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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.

This is the pre-peer reviewed version of the following article: Wang, M., Hamborg, Z., Ma, X., Blystad, D., & Wang, Q. (2021). Double‐edged effects of the cryogenic technique for virus eradication and preservation in shallot shoot tips. Plant Pathology, 71(2), 494–504. Portico, which has been published in final form at https://doi.org/10.1111/ppa.13466. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions." 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 36℃ 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.

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.

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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.

Additional file 1. Supplementary Material.

Additional file 2. 387 genes selected for sequencing in this study.

Kiwifruit (Actinidia spp.) is an economically important fruit crop in the world. China is the largest producer of kiwifruit in terms of both acreage and yield. In this study, a novel virus, tentatively named Actinidia virus C (AcVC), was discovered in Actinidia deliciosa ‘Xuxiang’ grown in commercial kiwifruit fields in the Shaanxi province, a major kiwifruit‐growing region in China. Sequencing of the complete genome showed that AcVC has 7,668 nucleotides (not including the poly‐(A) sequence). Complete genome comparison and phylogenetic analysis showed that AcVC is most closely related to AcVA and then to AcVB. AcVC shares 69.2% nucleotide identity with AcVA (GenBank accession number JN427014). Based on these data, Actinidia virus C is assigned as a new tentative member of the genus Vitivirus in the family Betaflexiviridae. AcVC was found to infect A. deliciosa and A. chinesis, the two major cultivated kiwifruits. Infection frequency of AcVC varied with the kiwifruit‐growing regions and cultivars in the Shaanxi province of China.

Drought-tolerant rootstocks have been used to improve production efficiency of graft-propagated crops. Kiwifruit is a high-valued fruit crop of the world. Drought stress widely occurs in many of kiwifruit-growing regions including China. The present study used micrografting for evaluation of drought tolerance in four kiwifruit cultivars micrografted on the drought-tolerant rootstock MX1. When stressed by PEG-induced drought, the micrografted kiwifruit cultivars showed significant differences in accumulation of ROS (O2·− and H2O2), physiological metabolism (total soluble sugar, total soluble protein and free proline), activities of antioxidants (SOD and CAT), endogenous ABA levels and expression levels of ABA biosynthetic genes (DREB1 and DREB2). Drought tolerance of the micrografts varied with the four kiwifruit cultivars, with the highest and lowest drought tolerance found in ‘Yuxiang’ and ‘Hayward’, respectively. Micrografting provides an alternative strategy for fast and efficient evaluation of drought tolerance of plant cultivars, thus assisting sustainable development of production of the graft-propagated plant species. In vitro micrografting provided a fast and efficient approach to evaluate drought tolerance of kiwifruit, and has potential applications to other graft-propagated plant species.

The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 µl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species. Establishment of a droplet-vitrification cryopreservation of shallot shoot tips provided technical supports for long-term preservation of diverse genetic resources and cryotherapy for virus eradication in shallot plants.

The present study described a micrografting method for kiwifruits and evaluated graft-compatibility, shoot growth, and root formation and growth of the micrografts. Although time durations of callus formation and initiation of new cambial cells varied with the scion cultivars, vascular bundles were established at about day 21 of micrografting in the four kiwifruit scion cultivars tested. Early callus formation and initiation of new cambial cells promoted bud break in the scions and root formation in rootstocks. These promotive effects enhanced shoot growth, and root formation and growth of the micrografts. This micrografting method has potential for micropropagation of kiwifruit. Shoot growth, and root formation and growth of the micrografts varied with the scion cultivars. Micrografting method has potential for micropropagation of kiwifruit.

