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.

Vitrification cryo-foil method for shoot tip cryopreservation and virus eradication in apple.

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.

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.

ROS-induced oxidative stress in plant cryopreservation: occurrence and alleviation.

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.

Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

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.

In vitro tissue culture of apple and other Malus species: recent advances and applications.

MAIN CONCLUSION: Studies on the tissue culture of apple have allowed for molecular, biotechnological and applied breeding research to advance. In the past 8 years, over 100 papers advancing basic biology, genetic transformation and cryobiology have emerged. Apple (Malus × domestica Borkh.; Rosaceae) is an important fruit crop grown mainly in temperate regions of the world. In vitro tissue culture is a biotechnological technique that has been used to genetically improve cultivars (scions) and rootstocks. This updated review presents a synthesis of findings related to the tissue culture of apple and other Malus spp. between 2010 and 2018. Increasingly complex molecular studies that are examining the apple genome, for example, in a bid to identify the cause of epigenetic mutations and the role of transposable elements in this process would benefit from genetically stable source material, which can be produced in vitro. Several notable or curious in vitro culture methods have been reported to improve shoot regeneration and induce the production of tetraploids in apple cultivars and rootstocks. Existing studies have revealed the molecular mechanism underlying the inhibition of adventitious roots by cytokinin. The use of the plant growth correction factor allows hypothetical shoot production from leaf-derived thin cell layers relative to conventional leaf explants to be determined. This updated review will allow novices and established researchers to advance apple and Malus biotechnology and breeding programs.

Occurrence and Molecular Variability of Kiwifruit Viruses in Actinidia deliciosa 'Xuxiang' in the Shaanxi Province of China.

Kiwifruit (Actinidia spp.) is an economically substantial fruit crop with China the main producer. China is the primary source of wild kiwifruit and the largest producer of kiwifruit in terms of both production and planting area, and Shaanxi province is the largest kiwifruit producer in China. Previous studies reported presence of kiwifruit viruses in Actinidia chinensis. In this study, six viruses were identified in kiwifruit 'Xuxiang' (A. deliciosa) in Shaanxi, China. The incidence, distribution, and genetic diversity of these viruses were studied. The results showed that Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), cucumber mosaic virus (CMV), apple stem grooving virus (ASGV), and potato virus X (PVX) were the main viruses infecting Xuxiang kiwifruit in Shaanxi, China. Incidence of the various viruses with both single and multiple infection varied with different kiwifruit-growing counties. For single virus infection, the highest and the lowest numbers of samples infected were about 22 for AcCRaV and 0 for AcVB in Meixian out of 170 samples, 12 for AcVA and 0 for CMV in Zhouzhi out of 120 samples, 10 for AcVA and 0 for AcVB, AcCRaV, ASGV, PVX, and CMV in Yangling out of 70 samples, and 8 for AcCRaV and CMV and 0 for AcVA, AcVB, ASGV, and PVX in Hanzhong out of 80 samples, respectively. Samples which were multiply infected with two or more viruses were also detected. Analysis of the phylogenetic tree of these viruses showed some genetic variability in the AcVA, AcVB, and AcCRaV isolates of Shaanxi kiwifruit. There was no obvious molecular variation in the coat protein genes of ASGV, CMV, and PVX virus isolates from Shaanxi kiwifruit. The present study is the first large-scale survey of kiwifruit viruses in Shaanxi, China. To our knowledge, this is the first report of PVX infecting kiwifruit and the first report of molecular variability of AcVA, AcVB, and AcCRaV. These results provide important data for studying the genetic evolution of AcVA, AcVB, AcCRaV, ASGV, CMV, and PVX.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

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.

Drought Stress Enhances Up-Regulation of Anthocyanin Biosynthesis in Grapevine leafroll-associated virus 3-Infected in vitro Grapevine (Vitis vinifera) Leaves.

