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Availability of the methods for long-term virus preservation facilitates easy acquirement of viruses, which are needed in many basic and applied virological studies. Cryopreservation is currently considered an ideal means for long-term preservation of plant germplasm. Recent studies have shown that cryopreservation provided an efficient and reliable method for long-term preservation of plant viruses. Here, we describe the detailed procedures of droplet vitrification for long-term preservation of apple stem grooving virus (ASGV), which represents a type of viruses that can invade meristematic cells of the shoot tips, and potato leafroll virus (PLRV), which is a phloem-limited virus that does not infect the apical meristem. Shoot tip cryopreservation provides an advantageous strategy for the long-term preservation of plant viruses., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Main Conclusion: Reactive oxygen species (ROS)-induced oxidative stress results in low success or even total failure of cryopreservation. Better understanding of how the plant establishes resistance/tolerance to ROS-induced oxidative stress facilitates developments of robust cryopreservation procedures. Cryopreservation provides a safe and efficient strategy for long-term preservation of plant genetic resources. ROS-induced oxidative stress caused damage to cells and reduced the ability of the plant to survive following cryopreservation, eventually resulting in low success or even total failure. This paper provides updated and comprehensive information obtained in the past decade, including the following: (1) ROS generations and adaptive responses of antioxidant systems during cryopreservation; (2) expressions of oxidative stress-associated genes and proteins during cryopreservation; (3) ROS-triggered programmed cell death (PCD) during cryopreservation; and (4) exogenous applications of enzymatic and non-enzymatic antioxidants in improving success of cryopreservation. Prospects for further studies are proposed. The goal of the present study was to facilitate better understanding of the mechanisms by which the plant establishes resistance/tolerance to oxidative stress during cryopreservation and promote further studies toward the developments of robust cryopreservation procedures and wider application of plant cryobiotechnology., (© 2021. The Author(s).)

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit 'Yuxiang' (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis 'Jinmi', respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We previously reported successful cryopreservation of shoot tips of potato 'Zihuabai' by three vitrification-based protocols. In the present study, cryo-injury to shoot tips and genetic stability in regenerants recovered from cryopreserved shoot tips by the three vitrification-based protocols were further investigated. The results showed that sucrose preculture caused no obviously different injuries, while dehydration with plant vitrification solution 2 (PVS2) was the step causing major damage to cells of shoot tips, regardless of the cryogenic procedures. Compared with droplet-vitrification and encapsulation-vitrification, vitrification caused the most severe injury to cells of the shoot tips, thus resulting in much longer time duration for shoot recovery and much lower shoot regrowth rate. Cells in apical dome and the youngest leaf primordia were able to survive and subsequently some of them regrew into shoots following all three vitrification-based cryopreservation procedures. Analyses using inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers in shoots regrown from all three vitrification-based protocols did not find any polymorphic bands. The results reported here suggest that vitrification-based cryo-procedures can be considered promising methods for long-term preservation of potato genetic resources., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Rapid increases in human populations provide a great challenge to ensure that adequate quantities of food are available. Sustainable development of agricultural production by breeding more productive cultivars and by increasing the productive potential of existing cultivars can help meet this demand. The present paper provides information on the potential uses of cryogenic techniques in ensuring food security, including: (1) long-term conservation of a diverse germplasm and successful establishment of cryo-banks; (2) maintenance of the regenerative ability of embryogenic tissues that are frequently the target for genetic transformation; (3) enhancement of genetic transformation and plant regeneration of transformed cells, and safe, long-term conservation for transgenic materials; (4) production and maintenance of viable protoplasts for transformation and somatic hybridization; and (5) efficient production of pathogen-free plants. These roles demonstrate that cryogenic technologies offer opportunities to ensure food security., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Root and tuber crops (RTCs) are the second-most important carbohydrate commodity after cereals. Many species of the RTCs are vegetatively propagated, making their shoot tips the preferred material to be conserved for future uses. Shoot tip cryopreservation provides an important tool to support the long-term conservation of plant genetic resources. Over the past four decades, significant efforts have been undertaken to move shoot tip cryopreservation of RTCs from research projects to full-scale implementation in cryobanks. This comprehensive review focuses on the history of cryopreservation protocols developed in RTCs. The encapsulation and vitrification solution-based cryopreservation techniques followed by ultra-rapid freezing and thawing have been highly successful. Additionally, different strategies for improving the cryotolerance of shoot tips have been introduced to further increase post-cryopreservation recovery. Finally, the research conducted to explain the mechanism underlying cryoprotection and differential cryotolerance including the use of histological studies are highlighted. [ABSTRACT FROM AUTHOR]