Improvements of existing cryopreservation protocols are necessary to facilitate long-term preservation of plant germplasm and the cryotherapy-effect of pathogen eradication. This study reported a vitrification (V) cryo-foil/plate methods for cryopreservation of shoot tips and cryotherapy effect in ‘Pink Lady’ apple. In V cryo-foil/plate protocols, shoot tips were first attached onto aluminum foils/plates using calcium alginate before other procedures. Shoot tips cryopreserved by V cryo-foil required 6.1 weeks to fully recover and 53% of shoot regrowth was obtained, comparable to the Dv cryopreservation. Similar regrowth levels were produced between applying V cryo-foil and Dv cryopreservation to another 4 Malus genotypes. Histological observations in shoot tips cryopreserved by Dv and V cryo-foil found only those with surviving apical dome and leaf primordia (LPs) could recover after cryopreservation. In apical meristem of shoot tips cryopreserved by Dv and V cryo-foil, higher surviving probability was detected from the V cryo-foil protocol, and the young LPs showed the highest level of surviving. Virus detection in cryo-derived plants showed apple stem grooving virus and apple chlorotic leaf spot virus were all preserved after cryopreservation, and higher eradication efficiency of apple stem pitting virus (70%) was produced by Dv than the 55% of V cryo-foil. These results supported applying V cryo-foil as an improvement to the widely applied Dv method in shoot tip cryopreservation, and also revealed a seesaw mode between shoot recovery and cryotherapy effect. Once the seesaw moves to increase the recovery after cryopreservation, the cryotherapy-effect on the other side would be decreased.

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.

Background and Aims Grafted grapevines are a key component of modern viticulture. Grafting provides not only resistance to soil‐borne pests, but can also improve adaptability to the environment and can improve fruit yield and quality. Graft incompatibility induced by grapevine viruses causes damage to vineyards worldwide. The aim of the present study was to create a rapid and reliable means of evaluating graft incompatibility resulting from grafting virus‐infected scions onto healthy grape rootstocks. Methods and Results Scions of Control (healthy Cabernet Franc), LR131 (Cabernet Franc infected with GLRaV‐1) and LR132 (Cabernet Franc infected with GLRaV‐1 and GVA) were grafted onto different rootstocks by micrografting, green grafting and bench grafting methods. All the micrografts infected with LR132 failed, while LR131 and Control micrografts had similar survival rates. The impact of LR132 was less severe on green grafted plants, followed by bench grafts. Analysis of variance showed an interaction of grafting method × virus variety × rootstock, affecting grafting survival. When grafted onto Freedom and 101‐14, LR131 inhibited the growth of both scion and rootstock, but no significant impact was seen when LR131 was grafted onto St. George and AXR1. Well‐established callus was visible between the surfaces of LR131 and the rootstocks. The virus GLRaV‐1 was detected moving from the LR131 scion source to rootstocks 7 days after micrografting. Callus formation was delayed by LR132 between scion and rootstock surfaces, and a scion‐rootstock vascular connection did not establish, resulting in the death of all micrografts. Conclusions Micrografting is a reliable and sensitive method to screen for virus tolerant graft combinations compared with green and bench grafting. Significance of the Study The results of the present study validate micrografting as a means of producing credible information for selection of virus tolerant rootstocks, and ultimately vineyards with higher rates of compatible grafts.

Shallot (Allium cepa var. aggregatum) is an important vegetable crop belonging to the genus Allium. The present study attempted to develop an efficient droplet-vitrification cryopreservation method for shallot ‘10603’ shoot tips. In vitro stock shoots were maintained on Murashige and Skoog (1962) medium (MS) supplemented with 30 g L-1 sucrose, 0.5 mg L-1 BAP, 0.1 mg L-1 NAA and 8 g L-1 agar (pH=5.8). Shoot tips (2.0-3.0 mm in length) were excised from 4-week-old stock shoots and stepwise precultured with increased sucrose concentrations from 0.3 to 0.5 M, each concentration for 1 day. The precultured shoot tips were then loaded for 20 min with a solution composed of 2 M glycerol and 0.5 M sucrose, before exposure to PVS3 for 3 h at room temperature. Dehydrated shoot tips were transferred onto aluminum foils (2×0.8 cm), prior to direct immersion into liquid nitrogen (LN) for cryostorage. For thawing, frozen aluminum foils were moved from LN and immediately transferred into unloading solution composed of liquid MS containing 1.2 M sucrose. After incubation at room temperature for 20 min, shoot tips were post-cultured on solidified MS medium containing 0.3 M sucrose for 2 days and then transferred onto a recovery medium for shoot regrowth. With this procedure, 94% shoot tips survived, and 58% shoot tips regenerated into shoots following cryopreservation. Cryopreservation of shallot (Allium cepa var. aggregatum) shoot tips by droplet-vitrification

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.

