Rapid population growth poses a major challenge to global food security. Promoting sustainable agricultural production is necessary to ensure global food security. Horticultural plants are a high-valued part of agricultural production. Virus and viroid diseases have long been a key factor limiting horticultural production. Cultivating and distributing pathogen-free plants is currently the most efficient practice for managing virus and viroid diseases and their spread in the landscape. Cryotherapy-based methods are recently developed novel biotechnologies for the efficient production of pathogen-free plants. This review outlines updated information on the development and advances in cryotherapy-based methods for efficiently eradicating viruses and viroids in horticultural plants. Mechanisms underlining cryotherapy-based methods for improved pathogen eradication are discussed, and suggestions for further studies are proposed.

We report the successful cryopreservation of three economically important Rubus viruses: raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), and Rubus yellow net virus (RYNV), using shoot tip cryopreservation in four raspberry cultivars. Virus-infected shoot tips (approximately 1.0 mm in length) containing 3–4 leaf primordia (LPs) were cryopreserved using the droplet-vitrification technique. In the cultivars ‘Zlatá Královna (ZK)’ and ‘Tulameen (TUM)’, over 90

In vitro tissue culture technologies provide novel tools for improving plant production [...]

By optimizing size of shoot tips, preculture medium and exposure duration to PVS2, we established an efficient and wide-spectrum droplet-vitrification cryopreservation for shoot tips of raspberry (Rubus idaeus L.). This protocol yielded 80–100

Background of the study- Cryopreservation is considered to be a valuable method for long-term preservation of plant germplasm and recently it has been shown to be a reliable method for preserving obligate pathogens including plant viruses. Objectives- (1) Droplet-vitrification cryopreservation of strawberry genotypes in Norway; (2) Preservation efficiency of aphid-transmitted strawberry mild yellow edge virus (SMYEV) and strawberry vein banding virus (SVBV) following cryopreservation. Methods- Excised shoot tips of cv. 'Korona' were cryopreserved with different durations of PVS2 varying from 10 to 60 min, whereas virus-infected shoot tips were cryopreserved using either 10, 40 or 60 min of PVS2. Results- The results showed that 40-60 minutes of PVS2 treatment was more efficient for preserving strawberry germplasm than lower duration times (10-30 min). Thirtytwo strawberry genotypes have been successfully cryopreserved through droplet-vitrification with regeneration rates ranging from 45% to 100% with 40 min PVS2 treatment. Cryopreserved viruses were quantitatively analyzed by Reverse Transcription- quantitative polymerase chain reaction (RT-qPCR). SVBV was successfully cryopreserved in all the regenerated shoots following cryopreservation with all the three durations of PVS2 examined. SMYEV, however, was more efficiently preserved in shoot tips exposed to 40 min (90%) of PVS2, in comparison to 60 min (33%). Conclusion- This demonstrates that SMYEV and SVBV can be successfully cryopreserved in living cells of Fragaria ssp. by droplet vitrification. The results indicate that cryopreservation has great potential for long-time preservation of both strawberry germplasm and aphid-transmitted strawberry-infecting viruses.

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.

Establishment of a new method for improved shoot tip cryopreservation is crucial to facilitate the long-term preservation of plant germplasm as well as the use of cryotherapy for pathogen eradication. The present study reported a vitrification (V) cryo-foil method for shoot tip cryopreservation and virus eradication in apple. Shoot tip regrowth levels after cryopreservation were comparable among V cryo-foil (53 %), V cryo-plate (46 %) and conventional droplet vitrification (Dr-vi, 48 %). The V cryo-foil is more efficient to perform than Dr-vi as more shoot tips can be cryopreserved by one person. In the histological study applying an image-overlaying strategy, shoot tips cryopreserved by V cryo-foil showed a higher survival chance in the youngest leaf primordia than in the apical dome. When V cryo-foil was tested for virus eradication, fifty-five percent (55 %) of cryo-derived shoots were free of the apple stem pitting virus (ASPV), while none and less than 10 % were free of the apple stem grooving virus (ASGV) and the apple chlorotic leaf spot virus (ACLSV), respectively. Thus, these two viruses were efficiently preserved by V cryo-foil cryopreservation. Noticeably, although the shoot regrowth level was reduced to 27 %, a higher frequency (81 %) of ASPV eradication was achieved when a reduced duration of cryoprotectant exposure was applied in V cryo-foil, supporting the use of insufficient cryoprotection for improved virus eradication.

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.

Diseases caused by plant pathogens such as viruses, viroids and phytoplasmas cause huge economic losses of agricultural production and limit the safe movement of plant materials across borders. The use of pathogen-free planting materials provides a strategy for efficient management of these diseases and facilitates the global exchange of genetic resources. Shoot tip cryotherapy is a novel biotechnology method that uses cryogenic procedures to eradicate plant pathogens from the diseased plants. Combining thermotherapy or chemotherapy with shoot tip cryotherapy has further enhanced pathogen eradication efficiency. This review provides updated and comprehensive information on shoot tip cryotherapy and the combination of thermotherapy or chemotherapy with shoot tip cryotherapy for pathogen eradication. Prospects are proposed for future studies.

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.

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.

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.

