were used as the plant material. The encapsulation-vitrification cryopreservation protocol included preculture, encapsulation, dehydration, storage in liquid nitrogen, rewarming, and recovery steps. Gold (AuNPs), silver (AgNPs), or zinc oxide (ZnONPs) nanoparticles were added at various concentrations either into the preculture medium or the protective bead matrix during encapsulation. The explant survival and further morphogenic and biochemical events were studied. Results showed that the impact of NPs on cryopreservation outcomes was cultivar-specific. In the 'Valentine' cultivar, incorporating 5 ppm AgNPs within the alginate bead matrix significantly improved cryopreservation efficiency by up to 12%. On the other hand, the 'Gold Heart' cultivar benefited from alginate supplementation with 5 ppm AgNPs and 5-15 ppm ZnONPs, leading to an over 28% increase in the survival rate of shoot tips. Interestingly, adding NPs to the preculture medium was less effective and sometimes counterproductive, despite promoting greater shoot proliferation and elongation in 'Valentine' explants compared to the control. Moreover, nanoparticles often induced oxidative stress (and enhanced the activity of APX, GPOX, and SOD enzymes), which in turn affected the biosynthesis of plant primary and secondary metabolites. It was found that supplementation of preculture medium with higher concentration (15 ppm) of gold, silver and zinc oxide nanoparticles stimulated the production of plant pigments, but in a cultivar-dependent matter. Our study confirmed the beneficial action of nanoparticles during cryopreservation of plant tissues.

Effect of nanoparticles on the ex-vitro performance of cryopreservation-derived plant material.

The integration of nanoparticles into plant cryopreservation protocols holds great promise for improving the survival rates and recovery potential of explants. This study aimed to verify the effect of nanoparticles on the ex-vitro performance of cryopreservation-derived plants. Lamprocapnos spectabilis (L.) Fukuhara (bleeding heart) 'Gold Heart' and 'Valentine' cultivars were used as the plant material. The encapsulation-vitrification cryopreservation protocol of shoot tips included the preculture, encapsulation, dehydration, storage in liquid nitrogen, rewarming, and recovery steps. Gold (AuNPs), silver (AgNPs), or zinc oxide (ZnONPs) nanoparticles were added at varying concentrations, either into the preculture medium or the protective bead matrix during encapsulation. After the in vitro recovery, the plants were transferred to the glasshouse and subjected to detailed biometrical, biochemical and cytogenetic analyses. Nanoparticles had no evident effect on the acclimatization efficiency (80-100% survival) and leaf number in L. spectabilis 'Gold Heart'. Nonetheless, shoots developed from alginate beads supplemented with 5 ppm AuNPs were twice as long as the control, while the leaves of plants grown on the preculture medium with ZnONPs contained significantly more chlorophyll and had higher Leaf Soil-Plant Analysis Development (SPAD) values. Moreover, several NPs treatments stimulated the development of leaves, including their surface area, length, and perimeter. Higher ZnONPs levels enhanced also the replication process, resulting in higher nuclear DNA content. As for L. spectabilis 'Valentine', alginate augmentation with 5 ppm AgNPs or 5 ppm ZnONPs stimulated the elongation of shoots. There was also a tendency suggesting a positive influence of 5 ppm AgNPs in the alginate bead matrix on foliar growth. The effect of nanoparticles on the content of flavonoids, anthocyanins, and stress markers in the plants varied depending on the treatment and cultivar, but also on the organ studied (leaf or stem). Overall, L. spectabilis 'Gold Heart' was more stress-tolerant and genetically stable than 'Valentine' judging by the activity of Photosystem II (PSII) and flow cytometric analyses, respectively. The complex effects of nanoparticles on survival, biometric parameters, physiological responses, and cytogenetic events underscore the intricate interplay between nanoparticles and plant systems. Nonetheless, our research confirmed the positive effect of nanoparticles on the ex-vitro growth and development of L. spectabilis plants after cryostorage.

The Cryopreservation of Medicinal and Ornamental Geophytes: Application and Challenges.

