Zhibo Hamborg
Research Scientist
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ResearchGateAbstract
Potato (Solanum tuberosum L.) is one of the most important crops grown in Norway, and virus-free plants are required for commercial potato production and for preservation of potato germplasm. The present study evaluates three in vitro therapies – meristem culture, cryotherapy, and chemotherapy combined with thermotherapy – to eliminate viruses from eight historically valuable potato cultivars belonging to the Norwegian potato germplasm. Potato virus Y, potato virus M, potato virus X and potato virus S were present in eight selected old potato cultivars due to long-term conservation in open field. Double-antibody sandwich enzyme-linked immunological assay (DAS-ELISA) and biological indicators were the standard tests used to confirm virus infection in our study. Six virus-free plants from four potato cultivars were obtained after meristem culture, and no virus-free potato cultivars were obtained after cryotherapy. Virus-free frequency for eight different potato cultivars after combining chemotherapy with thermotherapy varied from 36.4% to 100%, with single virus elimination rates of between 74.2% and 92.9%. Chemotherapy compared with thermotherapy was the most effective of the three in vitro therapies used in this study. Highly sensitive small RNA high-throughput sequencing (HTS) was used to evaluate the virus status of potato virus-free materials after virus eradication, and no virus was found, which was consistent with the results of DAS-ELISA and biological indicators. Small RNA HTS has been reported for the first time to evaluate the virus status after virus elimination and to control virus-free potato nuclear stocks.
Abstract
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.
Authors
Zhibo Hamborg YeonKyeong Lee Carl Jonas Jorge Spetz Jihong Liu Clarke Qiaochun Wang Dag-Ragnar BlystadAbstract
No abstract has been registered
Authors
Zhibo Hamborg Ada Konstanse Kristensen Xiaoyan Ma Sissel Haugslien Carl-Henrik Lensjø Alvin Peter van der Ende Øyvor Stensbøl Qiaochun Wang Jana Fránová Dag-Ragnar BlystadAbstract
Background of the study – Cryopreservation is considered to be a valuable method for long-term preservation of plant germplasm and recently it has been shown to be a reliable method for preserving obligate pathogens including plant viruses. Objectives – (1) Droplet-vitrification cryopreservation of strawberry genotypes in Norway; (2) Preservation efficiency of aphid-transmitted strawberry mild yellow edge virus (SMYEV) and strawberry vein banding virus (SVBV) following cryopreservation. Methods – Excised shoot tips of cv. ‘Korona’ were cryopreserved with different durations of PVS2 varying from 10 to 60 min, whereas virus-infected shoot tips were cryopreserved using either 10, 40 or 60 min of PVS2. Results – The results showed that 40–60 minutes of PVS2 treatment was more efficient for preserving strawberry germplasm than lower duration times (10–30 min). Thirty-two strawberry genotypes have been successfully cryopreserved through droplet-vitrification with regeneration rates ranging from 45% to 100% with 40 min PVS2 treatment. Cryopreserved viruses were quantitatively analyzed by Reverse Transcription-quantitative polymerase chain reaction (RT-qPCR). SVBV was successfully cryopreserved in all the regenerated shoots following cryopreservation with all the three durations of PVS2 examined. SMYEV, however, was more efficiently preserved in shoot tips exposed to 40 min (90%) of PVS2, in comparison to 60 min (33%). Conclusion – This demonstrates that SMYEV and SVBV can be successfully cryopreserved in living cells of Fragaria ssp. by droplet vitrification. The results indicate that cryopreservation has great potential for long-time preservation of both strawberry germplasm and aphid-transmitted strawberry-infecting viruses.
Authors
Alois Bilavčík Stacy Denise Hammond Hammond Jana Fránová Igor Koloniuk Zhibo Hamborg Dag-Ragnar Blystad Milos Faltus Jiri ZamecnikAbstract
This study aims to establish a cryopreservation protocol for in vitro grown raspberry (Rubus idaeus L.) without the use of dimethyl sulfoxide (DMSO). The protocol exploits cryotherapy for virus eradication in selected favourable genotypes and will facilitate safe preservation of raspberry genotypes in the Czech national cryobank. Raspberries are highly valuable perennial berries grown in temperate countries including the Czech Republic. Raspberry cultivars and elite breeding lines must be maintained as plant collections either in the field or as in vitro cultures. Both maintenance methods are suboptimal primarily because of the many viral and other pathogens transmitted in raspberry field collections, the industriousness, the maintenance costs, and possible somaclonal variation associated with maintenance of in vitro cultures. For these reasons, cryopreservation is currently considered as a prospective method for the long-term preservation of germplasm. One of the best-performing vitrification procedures for plant cryopreservation is based on DMSO solutions. However, due to the potentially mutagenic effect of DMSO, which is contradictory to the principle of germplasm preservation, we focused on testing a DMSO-free procedure using two raspberry varieties. Regrowth rate of the raspberry variety ‘Tulameen’ after application of Plant Vitrification Solution 3 (PVS3) followed by immersion in liquid nitrogen (LN) was 86%. For the raspberry variety ‘Autumn First’, the regrowth level was 73%. In vitro shoots of the cryopreserved variety ‘Tulameen’ were multiplied in a standard cultivation medium and RT-PCR screened for eradication of Black raspberry necrosis virus (BRNV). The eradication rate of BRNV from in vitro cultures of ‘Tulameen’ by cryopreservation was 94%. The eradication of BRNV from control samples, not subjected to LN, was not significantly different (92%). It can be concluded that the DMSO-free tested method is suitable for efficient cryoconservation. Although the LN phase of the cryoprotocol is not required, the PVS3 protocol alone represents a valuable tool for eradication of BRNV from in vitro cultures of tested raspberry shoots. The suggested protocol will be used for the safe backup of healthy plant material.
Authors
Xiao-Yan Ma Dag-Ragnar Blystad Qiao-Chun Wang Lu Tong Øyvor Stensbøl Dong Zhang Zhibo HamborgAbstract
By optimizing size of shoot tips, preculture medium and exposure duration to PVS2, we established an efficient and wide-spectrum droplet-vitrification cryopreservation for shoot tips of raspberry (Rubus idaeus L.). This protocol yielded 80–100% and 67–100% of survival and shoot regrowth levels in cryopreserved shoot tips across 23 raspberry genotypes. Genetic integrity was assessed in cryo-derived regenerants after 3 months of post-cryopreservation using inter simple sequence repeat (ISSR), single nucleotide polymorphism (SNPs), and insertions and deletions (InDels). ISSR did not detect any polymorphic bands in the cryo-derived regenerants. Although the number of SNPs and InDels decreased in the cryo-derived regenerants, variation trends were similar between the cryo-derived regenerants and the control. Plant vegetative growth and root growth were assessed in the cryo-derived plants after 9 weeks of growth in greenhouse. There were no significant differences in plant vegetative growth measured by plant height, number of fully-opened leaved, leaf area, and fresh and dry weight between the cryo-derived plants and the control, although significant differences were observed in root growth measured by root total length, root average diameter and root volume between the two types of plants. The results obtained in the present study indicate that the droplet-vitrification method has great potential for cryopreservation of raspberry germplasm.