Plant virus eradication is a prerequisite for the use of virus-free propagules for sustainable crop production. In contrast, virus preservation is required for all types of applied and basic research of viruses. Shoot tip cryopreservation can act as a double-edged strategy, facilitating either virus eradication or virus preservation in cryoderived plants. Here, we tested the efficacies of shoot tip cryopreservation for virus eradication and preservation in shallot (Allium cepa var. aggregatum). In vitro stock shallot shoots infected with onion yellow dwarf virus (OYDV) and shallot latent virus were thermotreated for 0, 2, and 4 weeks at a constant temperature of 36celcius before shoot tip cryopreservation. Results showed that viruses were preserved in recovered shoots when thermotherapy was not applied. Although thermotherapy lowered the regrowth levels of cryotreated shoot tips, the efficiency of virus eradication increased from 5% to 54%. Immunolocalization of OYDV and histological observation of cryotreated shoot tips showed the high frequency of virus preservation was due to the viral invasion of cells close to the apical meristem and the high proportion of cells surviving. Four weeks of thermotherapy drastically decreased the distribution of OYDV, as well as the percentage of surviving cells within the shoot tips, thereby promoting virus eradication. Virus-free plants obtained from combining thermotherapy with cryotherapy showed significantly improved vegetative growth and bulb production. The present study reports how thermotherapy can act as a trigger to facilitate either the safe preservation of Allium viruses or the production of virus-free shallot 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.

Grapevine (Vitis spp.) is one of the most economically important temperate fruit crops. Grapevine breeding programs require access to high-quality Vitis cultivars and wild species, which may be maintained within genebanks. Shoot tip cryopreservation is a valuable technique for the safe, long-term conservation of Vitis genetic resources that complements traditional field and in vitro germplasm collections. Vitis is highly susceptible to virus infections. Virus-free plants are required as propagation material for clonally propagated germplasm, and also for the global exchange of grapevine genetic resources. Shoot tip cryotherapy, a method based on cryopreservation, has proven to be effective in eradicating viruses from infected plants, including grapevine. This comprehensive review outlines/documents the advances in Vitis shoot tip cryopreservation and cryotherapy that have resulted in healthy plants with high regrowth levels across diverse Vitis species.

Background and Aims Virus detection is essential in programs in which virus-tested plants are required. The aim of this study was to establish in vitro biological methods for indexing grapevine leafroll-associated virus-3 (GLRaV-3) in red- and white-berried grapevines. Methods and Results Healthy, red-berried grapevine Cabernet Sauvignon [CaSa (H)] was used as a rootstock in micrografting experiments with virus-infected Cabernet Sauvignon [CaSa (VI)] and Chardonnay [Ch (VI)] scions. Vascular bundles connecting the rootstock and scions developed, and 100% of the micrografts survived 3 and 4 weeks after micrografting, respectively. Symptoms of GLRaV-3 were expressed in 80 and 20% of CaSa (H) rootstocks micrografted with CaSa (VI) and with Ch (VI) scions, respectively, 12 weeks after micrografting. The rootstock CaSa (H) that had been micrografted with CaSa (VI) and Ch (VI) scions for 5 weeks was subsequently cultured on half-strength Murashige and Skoog medium supplemented with 75 mmol/L NaCl to induce salt stress. The GLRaV-3 symptoms were expressed in 88% of micrografts CaSa (VI)/CaSa (H) and 85% of micrografts Ch (VI)/CaSa (H) after 5 weeks of salt stress. Reddish-purple leaf coloration and downward rolling were observed in CaSa (H) rootstocks that were micrografted with CaSa (VI) scions, while leaf downward rolling and yellowing were seen in CaSa (H) rootstocks that were micrografted with Ch (VI) scions. Conclusions Salt stress improved in vitro biological indexing of GLRaV-3 in grapevines. Significance of the Study Establishment of in vitro micrografting and salt stress provided alternative methods for indexing of GLRaV-3 in red- and white-berried grapevines.

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. Abstract 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.

Viral diseases (biotic stress) and salt stress (abiotic stress) are the two major stress factors limiting the sustainable development of the grape industry. These two stresses often occur simultaneously in many grape-growing regions. The present study investigated crosstalk between grapevine leafroll-associated virus-3 (GLRaV-3) and NaCl-induced salt stress in in vitro plantlets of the red-fruited grape ‘Cabernet Sauvignon’. Results showed that although the healthy plantlets had greater vegetative growth than the virus-infected plantlets when grown without salt stress, the latter produced better vegetative growth than the former when cultured under salt stress, indicating that the virus-infected plantlets were more tolerant to NaCl-induced salt stress than the healthy ones. Analyses of physiological metabolites found that contents of total soluble sugar and free proline were generally much higher in the latter than in the former under salt stress. The virus-infected plantlets responded to salt stress by significantly increasing activities of SOD, POD and CAT, compared with the healthy ones. Salt stress induced much higher levels of ABA in the virus-infected plantlets than the healthy ones. To the best of our knowledge, this is the first study reporting virus infection improves plant tolerance to salt stress. The results reported here provide new insights into better understanding of improvement of plant salt tolerance by virus infection. The red-fruited grape 'Cabernet Sauvignon' infected with grapevine leafroll-associated virus-3 improved salt tolerance of the infected in vitro plantlets. In vitro culture system provided an alternative strategy for studying crosstalk between plants and combined stresses by abiotic and biotic factors.