Sissel Haugslien
Senioringeniør
Forfattere
Ana-Maria Madalina Pantazica Andre van Eerde Mihaela-Olivia Dobrica Iuliana Caras Irina Ionescu Adriana Costache Catalin Tucureanu Hege Særvold Steen Catalin Lazar Inger Heldal Sissel Haugslien Adrian Onu Crina Stavaru Norica Branza-Nichita Jihong Liu ClarkeSammendrag
The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with “humanized” N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of β-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.
Forfattere
Hang Su Andre van Eerde Hege Særvold Steen Inger Heldal Sissel Haugslien Irene Ørpetveit Stefanie Caroline Wüstner Makoto Inami Marie Løvoll Espen Rimstad Jihong Liu ClarkeSammendrag
Cardiomyopathy syndrome (CMS) is a severe cardiac disease occurring in the grow-out sea phase of farmed Atlantic salmon with approximately 100 outbreaks annually in Norway. Piscine myocarditis virus (PMCV) is believed to be the causative agent of CMS. There is no vaccine available to control CMS, partially because PMCV withstands propagation in known cell cultures. In the present study, we selected the putative capsid protein of PMCV as the candidate antigen for immunization experiments and produced it in the plant Nicotiana benthamiana by transient expression. The recombinant PMCV antigen formed virus-like particles (VLPs). To evaluate the efficacy of the plant made VLP vaccine, a PMCV infection model was established. In an experimental salmon vaccination trial, the VLP vaccine triggered innate immunity, and indicative but not significant inhibition of viral replication in heart, spleen and kidney tissues was observed. Similarly, a reduction of inflammatory lesions in cardiomyocytes and subendocardial infiltration by mononuclear leukocytes were observed. Therefore, there was no difference in efficacy or immune response observed post the plant made PMCV VLP antigen vaccination. Taken together, this study has demonstrated that plant made VLP antigens should be investigated further as a possible platform for the development of PMCV antigens for a CMS vaccine.
Forfattere
Mihaela-Olivia Dobrica Andre van Eerde Catalin Tucureanu Adrian Onu Lisa Paruch Iuliana Caras Ene Vlase Hege Særvold Steen Sissel Haugslien Dominic Alonzi Nicole Zitzmann Ralph Bock Jean Dubuisson Costin-Ioan Popescu Crina Stavaru Jihong Liu Clarke Norica Branza-NichitaSammendrag
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Forfattere
Min-Rui Wang Zhibo Hamborg Rune Slimestad Abdelhameed Elameen Dag-Ragnar Blystad Sissel Haugslien Gry Skjeseth Qiao‑Chun WangSammendrag
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.
Forfattere
Min-Rui Wang Zhibo Hamborg Sissel Haugslien Astrid Sivertsen Morten Rasmussen Qiaochun Wang Dag-Ragnar BlystadSammendrag
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.
Forfattere
Min-Rui Wang Zhibo Hamborg Jiří Zámečník Alois Bilavčík Dag-Ragnar Blystad Sissel Haugslien Qiao-Chun WangSammendrag
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.
Forfattere
Mihaela-Olivia Dobrica Catalin Lazar Lisa Paruch Hanne Skomedal Hege Særvold Steen Sissel Haugslien Catalin Tucureanu Iuliana Caras Adrian Onu Sonya Ciulean Alexandru Branzan Jihong Liu Clarke Crina Stavaru Norica Branza-NichitaSammendrag
Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most costeffective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.
Forfattere
Jihong Liu Clarke Lisa Paruch Mihaela-Olivia Dobrica Iuliana Caras Catalin Tucureanu Adrian Onu Sonya Ciulean Crina Stavaru Andre van Eerde Hege Særvold Steen Sissel Haugslien Catalina Petrareanu Catalin Lazar Ioan Popescu Ralph Bock Jean Dubuisson Norica Branza-NichitaSammendrag
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Forfattere
Jihong Liu Clarke Lisa Paruch Mihaela-Olivia Dobrica Iuliana Caras Catalin Tucureanu Adrian Onu Sonya Ciulean Crina Stavaru Andre van Eerde Yanliang Wang Hege Særvold Steen Sissel Haugslien Catalina Petrareanu Catalin Lazar Costin-loan Popescu Ralph Bock Jean Dubuisson Norica Branza-NichitaSammendrag
The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.