Cylindrical density depressions generated by femtosecond laser pulses filamenting in air for different energy depositions is investigated numerically, by using a set of hydrodynamic equations. The evolution of density profile is calculated for different temperature elevations, the results indicate that the gas density hole is getting shallower and wider with the increasing temperature elevations. A simulation of the propagation inside low-density channel implies a new way to generate a type of bottle beam., Comment: 4 pages,5 figures

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.

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Viral diseases (a biotic stress) and salinity (an abiotic stress) have been/are the two major constraints for sustainable development of the world’s agricultural production including potato. Crops grown in field are often exposed simultaneously to abiotic and biotic stress, and responses of plants to co-stress by two or more factors may differ from those to each of the multiple stresses. Using in vitro cultures, we demonstrated that virus infection (singly and in combination) or salt, and co-stress by virus infection (singly and in combination) and salt significantly reduced growth and microtuber production, and caused severely oxidative cell damage determined by levels of O2·− and methane dicarboxylic aldehyde, and H2O2 localization in situ. Alterations in physiological metabolism by increasing total soluble sugar and free proline, and by decreasing chlorophyll content are responses of potato plantlets to virus infection (singly and in combination) or salt stress and co-stress by virus infection (singly and in combination) and salt. Oxidative cell damage and reduced chlorophyll content caused by virus and/or salt are believed to be responsible for the reduced growth, eventually resulting in decreased tuber yield. Results reported here would help us to better understand possible mechanism of reduced tuber yield by virus infection and/or salt stress.

We report a new cryopreservation method for highbush blueberry (Vaccinium corymbosum) using small leaf squares-bearing adventitious buds. Leaf explants were cultured on adventitious bud regeneration medium composed of Woody Plant Medium (WPM) supplemented with 20 µM zeatin. After 21 days of adventitious bud regeneration, small leaf squares (SLSs, 2 × 3 mm), each bearing multiple adventitious buds, were cut from the leaf explant, precultured on WPM containing 0.3 M sucrose for 24 h and were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 at 0 °C for 40 min. Each of dehydrated SLS was then transferred onto an aluminum foil with small holes and PVS2 was dropped until it covered the SLS, prior to a direct immersion in liquid nitrogen. Cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, more than 23 adventitious buds were produced in each leaf explant, and 100% of SLSs were able to survive and resume shoot regrowth, with more than six shoots per SLS obtained following cryopreservation in three highbush blueberry cultivars. In ‘Misty’, the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones. No polymorphic bands were detected using inter-simple sequence repeat markers and random amplified polymorphic DNA in plantlets of ‘Misty’ recovered from cryopreservation. The use of SLSs-bearing adventitious buds for cryopreservation reported in the present study eliminates shoot tip excision. This cryopreservation method can be considered an efficient cryopreservation of plant shoot tips, and has potential applications to other plant species.

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.

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.

