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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 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.

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

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.