Forfattere
Jihong Liu Clarke Andre van Eerde Lisa Paruch Inger Heldal Hege Særvold Steen Yanliang Wang Astrid Sivertsen Sissel HaugslienSammendrag
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Forfattere
Zhibo Hamborg YeonKyeong Lee Carl Jonas Jorge Spetz Jihong Liu Clarke Astrid Sivertsen Gry Skjeseth Sissel Haugslien Qiaochun* Wang Dag-Ragnar BlystadSammendrag
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Forfattere
Zhibo Hamborg YeonKyeong Lee Astrid Sivertsen Gry Skjeseth Sissel Haugslien Jihong Liu Clarke Qiao-Chun Wang Dag-Ragnar BlystadSammendrag
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Forfattere
Jihong Liu Clarke Lisa Paruch Hege Særvold Steen Andre van Eerde Yanliang Wang Astrid Sivertsen Sissel Haugslien Inger HeldalSammendrag
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Sammendrag
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Forfattere
Zhibo Hamborg Gry Skjeseth Abdelhameed Elameen Sissel Haugslien Astrid Sivertsen Jihong Liu Clarke Qiao-Chun Wang Dag-Ragnar BlystadSammendrag
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Jihong Liu Clarke Johanna Gottschamel Hege Særvold Steen Sissel Haugslien Anna-Marja Aura Stephanie Ruf Ralph BockSammendrag
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Jihong Liu Clarke Johanna Gottschamel Hege Særvold Steen Sissel Haugslien Anna-Marja Aura Stephanie Ruf Ralph BockSammendrag
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Dag-Ragnar Blystad Zhibo Hamborg Kari Ørstad Eva Borowski Sissel Haugslien Tor Munthe Åsmund AsdalSammendrag
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Zhibo Hamborg Sissel Haugslien Jihong Liu Clarke Carl Jonas Jorge Spetz Dag-Ragnar Blystad Qing Wang YeonKyeong Lee Astrid H Sivertsen Gry SkjesethSammendrag
Chrysanthemum stunt viroid (CSVd) was first reported in US in the 1940s and is widespread in the world wherever chrysanthemum is grown. Cryotherapy of shoot tips, a new biotechnology developed in the recent years, is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. Existing studies have proven that this technique can efficiently eradicate pathogens such as virus, phytoplasma and bacterium. However, up to now, there has been no report on viroid eradication. In the present study, we attempted to establish a droplet vitrification cryotherapy method for Argyranthemum and to apply it to eradicate CSVd. Results obtained so far demonstrated that cryotherapy of shoot tips alone failed to eradicate CSVd from the infected shoot tips of Argyranthemum maderense ‘Yellow Empire’. Using in situ hybridization of CSVd and histological analysis, we found that CSVd can invade meristematic cells and at the same time, these cells were able to survive following cryotherapy. These findings explained why cryotherapy of shoot tips alone could not be efficient enough to eradicate CSVd from the diseased materials. Further studies combining cold treatment with cryotherapy are under investigation for CSVd eradication.