Buds of in vitro-grown shoots of two purple-fleshed potato genotypes were successfully cryopreserved by encapsulation-vitrification (Encap-vitri) and droplet-vitrification (Drop-vitri). Optimal time durations of exposure to PVS2 for shoot regrowth of cryopreserved buds were 5–7 h and 6 h for ‘E03-2677’ and for ‘Blue Congo’, respectively, in Encap-vitri, and 30–50 min and 40 min for the former and the latter, respectively, in Drop-vitri. Higher rates of shoot regrowth were obtained in 1.5–2.0 mm-buds than in 1.0–1.4 mm-ones in Encap-vitri for ‘E03-2677’ and ‘Blue Congo’, while opposite results were found in Drop-vitri. In ‘Blue Congo’, only apical shoot tips survived and developed into shoots, with one shoot produced in one cryopreserved bud in Encap-vitri and Drop-vitri. In ‘E03-2677’, survival and shoot regrowth patterns were similar to those of ‘Blue Congo’ in Encap-vitri. However, both apical and axillary shoot tips survived and developed into two shoots in one bud in Drop-vitri. In ‘E03-2677’, histological observations revealed only apical shoot tips survived following Encap-vitri, while both apical and axillary shoot tips survived following Drop-vitri. Vegetative growth in shoots regenerated from Encap-vitri and Drop-vitri after 3 weeks of post-thaw culture was significantly lower than that from control, but markedly increased after 6 months of post-thaw culture. In both ‘E03-2677’ and ‘Blue Congo’, number of microtubers per shoot, per vessel and ≥3 mm in diameter were significantly greater in shoots regenerated from Encap-vitri than in those from the control. Assessments by ISSR and RAPD of genetic stability did not find any polymorphic bands in regenerants recovered from Encap-vitri and Drop-vitri. To the best of our knowledge, this is the first report on cryopreservation of purple-fleshed potato by vitrification-based procedures. Results reported here would provide valuable basic and technical information on cryopreservation of purple-fleshed potato.

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

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.

Kiwifruit (

In Meixian County of Shaanxi Province, China, during the summer of 2002, mature corn plants in a field plot showed severe leaf spot symptoms. The lesions were narrow (3.5 to 18 mm long and 0.4 to 1.5 mm wide), grayish tan, and surrounded by a light- to dark-pigmented border. Leaves wilted when lesions coalesced. From 2002 to 2005, the disease was observed in other Shaanxi Province counties, including Yangling, Wugong, Qianxian, Longxian, and Qianyang, although in most cases, symptom development was less severe than it was in Meixian. Seven isolates from four counties were obtained by isolation from host tissue on potato dextrose agar (PDA), followed by single-spore culturing and incubation on PDA at 25°C in the dark for 7 days. Conidial suspensions were prepared from a single-spored culture on PDA plates. Pathogenicity tests were performed by spraying five corn seedlings (cv. Yuyu 22) at the three- to four-leaf stage in separate 10-cm-diameter pots with 10 ml of a conidial suspension (106 spores per ml) per plant. Each of three isolates was used in separate inoculations that were performed in different weeks. Controls were sprayed with sterile distilled water only. Plants were covered with plastic bags for 48 h and incubated at 23 to 25°C in a chamber. One week after inoculation, leaves on all inoculated plants developed characteristic lesions, whereas untreated controls had no symptoms. The pathogen was reisolated from diseased leaves on PDA after surface sterilization with 2% NaOCl. On PDA, proliferation of conidia usually occurred on all sides of the conidiophore. Conidiophores were cylindrical, simple, smooth, septate, and straight to flexuous. Conidia were 49 to 89 μm long and 11 to 17 μm wide, with 3 to 10 distosepta, straight or moderately curved, dark or olivaceous brown, and the cells on the ends sometimes appeared paler than those in the middle. These characteristics match those of Bipolaris zeicola (Stout) Shoemaker. On the basis of the arbitrary primers selected by Jones et al. (1), random amplified polymorphic DNA (RAPD) analysis was used for species and physiological race determination. A single DNA fragment approximately 1.2 kb, which is characteristic of B. zeicola, was amplified from all seven isolates with arbitrary primer A20 (5′CTTGGATTC3′). Analysis of PCR products obtained with arbitrary primer A03 (5′AGTCAGCCAC3′) showed that all seven isolates lacked 2,700- and 2,300-base bands, and therefore, sorted into B. zeicola race 3. On the basis of pathogenicity, morphology, and RAPD band patterns of primer A20, the fungus was confirmed as B. zeicola. The shape of leaf lesions and RAPD band patterns using primer A03 showed further that the pathogen was race 3 of B. zeicola. Bai et al. (2) reported race 1 and race 2 of B. zeicola in China, but to our knowledge, this is the first report of race 3 in China. References: (1) M. J. Jones and L. D. Dunkle. Phytopathology 83:366, 1993. (2) J. K. Bai et al. Acta Phytopathol. Sin. 12:61, 1982.