Nowadays, plant genetic resources are often at risk of loss and destruction. Geophytes are herbaceous or perennial species that are annually renewed by bulbs, rhizomes, tuberous roots, or tubers. They are often subject to overexploitation, which, combined with other biotic and abiotic stresses, can make these plants more vulnerable to a decline in their diffusion. As a result, multiple endeavors have been undertaken to establish better conservation strategies. Plant cryopreservation at ultra-low temperatures in liquid nitrogen (-196 °C) has proven to be an effective, long-term, low-cost, and suitable conservation method for many plant species. Over the last two decades, major advances in cryobiology studies have enabled successful explants of multiple genera and types, including pollen, shoot tips, dormant buds, and zygotic and somatic embryos. This review provides an update on recent advances and developments in cryopreservation and its application to medicinal and ornamental geophytes. In addition, the review includes a brief summary of factors limiting the success of bulbous germplasm conservation. The critical analysis underpinning this review will benefit biologists and cryobiologists in their further studies on the optimization of geophyte cryopreservation protocols and will support a more complete and wider application of knowledge in this area.

Cryopreservation of Plant Tissues in Poland: Research Contributions, Current Status, and Applications

Abstract Cryopreservation of vegetatively propagated plant material is an increasingly widely used method for the efficient and safe storage of germplasm resources around the world. In Poland, there are currently four cryobanks in use for long-term plant protection programs. However, plant tissues propagated in vitro constitute only a small portion of the accessions stored in them. To date, cryogenic storage techniques have been developed and adopted in this country for ornamental plants (roses, chrysanthemums, and geophytes), crop species (potato and garlic), forest tree species (the genera Quercus and Fraxinus ), and some ferns. Polish researchers have used suspension cultures of Gentiana spp. and shoot tips of Lamprocapnos spectabilis to improve cryopreservation knowledge. A better understanding of the benefits of cryopreservation and its widespread implementation in plant biodiversity conservation programs is required. The objective of this review is to provide a concise synthesis of the scientific contributions, current status, and applications of cryogenic techniques for the conservation of in vitro culture-derived plant tissues in Poland. First, the results contributing to research that has been achieved using cell suspensions and advances related to the use of nanoparticles and plant extracts to improve cryopreservation efficiency are discussed. Then, the applications and advances in cryopreservation of ornamental plants (roses, radiomutants, plant chimeras, Lamprocapnos spp., and geophytes), crop species (potato and garlic), forest trees, and ferns are summarized.

The world's largest potato cryobank at the International Potato Center (CIP) - Status quo, protocol improvement through large-scale experiments and long-term viability monitoring.

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP's cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

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.

The Tip of the Iceberg: Cryopreservation Needs for Meeting the Challenge of Exceptional Plant Conservation.

Cryopreservation is increasingly important as a conservation tool, particularly for threatened exceptional species. The goal of this study was to investigate the current knowledge of plant cryopreservation through a search of the literature in Web of Science and align that with the 775 species currently identified on the Working List of Exceptional Plants. While there is a good foundation in plant cryopreservation research, particularly with economically important species, there are significant gaps in research on families that contain the largest numbers of currently known exceptional species, including the Dipterocarpaceae, Rhizophoraceae, and Pittosporaceae. Even families well represented in both in the literature and on the List of Exceptional Plants had much less overlap at the level of genus. Tropical trees, a significant portion of exceptional species, were not as well represented in the literature as herbaceous species. Over 70% of all articles dealt with in vitro cryopreservation, with much less emphasis on other methods (seed, embryo, dormant bud, and pollen) that will be more cost-effective for species where they can be applied. While the research on plant cryopreservation to date provides a strong foundation and is being utilized effectively for conserving the diversity of a number of economically important species, this study revealed significant gaps that can help prioritize future research to more effectively conserve the diversity of threatened exceptional species.

Plant cryopreservation: implementation and outreach

ISHS IX International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (physiological, biochemical, embryological, genetic and legal aspects) Plant cryopreservation: implementation and outreach

In vivo protective effect of late embryogenesis abundant protein (ApSK3 dehydrin) on Agapanthus praecox to promote post-cryopreservation survival