Forfattere
Yan-liang Wang Sissel Haugslien Marit Almvik Nicholas Clarke Anne Falk Øgaard Jihong Liu ClarkeSammendrag
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Yanliang Wang Sissel Haugslien Marit Almvik Nicholas Clarke Anne Falk Øgaard Jihong Liu ClarkeSammendrag
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Yanliang Wang Sissel Haugslien Marit Almvik Nicholas Clarke Anne Falk Øgaard Jihong Liu ClarkeSammendrag
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Yanliang Wang Sissel Haugslien Marit Almvik Nicholas Clarke Anne Falk Øgaard Jihong Liu ClarkeSammendrag
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Zhibo Zhang Y Lee Sissel Haugslien Astrid H Sivertsen Gry Skjeseth Jihong Liu Clarke Carl Jonas Jorge Spetz Q Wang Dag-Ragnar BlystadSammendrag
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Zhibo Hamborg Sissel Haugslien Y Lee Astrid H Sivertsen Gry Skjeseth Jihong Liu Clarke Carl Jonas Jorge Spetz QC Wang Dag-Ragnar BlystadSammendrag
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Forfattere
Jihong Liu Clarke Johanna Gottschamel Hege Særvold Steen Sissel Haugslien Hanne Skomedal Andreas G. Lössl Stephanie Ruf Ralph BockSammendrag
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Forfattere
Dag-Ragnar Blystad Jihong Liu Clarke Sissel Haugslien Astrid H Sivertsen Gry Skjeseth Zhang Zhibo YeonKyeong Lee John Harald Rønningen Helen Otter Søgnen Peter van der Ende Bjørnar BjellandSammendrag
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Dag-Ragnar Blystad Jihong Liu Clarke Sissel Haugslien Astrid H Sivertsen Gry Skjeseth Zhibo Zhang YeonKyeong Lee John Harald Rønningen Helen Otter Søgnen Peter van der Ende Bjørnar BjellandSammendrag
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Yanliang Wang Sissel Haugslien Marit Almvik Nicholas Clarke Anne Falk Øgaard Jihong Liu ClarkeSammendrag
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Jihong Liu Clarke Gottschamel Johanna Hege Særvold Steen Sissel Haugslien Andreas G. Lössl Stephanie Ruf Ralph BockSammendrag
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Ashraful Islam Henrik Lütken Sissel Haugslien Sissel Torre Jorunn Elisabeth Olsen Dag-Ragnar Blystad Søren K. Rasmussen Jihong Liu ClarkeSammendrag
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M Ashraful Islam Henrik Lütken Sissel Haugslien Sissel Torre Jorunn Elisabeth Olsen Dag-Ragnar Blystad Søren K. Rasmussen Jihong Liu ClarkeSammendrag
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Ashraful Islam Henrik Lütken Sissel Haugslien Hege Særvold Steen Dag-Ragnar Blystad Sissel Torre Jakub Rolcik Søren K. Rasmussen Jorunn Elisabeth Olsen Jihong Liu ClarkeSammendrag
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M Ashraful Islam Henrik Lütken Sissel Haugslien Dag-Ragnar Blystad Sissel Torre Jakub Rolcik Søren Rasmussen Jorunn Elisabeth Olsen Jihong Liu ClarkeSammendrag
Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21–52%) and internode lengths (31–49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11–31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.
Forfattere
Ashraful Islam Sissel Haugslien Dag-Ragnar Blystad Sissel Torre Jorunn Elisabeth Olsen Henrik Lütken Jihong Liu ClarkeSammendrag
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Ashraful Islam Sissel Haugslien Dag-Ragnar Blystad Sissel Torre Jorunn Elisabeth Olsen Henrik Lütken Jihong Liu ClarkeSammendrag
Poinsettia is one of the most important potted ornamentals for Christmas. Appropriate plant height is an important trait in poinsettia production. Concern about the negative effects of chemical growth retardants has limited their availability and use. Generation of compact poinsettia plants by genetic transformation has become a promising approach.
Forfattere
Dag-Ragnar Blystad Jihong Liu Clarke Sissel Haugslien Astrid H Sivertsen Gry Skjeseth Zhang Zhibo John Harald Rønningen Helen Otter Søgnen Peter van der Ende Bjørnar BjellandSammendrag
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M. Ashraful Islam Henrik Lütken Sissel Haugslien Sissel Torre Jorunn Elisabeth Olsen Dag-Ragnar Blystad Søren K. Rasmussen Jihong Liu ClarkeSammendrag
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M. Ashraful Islam Henrik Lütken Sissel Haugslien Sissel Torre Jorunn Elisabeth Olsen Dag-Ragnar Blystad Søren Rasmussen Jihong Liu ClarkeSammendrag
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Carl Jonas Jorge Spetz Jihong Liu Clarke Merete Dees Sissel Haugslien Roar Moe Dag-Ragnar BlystadSammendrag
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Jihong Liu Clarke Carl Jonas Jorge Spetz Sissel Haugslien Merete Dees Roar Moe Dag-Ragnar BlystadSammendrag
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Kirsten Bundgaard Muath K Alsheikh Sissel Haugslien G Adam F. Dale Dag-Ragnar Blystad Carl Jonas Jorge SpetzSammendrag