In vitro micrografting is an important technique supporting the micropropagation of a range of plant species, particularly woody plant species. Over the past several decades, in vitro micrografting has become a strategy to facilitate shoot recovery and acclimatization of in vitro-grown horticultural species. This review focuses on studies on horticultural crops over the past two decades that cover the establishment of in vitro micrografting, discusses factors affecting the success of in vitro micrografting, and provides commentary on the contribution of micrografting applications to the field of micropropagation. Considering the important roles of micrografting in the restoration of vigor and rooting competence, in promotion of shoot recovery following somatic embryogenesis and organogenesis, and in facilitation of shoot regrowth after cryopreservation, the potential use of this technique in facilitation of genetic engineering and safe conservation of horticultural species are specially highlighted. [ABSTRACT FROM AUTHOR]

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.Abstract: 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. [ABSTRACT FROM AUTHOR]

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L−1 α-naphthalene acetic acid and 0.5 mg L−1 thidiazuron. Small leaf squares (SLSs, 3 × 4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • We could successfully cryopreserve two-viroid infected chrysanthemum. • We discover that stock hygiene significantly affects cryopreservation success. • We could provide explanations as to why cryopreservation failed to eradicate viroids. Abstract Chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) are the two viroids most frequently infecting chrysanthemum. This study attempted to cryopreserve in vitro shoot tips of Chrysanthemum morifolium 'Piato' infected with high or low titers of CSVd, and co-infected with CSVd and CChMVd. By optimizing several key factors including the time and light regimes during cold-hardening of stock shoots, and the size of shoot tips, an encapsulation-vitrification procedure was established for cryopreservation of viroid-infected shoot tips. Viroid-infected stock shoots were cold-hardened in vitro at 4 °C in a 16-h photoperiod for 6 weeks. Shoot tips (1.5 mm in size) containing 3-4 LPs were excised from the cold-hardened stock shoots and subjected to encapsulation-vitrification cryopreservation. With this protocol, about 65%, 45% and 42% of shoot regrowth levels were obtained in cryopreserved shoot tips derived from in vitro stock shoots infected with low or high titers of CSVd, and co-infected with CSVd and CChMVd, respectively. All regenerants from cryopreservation maintained their viroid-infected status, identical to those of their in vitro stock shoots. Histological observations and in situ hybridization elucidated why cryopreservation could maintain viroids in cryopreserved shoot tips. Cryopreservation of viroid-infected plant materials has potential applications to all types of viroid-related basic and applied studies. [ABSTRACT FROM AUTHOR]

We report a droplet-vitrification for efficient cryopreservation of valuable germplasm of blueberry. In this protocol, in vitro stock shoots were cold-hardened in the dark at 4 °C for 3 weeks. Shoot tips (1.5–2.0 mm in length) including 4–5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3 M sucrose for 1 day. Precultured shoot tips were treated with a loading solution containing 1.0 M sucrose and 2 M glycerol for 30 min, followed by exposure to PVS2 for 40 min at 0 °C. Dehydrated shoot tips were then transferred onto 3.5 μL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen foils with shoot tips were re-warmed in 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for a recovery medium containing 0.3 mg L −1 zeatin for shoot regrowth. With optimized parameters, shoot regrowth levels were 46.7% for ‘O’ Neal’ and 91.7% for ‘Misty’, with a mean shoot regrowth level of 66.5% obtained across the five valuable blueberry genotypes tested. Histological observations showed that many cells in the upper part of apical dome and some in leaf primordia 1–3 survived, while those in lower part of apical dome and leaf primordia 4–5 were seriously damaged, following cryopreservation. No polymorphic bands were detected using inter-simple sequence repeat markers (ISSR) and random amplified polymorphic DNA (RAPD) in regenerants of ‘O’ Neal’ and ‘Misty’ recovered from cryopreservation. To the best our knowledge, this is the first report on blueberry cryopreservation by droplet-vitrification, which would provide a technical platform for establishment of cryo-banks of valuable blueberry germplasm. [ABSTRACT FROM AUTHOR]