Dehydrins (DHNs), as members of the late embryogenesis abundant protein family, play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses. Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage. In this study, ApSK3 type DHN was genetically transformed into embryogenic calluses (EC) of Agapanthus praecox by overexpression (OE) and RNA interference (RNAi) techniques to evaluate the in vivo protective effect of DHNs during cryopreservation. The cell viability showed a completely opposite trend in OE and RNAi cell lines, the cell relative death ratio was decreased by 20.0% in ApSK3-OE EC and significantly increased by 66.15% in ApSK3-RNAi cells after cryopreservation. Overexpression of ApSK3 increased the content of non-enzymatic antioxidants (AsA and GSH) and up-regulated the expression of CAT, SOD, POD, and GPX genes, while ApSK3-RNAi cells decreased antioxidant enzyme activities and FeSOD, POD, and APX genes expression during cryopreservation. These findings suggest that ApSK3 affects ROS metabolism through chelating metal ions (Cu2+ and Fe3+), alleviates H2O2 and OH· excessive generation, activates the antioxidant system, and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation. DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.

Determination of The Potential of Fructants In Plant Cryopreservation Applications

Determination of The Potential of Fructants In Plant Cryopreservation Applications

Vitrification Solutions for Plant Cryopreservation: Modification and Properties.

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration.

Epigenetic and Genetic Integrity, Metabolic Stability, and Field Performance of Cryopreserved Plants

Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 μM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.

Gold nanoparticles affect the cryopreservation efficiency of in vitro-derived shoot tips of bleeding heart

The popularity of nanoparticles (NPs) is continuously increasing. To date, however, there has been little research on the application of NPs in plant cryopreservation, i.e. storage of tissues in liquid nitrogen (LN). The aim of this study is to analyze the effect and evaluate the usefulness of gold nanoparticles (AuNPs) in regard to cryobiology studies. In vitro-derived shoot tips of Lamprocapnos spectabilis ‘Valentine’ were cryopreserved with the encapsulation-vitrification protocol. Gold nanoparticles (at 10–30 ppm concentration; 13 nm in size) were added either into the preculture medium; to the protective bead matrix during encapsulation; or to the recovery medium after rewarming of samples. The control plants were produced from cryopreserved explants non-treated with nanoparticles or treated with colloid dispersion medium without NPs. A non-LN-treated standard was also considered. The influence of AuNPs on the cryopreservation efficiency was determined by evaluating the recovery rate of explants and their morphogenic response; the membrane stability index (MSI); the concentration of pigments in shoots; and the antioxidant enzymes activity. The genetic stability of the plant material was evaluated using Start Codon Targeted Polymorphism (SCoT) markers. It was found that 10 ppm of AuNPs added into the alginate bead matrix improved the recovery level of LN-derived shoot tips (70.0%) compared to the non-NPs-treated cryopreserved control (50.5%). On the other hand, the presence of nanoparticles in the recovery medium had a deleterious effect on the survival of explants. AuNPs usually had no impact on the MSI (73.9–85.9%), except for those added into the recovery medium at the concentration of 30 ppm (decline to 55.8%). All LN-derived shoots were shorter and contained less chlorophyll and carotenoids than the untreated standard. Moreover, the application of AuNPs affected the enzymatic activity in L. spectabilis. Minor genetic variation was found in 8.6% of plants if AuNPs were added either into the preculture medium (at 10 and 20 ppm) or to the alginate matrix (at 30 ppm). In conclusion, AuNPs added at a lower concentration (10 ppm) into the protective bead matrix can significantly improve the cryopreservation efficiency in L. spectabilis with no alternation in the DNA sequence.

ApSerpin-ZX from Agapanthus praecox, is a potential cryoprotective agent to plant cryopreservation

Cryopreservation-induced cell death is regarded as an important problem faced by cryobiologists. Oxidative stress and programmed cell death are detrimental to cell survival. Serine protease inhibitors (serpins) inhibit pro-cell-death proteases and play a pro-survival role in excessive cell death induced by abiotic stress. In this study, ApSerpin-ZX was isolated from Agapanthus praecox and characterized as a protective protein in plant cryopreservation. The mRNA level of ApSerpin-ZX was elevated under abiotic stress, such as salt, osmosis, oxidative, cold, and cryoinjury. The purified recombinant protein expressed in E. coli was added to the plant vitrification solution and used for A. praecox embryogenic callus cryopreservation. The concentration of 0.6–4.8 mg∙L−1 of ApSerpin-ZX protein was beneficial to the survival of cryopreserved embryogenic callus of A. praecox. The most effective concentration was 1.2 mg∙L−1, which elevated the survival by 37.15%. Subsequently, the cryopreservation procedure with 1.2 mg∙L−1 of ApSerpin-ZX protein was regarded as the treated group, compared to standard procedure, to determine the physiological mechanism of ApSerpin-ZX protein on cryopreserved cell. The MDA and H2O2 contents were significantly decreased in the treated group, along with reduced OH· generation activity in the recovery stage. After the addition of ApSerpin-ZX, the POD and CAT activities keep increased, while SOD activity increased only after dehydration. Besides, the caspase-1-like and caspase-3-like activities were lower than the standard procedure. This study indicated that ApSerpin-ZX was a potential cryoprotective agent that alleviated oxidative stress and cell death induced by cryopreservation.