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Jihong Liu Clarke Carl Jonas Jorge Spetz Sissel Haugslien Merete Dees Roar Moe Dag-Ragnar BlystadSammendrag
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Carl Jonas Jorge Spetz Jihong Liu Clarke Merete Dees Sissel Haugslien Roar Moe Dag-Ragnar BlystadSammendrag
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Dag-Ragnar Blystad Randi Knudsen Carl Jonas Jorge Spetz Sissel Haugslien Kari Ørstad Mariano Cambra Tor MuntheSammendrag
In 1998 Plum pox virus (PPV) wasdetected for the first time in Norway. Virus-like symptoms were observed on several trees in a collection ofplum cultivars at Njøs Research Station in the Sogn og Fjordane County in WestNorway. The Norwegian Food SafetyAuthority and the Norwegian Crop Research Institute immediately startedsurveying other variety collections around the country, nuclear stock materialand orchards in all important plum-growing areas. Since 1998 we have surveyed themain part of the commercial plum orchards in Norway. About 75 000 individualtrees have been tested. About 1 % of the trees have been found infected by PPV.Only the PPV-D strain has been found. It is suspected that the main infectionsource was infected plums or apricots imported to Njøs around 1970 or earlier.In most plum orchards in Norway,the spread of PPV by aphids is relatively slow. Therefore, we expect to be ableto eradicate PPV from commercial plum orchards in the near future.The eradication work iscontinuing.
Forfattere
Dag-Ragnar Blystad jihong liu clarke Sissel Haugslien Merete Wiken Dees Erling Fløistad Shaochen Xing Carl Jonas Jorge SpetzSammendrag
Virus og fytoplasma i julestjerne har vært et forskningsfelt ved Bioforsk Plantehelse siden 1990-tallet. Vi har kombinert dette med ny kunnskap om gen-transformering. I 2009, etter flere års FoU-arbeid, er vi kommet så langt at vi har dyrket virusresistent julestjerne i et mindre forsøk i et gartneri. Nå er det naturlig å summere opp resultater vi har oppnådd og muligheter som åpner seg.
Forfattere
Carl Jonas Jorge Spetz jihong liu clarke Merete Wiken Dees Sissel Haugslien Roar Moe Dag-Ragnar BlystadSammendrag
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Sammendrag
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Forfattere
jihong liu clarke Carl Jonas Jorge Spetz Sissel Haugslien Dag-Ragnar Blystad Merete Wiken Dees Shaochen Xing Roar MoeSammendrag
Abstract Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Poinsettia mosaic virus (PnMV) can cause diseases in modern poinsettia cultivars. PnMV is a single-stranded, positive-sense RNA virus that belongs to the family Tymoviridae. Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season, which in turn decreases the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. There is a need for a new and effective alternative approach, like Agrobacterium-mediated transformation, which can overcome these difficulties. Therefore, we have developed an Agrobacterium-mediated transformation approach for poinsettia for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring three hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75 % of the explants produced somatic embryos. In five experiments utilizing 868 explants, 18 independent transgenic lines were generated. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were detected among transformants. Northern blot analysis confirmed the production of transgene-derived small interfering RNAs (siRNAs). Transgenic lines showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA). The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.
Forfattere
Jihong Liu Clarke Carl Jonas Jorge Spetz Sissel Haugslien Shaochen Xing Merete Dees Roar Moe Dag-Ragnar BlystadSammendrag
Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.
Sammendrag
Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Poinsettia mosaic virus (PnMV) is a RNA virus that belongs to the family Tymoviridae. Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season and decrease the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. There is thus an urgent need for a new approach, like Agrobacterium-mediated transformation, which can overcome these difficulties. We have therefore developed an Agrobacterium-mediated transformation approach for poinsettia. Transgenic poinsettia plants with improved resistance against PnMV by expressing hairpin RNA constructs which targeted various regions of the virus genome were produced. Mechanical inoculation of PnMV and subsequent enzyme-linked immunosorbent assay (ELISA) confirmed the PnMV resistance. The siRNA analysis has demonstrated gene silencing mediated resistance. The PnMV resistant transgenic poinsettia lines produced are in the process of being commercialized. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.