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.

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.

Recent breakthroughs in in vitro culturing of plant cell tissue have helped to overcome the challenges that face important economic and medicinal plant species. Micropropagation and encapsulation techniques have been combined to develop a new tool, known as “synseed,” which has the advantages of both technologies. Synseeds or artificial seeds are alginate encapsulated somatic embryos, vegetative buds, or any other micropropagules that can be used as seeds and germinated into plantlets after propagating under in vitro or in vivo conditions and that can also sustain the regeneration potential after low temperature storage. Encapsulated propagules may be used for germplasm preservation of elite plant species and exchange of plant materials between national and international laboratories. In addition, the technology has been successfully utilized for cryopreservation via encapsulation-dehydration, and encapsulation–vitrification for the germplasm storage of elite plant species. In this paper, we provide updated and comprehensive information on synseed technology, with a particular focus on the importance of explant selection for successful synseed production and on the matrices used as an encapsulation material for synseeds. Furthermore, the limiting factors that hinder the progress of synseed technology and related future perspectives are also discussed.

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.

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from ‘Gala’ apple using shoot tips from of adventitious shoots derived from leaf segments. Leaf segments were excised from in vitro 4-week-old stock shoots and cultured on shoot regeneration medium containing 9.1 µM thidiazuron and 0.25 µM indole-3-butyric acid (IBA) to induce adventitious shoot formation. Shoot tips of different size and different developmental stage were excised from adventitious shoots and cultured on shoot tip culture medium containing 1.1 µM 6-benzyladenine and 0.05 µM IBA. Results showed size and developmental stage of shoot tips excised from adventitious shoots did not influence survival rate, but significantly affected shoot regrowth rate and ASPV-free frequency. Shoot regrowth rates increased from 10 to 15 % in 0.3 mm shoot tips containing two leaf primordia (LP) excised after 2–3 weeks of shoot regeneration, to 53–55 % in those containing three LP excised after 3–4 weeks. The highest shoot regrowth rate (82 %) was obtained in shoot tips of 0.4 mm shoot tips containing four LP excised after 4 weeks. ASPV-free frequencies (95–100 %) were high in 0.2–0.4 mm shoot tips containing two to three LP excised after 2–4 weeks, but low (20 %) in 0.4 mm shoot tips containing four LP excised after 4 weeks. None of the shoots regenerated from the shoot tips were ASGV-free, regardless of the size and developmental stage at which shoot tips were excised. Histological studies and virus localization provided explanations to the varying frequencies of the virus eradication using different size of shoot tips that were excised from adventitious shoots at different developmental stages. The protocol described here was characteristic of high frequency ASPV eradication and high efficiency of production of shoot tips that were used for virus eradication, and thus may have potential applications to virus eradication in other plant species.

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.

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.

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.

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.

Additional file 1: Table S1. Primers used for ASGV detection by RT-PCR, RT-qPCR and gene amplification in ASGV cryopreservation of virus-infected shoots of ‘Gala’ apple the present study. Figure S1. ASGV detection by RT-PCR for ASGV in in vitro stock shoots, and shoots regenerated from two cryopreservation methods and shoot tip culture in ‘Gala’ apple (A) and RT-PCR analysis for EF-1α gene as a reference (B). Figure S2. Gene fragments of ASGV genome used for gene sequencing in cryopreserved virus in in vitro shoots of ‘Gala’ apple. Figure S3. Comparison of gene fragments of A, B and C of ASGV genome preserved between cryopreservation and shoot tip culture.