Reddish-purple coloration on the leaf blades and downward rolling of leaf margins are typical symptoms of grapevine leafroll disease (GLD) in red-fruited grapevine cultivars. These typical symptoms are attributed to the expression of genes encoding enzymes for anthocyanins synthesis, and the accumulation of flavonoids in diseased leaves. Drought has been proven to accelerate development of GLD symptoms in virus-infected leaves of grapevine. However, it is not known how drought affects GLD expression nor how anthocyanin biosynthesis in virus-infected leaves is altered. The present study used HPLC to determine the types and levels of anthocyanins, and applied reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of genes encoding enzymes for anthocyanin synthesis. Plantlets of Grapevine leafroll-associated virus 3 (GLRaV-3)-infected Vitis vinifera 'Cabernet Sauvignon' were grown in vitro under PEG-induced drought stress. HPLC found no anthocyanin-related peaks in the healthy plantlets with or without PEG-induced stress, while 11 peaks were detected in the infected plantlets with or without PEG-induced drought stress, but the peaks were significantly higher in infected drought-stressed plantlets. Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress. The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress. Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation. This accumulation involved the up-regulation of two key genes, MYBA1 and UFGT, and their expression levels were further enhanced by drought stress.

Dual-Mode Controlled Self-Assembly of TiO2 Nanoparticles Through a Cucurbit[8]uril-Enhanced Radical Cation Dimerization Interaction.

The realization of controllable multicomponent self-assembly through reversible supramolecular interactions is a challenging goal, and is an important strategy for the fabrication of switchable nanomaterials. Herein we show that the self-assembly of TiO2 nanoparticles (NP) functionalized with methyl viologen can be controlled both by light irradiation and chemical reduction through cucurbit[8]uril-enhanced radical cation dimerization interactions. Moreover, the controlled assembly and disassembly of this system are accompanied by switchable photocatalytic activity of the TiO2  NPs, which shows potential application as a novel smart and recyclable photocatalyst.

Long-Term Preservation of Plant Viruses in Cryopreserved Shoot Tips.

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.

Grapevine Shoot Tip Cryopreservation and Cryotherapy: Secure Storage of Disease-Free Plants.

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.

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.

Development, progress and future prospects in cryobiotechnology of Lilium spp.

Lilium is one of the most popular flower crops worldwide, and some species are also used as vegetables and medicines. The availability of and easy access to diverse Lilium genetic resources are essential for plant genetic improvements. Cryopreservation is currently considered as an ideal means for the long-term preservation of plant germplasm. Over the last two decades, great efforts have been exerted in studies of Lilium cryopreservation and progress has been made in the successful cryopreservation of pollen, seeds and shoot tips in Lilium. Genes that exist in Lilium, including those that regulate flower shape, color and size, and that are resistant to cold stress and diseases caused by fungi and viruses, provide a rich source of valuable genetic resources for breeding programs to create novel cultivars required by the global floriculture and ornamental markets. Successful cryopreservation of Lilium spp. is a way to preserve these valuable genes. The present study provides updated and comprehensive information about the development of techniques that have advanced Lilium cryopreservation. Further ideas are proposed to better direct future studies on Lilium cryobiotechnology.

Cryotherapy: A Novel Method for Virus Eradication in Economically Important Plant Species.

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.

In vitro thermotherapy-based methods for plant virus eradication.

Production of virus-free plants is necessary to control viral diseases, import novel cultivars from other countries, exchange breeding materials between countries or regions and preserve plant germplasm. In vitro techniques represent the most successful approaches for virus eradication. In vitro thermotherapy-based methods, including combining thermotherapy with shoot tip culture, chemotherapy, micrografting or shoot tip cryotherapy, have been successfully established for efficient eradication of various viruses from almost all of the most economically important crops. The present study reviewed recent advances in in vitro thermotherapy-based methods for virus eradication since the twenty-first century. Mechanisms as to why thermotherapy-based methods could efficiently eradicate viruses were discussed. Finally, future prospects were proposed to direct further studies.