Sammendrag
Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Two viruses i.e. poinsettia mosaic virus (PnMV) and poinsettia cryptic virus (PnCV) can cause diseases in modern poinsettia cultivars. PnMV gives visible symptoms in poinsettia during parts of the growing season. Growers show great interest in the potential benefits of growing PnMV-free poinsettias. Traditionally, PnMV-free poinsettia plants were obtained by in vitro culture of apical meristems. However, this is a time-consuming method and the regenerated new PnMV-free poinsettia has sometimes lost the branching characteristic which is important for poinsettia. We have therefore developed an Agrobacterium-mediated transformation approach for poinsettia. Using this method, we have produced transgenic poinsettia with improved resistance against PnMV by expressing three hairpin (hp) RNA gene constructs which targeted various regions of the virus genome. Molecular analyses have confirmed the stable integration of transgenes into the poinsettia genome. This is the first report describing Agrobacterium-mediated transformation of poinsettia. The PnMV resistant transgenic poinsettia lines produced are of commercial potential. The methodology developed could also facilitate the further improvement of this ornamental plant with the aims of enhancing its disease resistance, quality traits, desirable colour and ornamental value. We have also transformed N. benthemiana to reveal the relationship of different vector constructs and the RNA silencing mediated PnMV resistance. This result will imrpove our understanding of RNA silencing mediated resistance through genetic engineering.
Forfattere
jihong liu clarke Sonja Klemsdal Erling Fløistad A.K. Hvoslef-Eide Sissel Haugslien Roe Moe Dag-Ragnar BlystadSammendrag
Poinsettia, Euphorbia pulcherrima Willd & Klotsch, is an ornamental flower which grows in many countries. Two viruses i.e. poinsettia mosaic virus (PnMV) and poinsettia cryptic virus (PnCV) can cause diseases on modern poinsettia cultivars. To produce virus-resistant poinsettia plants, genetic transformation has been considerated. Since there is no established method available for the transformation of poinsettia, we have chosen an electrophoresis-based transformation method to generate poinsettia transformants. The main advantage of this method is that we can avoid the time-consuming tissue culture and regeneration procedure. To develop a reliable transformation method, the green fluorescent protein (GFP) gene has been used as a reporter gene. Here, we report our preliminary results.
Forfattere
jihong liu clarke Sonja Klemsdal Erling Fløistad A.K. Hvoslef-Eide Sissel Haugslien R Moe Dag-Ragnar BlystadSammendrag
Poinsettia (Euphorbia pulcherrima) is a major ornamental pot plant in many countries. Two viruses, poinsettia mosaic virus (PnMV) and poinsettia cryptic virus, often infect modern poinsettia cultivars. To produce virus-resistant poinsettia plants, genetic engineering has been considered. Since there is no established method available for the transformation of poinsettia, we have chosen an electrophoresisbased transformation method to generate poinsettia transformants. The main advantage of this method is that we can avoid the time-consuming tissue culture and regeneration procedure. To develop a reliable transformation method, the green fluorescent protein (GFP) gene has been used as a reporter gene. Here we report our preliminary results.
Sammendrag
Blomster er både dekorative og viktig i hverdagslivet vårt. Euphorbia pulcherrima Willd. Ex Klotzsch, julestjerne (poinsettia på engelsk) er en av de blomstene som har stor symbolverdi og som dyrkes i mange land i verden (Ecke III et al. 2004). Julestjernen har blitt vår mest populære juleblomst. Det produseres faktisk mellom 5 og 6 millioner julestjerner årlig i Norge (Hagen 2004). Julestjernen framviser store variasjoner i forskjellige deler av verden, fra å være høye og busklignende til kompakte potteblomster med mange grener. Mange patogener og skadegjørere kan angripe julestjerne, blant annet virus. Poinsettia mosaic virus (PnMV) og poinsettia latent virus (PnLV) er to virus som ofte infiserer julestjerne. PnMV forårsaker symptomer på bladene slik at kvaliteten blir redusert i følsomme sorter. Vi ønsker å lage PnMV-resistent julestjerne for å kunne få til en julestjerne av ennå høyere kvalitet. I tillegg ønsker vi å bruke samspillet mellom PnMV og julestjerneplanta som et modellsystem for å bygge opp kompetansen på genmodifisering av planter.
Divisjon for bioteknologi og plantehelse
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