Background Preservation of plant virus is a fundamental requirement in all types of virus-related research and applied applications. Development of efficient, reliable strategies for long-term preservation of plant virus would largely assist these studies. Results The present study reported a novel biotechnology allowing cryopreservation of Apple stem grooving virus (ASGV) in living shoot tips. Following cryopreservation by droplet-vitrification or encapsulation-dehydration, about 62–67% of shoot regrowth and 100% of ASGV cryopreservation were obtained. Although shoot proliferation and virus concentration were reduced in cryopreserved diseased shoots after 8 weeks of shoot regeneration, continuous subculture for 4 times (16 weeks) increased shoot proliferation and virus concentration to comparative levels as those produced by shoot tip culture (as a control to shoot tip cryopreservation). Cryopreserved ASGV was efficiently transmitted to a woody plant by micrografting and to a herbaceous indicator by mechanical inoculation. Gene sequencing in three fragments of ASGV genome including coat protein and movement protein showed that cryopreserved ASGV shared 99.87% nucleotide identities with shoot tip culture-preserved virus, indicating cryopreserved virus is genetically stable. Conclusions The present study demonstrates ASGV, a representative virus that can infect meristematic cells of shoot tips, can be efficiently cryopreserved in shoot tips. To the best of our knowledge, this is the first report on plant virus cryopreservation in living tissues, and has great potential applications to long-term preservation of plant viruses. Electronic supplementary material The online version of this article (10.1186/s13007-018-0312-9) contains supplementary material, which is available to authorized users.

Field performance evaluation and genetic integrity assessment were conducted in Argyranthemum plants derived from cryopreserved shoot tips. Some variations in root formation and vegetative growth were found in the plants following cryopreservation, but morphologies of the leaves and flowers, and color, number and size of the flowers remained unchanged in the plants recovered from cryopreservation, compared with the control. Assessments of genetic integrity by the two molecular markers: inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands across the plants tested following cryopreservation. These data indicate that cryopreservation reduces, to a certain degree, root formation and vegetative growth, but it does not alter morphologies of leaves and flowers, may not cause any genetic alternations, and has no adverse effects on quantity and quality of the flowers. Therefore, the droplet vitrification cryopreservation can be considered promising for long-term preservation of Argyranthemum germplasm.

Plant regeneration via both shoot regrowth and de novo embryo-like structures was obtained from cryopreserved shoot tips of Lilium spp. Shoot tips (1.0–3.0 mm in length) of Lilium Oriental hybrid ‘Siberia’ containing two- to four-leaf primordia were excised from 4-wk-old adventitious shoots and cryopreserved by droplet vitrification. When post-cultured for 6 wk on recovery medium containing 0.1 mg/l α-naphthaleneacetic acid (NAA) in combination with different kinetin (KT) concentrations (0–0.4 mg/l), three types of regenerants were produced: (1) only embryo-like structures, (2) shoot regrowth with embryo-like structures, and (3) only shoot regrowth. The highest frequencies (≥75.0%) of total embryo-like structures were obtained from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.1–0.2 mg/l KT. Embryo-like structures proliferated within 4 wk of culture on medium containing 1.0 mg/l NAA and 0.05 mg/l thidiazuron (TDZ). Somatic embryo-like structures matured and converted into whole plantlets after 8 wk of culture on Murashige and Skoog (MS) medium without any plant growth regulators. The highest frequencies (≥25.0%) of total shoot regrowth were produced from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.05–0.4 mg/l KT. With the protocols reported here, frequencies of total embryo-like structures and shoot regrowth ranged from 45.0 to 90.0% and 25.0 to 52.5%, respectively, from six lily varieties belonging to five Lilium species or hybrids, representing diverse Lilium germplasm. The results reported here have potential applications to long-term conservation of genetic resources and genetic transformation in Lilium spp.