The present study described a combining thermotherapy with meristem culture for improved eradication of onion yellow dwarf virus (OYDV) and shallot latent virus (SLV) from co‐infected in ...vitro‐cultured shallot shoots. In vitro‐cultured shoots infected with OYDV and SLV were thermo‐treated at a constant temperature of 36°C for 0, 2 and 4 weeks, and then meristems (0.5 mm) containing 1–2 leaf primordia were excised and cultured for shoot regrowth. Meristem culture without thermotherapy produced much higher levels of survival (100%) and shoot regrowth (55%) than those (62% survival and 32% shoot regrowth) produced by the procedure combining 4 weeks of thermotherapy with meristem culture. However, much higher virus‐free frequencies (70% for OYSV, 80% for SLV and 50% for both viruses) were obtained in the latter than those (10% for OYSV, 15% for SLV and 10% for both viruses) obtained in the former. Histological and subcellular studies showed that thermotherapy imposed stress or damage to the cells of meristems, thus resulting in reduced meristem survival and shoot regrowth. Studies on virus location revealed considerable alternations of virus distribution patterns in the thermo‐treated meristems. The results of histological and subcellular studies and analysis of virus distribution pattern added valuable experimental data in the combining thermotherapy with meristem culture for virus eradication. These data provided explanations as to why combining thermotherapy with meristem culture improved the eradication of OYDV and SLV from the virus‐infected in vitro shallot shoots.
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.
Onion yellow dwarf virus and shallot latent virus were preserved in shallot recovered from shoot tip cryopreservation, while applying thermotherapy before cryopreservation promoted virus eradication.
Since Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic‐diseased Eurasian aspen (Populus ...tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA‐dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot‐associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT‐PCR in mosaic‐diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic‐diseased Eurasian aspen by virus‐specific RT‐PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic‐disease is graft‐transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft‐inoculated seedlings used as rootstocks as well as in the virus‐infected scions used for graft‐inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic‐associated virus.
Five genome segments of a novel virus from mosaic‐diseased Eurasian aspen (Populus tremula) have been completely determined. We could demonstrate that the mosaic‐disease is graft‐transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft‐inoculated seedlings used as rootstocks as well as in the virus‐infected scions used for graft‐inoculation. The virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic‐associated virus (AsMaV).
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.
Key message
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 estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated
spp. is European red raspberry (
L. subsp.
). Often cultivated for its high ...nutritional value, the red raspberry (
) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite,
, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.
High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in ...Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper
was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (
,
,
,
,
, and
), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically,
was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in
after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.
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.
Shallot (
Allium cepa
var.
aggregatum
), a small bulb onion, is widely grown in the world. We previously reported a droplet-vitrification for cryopreservation of in vitro-grown shoot tips of shallot ...genotype ‘10603’. The present study further evaluated rooting, vegetative growth, bulb production and contents of biochemical compounds, as well as genetic stability in cryo-derived plants. The results showed no significant differences in rooting, vegetative growth, bulb production and contents of soluble sugars and flavonols between the cryo- and in vitro-derived plants. Analyses of ISSR and AFLP markers did not detect any polymorphic bands in the cryo-derived plants. These results indicate rooting and vegetative growth ability, biochemical compounds and genetic stability were maintained in cryo-derived plants. The present study provides experimental evidences that support the use of cryopreservation method for long-term preservation of genetic resources of shallots and other
Allium
species.
Key message
Rooting, vegetative growth, bulb production, genetic stability and biochemical compounds were maintained in cryopreserved plants of shallot. Our results support use of cryopreservation for long-term preservation of shallot germplasm.
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.
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