Single-wall carbon nanotubes improve cell survival rate and reduce oxidative injury in cryopreservation of Agapanthus praecox embryogenic callus

BackgroundCryopreservation is the best way for long-term in vitro preservation of plant germplasm resources. The preliminary studies found that reactive oxygen species (ROS) induced oxidative stress and ice-induced membrane damage are the fundamental causes of cell death in cryopreserved samples. How to improve plant cryopreservation survival rate is an important scientific issue in the cryobiology field.ResultsThis study found that the survival rate was significantly improved by adding single-wall carbon nanotubes (SWCNTs) to plant vitrification solution (PVS) in cryopreservation of Agapanthus praecox embryogenic callus (EC), and analyzed the oxidative response of cells during the control and SWCNTs-added cryopreservation protocol. The SWCNTs entered EC at the step of dehydration and mainly located around the cell wall and in the vesicles, and most of SWCNTs moved out of EC during the dilution step. Combination with physiological index and gene quantitative expression results, SWCNTs affect the ROS signal transduction and antioxidant system response during plant cryopreservation. The EC treated by SWCNTs had higher antioxidant levels, like POD, CAT, and GSH than the control group EC. The EC mainly depended on the AsA-GSH and GPX cycle to scavenge H2O2 in the control cryopreservation, but depended on CAT in the SWCNTs-added cryopreservation which lead to low levels of H2O2 and MDA. The elevated antioxidant level in dehydration by adding SWCNTs enhanced cells resistance to injury during cryopreservation. The ROS signals of EC were balanced and stable in the SWCNTs-added cryopreservation.ConclusionsThe SWCNTs regulated oxidative stress responses of EC during the process and controlled oxidative damages by the maintenance of ROS homeostasis to achieve a high survival rate after cryopreservation. This study is the first to systematically describe the role of carbon nanomaterial in the regulation of plant oxidative stress response, and provided a novel insight into the application of nanomaterials in the field of cryobiology.

Root cryobanking: an important tool in plant cryopreservation

Cryopreservation in liquid nitrogen (− 196 °C) ensures safe and cost-effective conservation of plant genetic resources using diverse materials, including roots. To date, successful cryopreservation of root explants isolated from in vitro grown plants as well as adventitious and hairy root cultures has been reported in large numbers of plant species. In particular, significant progress has been made in cryopreserving in vitro cultures of adventitious and hairy roots that attract both scientific and commercial interest as an alternative source of plant-derived bioactive compounds with potential application in pharmaceutical, cosmetic, and natural health product industries. The process of cryopreservation has been shown to have little or no impact on growth, and biosynthetic and genetic stability of roots following recovery in an ambient growth environment. However, structural integrity of roots is critical for successful cryopreservation as thin and fragile root tips are very sensitive to stressful conditions associated with various steps of a cryopreservation protocol and a thorough optimization of each step, particularly the treatment with vitrification solution, is essential. This review summarizes the results of recent studies on cryopreservation of plant roots and highlights the importance of continued research in this area. Roots are uniquely suited for long term conservation of plant genetic resources due to their ease of excision, in vitro maintenance, and the ability to undergo morphogenesis. The root based regeneration and cryopreservation systems offer excellent opportunities to investigate a range of regulatory signalling and adaptation mechanisms that are activated in response to physical and chemical stresses. This review summarizes the results of recent studies on cryopreservation of plant roots, including hairy and adventitious root cultures used in biotechnology, and underpins their significance in biobanking of economically important food, ornamental, and medicinal crops, as well as, endangered plant species.