Til dokument

Sammendrag

Weeds are one of the biggest problems that modern agriculture is facing worldwide due to the impact they have on crop productivity. Thus, there is a necessity to develop crop varieties with herbicide resistance or tolerance, which would provide cost-effective tools for helping farmers control weeds in the field. Development of herbicide-tolerant crops was initially based on conventional plant breeding and transgenic technology. In recent years, the emerging genome technologies, including ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeat), provide us a new way for crop improvement through precise manipulation of endogenous genes in the plant genomes. Among these, CRISPR technologies, including nuclease systems, base editors, and prime editors, are really promising in creating novel crop germplasms with herbicide tolerance as they are simple, easy to use, and highly efficient. In this review, we briefly summarize the latest development and breakthroughs of CRISPR technologies in creating herbicide-tolerant crops. Finally, we discuss the future applications of CRISPR technologies in developing herbicide-tolerant crops.

Til dokument

Sammendrag

Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

Til dokument

Sammendrag

Sweet potato (Ipomoea batatas (L.) Lam.) is an important root crop for poor farmers in developing countries. Since the late 1980s, viral diseases have increasingly become a threat to sweet potato production in Ethiopia. This review paper presents the role of sweet potato production for ensuring food security, the level of sweet potato virus research, including the types of viral species identified and their current level of incidences in Ethiopia. Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato virus G (SPVG), and Cucumber mosaic virus (CMV) were reported in Ethiopia, where the first two are the most common and exist at high incidences. In addition, this paper discusses the virus vectors, virus transmission methods to new farms, factors exacerbating the rate of viral incidence and the methods used to reduce the incidences. Moreover, it highlights methods of sweet potato viruses’ detection and cleaning of infected materials in use and the challenges encountered towards the efficient utilization of the methods. Finally, we suggest major intervention techniques that will integrate all key players in managing the impact of the virus on sweet potato production to improve productivity and ensuring food security in Ethiopia. The findings obtained from this review could be an input for the current research on sweet potato improvement (both planting materials and routines) in Ethiopia.

Til dokument

Sammendrag

Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

Til dokument

Sammendrag

Wheat dwarf virus (WDV), a mastrevirus transmitted by the leafhopper Psammotettix alienus, causes a severe disease in cereal crops. Typical symptoms of wheat plants infected by WDV are yellowing and severe dwarfing. In this present study, RNA-Seq was used to perform gene expression analysis in wheat plants in response to WDV infection. Comparative transcriptome analysis indicated that a total of 1042 differentially expressed genes (DEGs) were identified in the comparison between mock and WDV-inoculated wheat plants. Genomes ontology (GO) annotation revealed a number of DEGs associated with different biological processes, such as phytohormone metabolism, photosynthesis, DNA metabolic process, response to biotic stimulus and defense response. Among these, DEGs involved in phytohormone and photosynthesis metabolism and response pathways were further enriched and analyzed, which indicated that hormone biosynthesis, signaling and chloroplast photosynthesis-related genes might play an important role in symptom development after WDV infection. These results illustrate the dynamic nature of the wheat-WDV interaction at the transcriptome level and confirm that symptom development is a complex process, providing a solid foundation to elucidate the pathogenesis of WDV.

Til dokument

Sammendrag

Sweet potato (Ipomoea batatas L. Lam) has become one of the staple crops in Africa in the last 20 years. In Ethiopia, sweet potato is the second most widely grown root crop and is the first regarding the production per hectare. Thus, there is a great demand of planting material throughout the country. Currently, planting material is usually obtained from own previous season harvest, local markets or from the neighboring fields since no certified clean planting material production scheme has been established in Ethiopia yet. Unfortunately, this practice has contributed to the spread of viral diseases throughout the country. Elimination of viruses from infected plants is a tedious job, which requires efficient methods to eliminate the virus and also to verify that the plants are indeed virus-free. In the case of sweet potato, it was observed that heat treatment, combined with meristem tip culture is an efficient method for virus elimination. Previous findings indicate that reverse transcription (RT) PCR is more efficient than ELISA to verify the efficiency of virus elimination. In this study, the use of next generation sequencing (NGS) was explored as a verification method and compared with RT-PCR. The results show that NGS seems to be more efficient than RT-PCR, although also prone to inconclusive results.

Sammendrag

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.

Sammendrag

Det ble sommeren 2017 og 2018 gjennomført et artleggingsprogram for å undersøke om plantemateriale av jordbær som var nylig importert, kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, i 2017 til sammen 150 prøver fra 20 bruk i fylkene Hedmark, Oppland, Vestfold, Buskerud, Agder, Rogaland og Sogn og Fjordane. I 2018 ble det tatt ut 156 prøver fra 14 bruk i fylkene Østfold, Akershus, Møre og Romsdal og Trøndelag. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus, SVBV), jordbær-mildmosaikkvirus (strawberry mottle virus, SMoV), jordbær-bladgulningvirus (strawberry mild yellow edge virus, SMYEV) og jordbær-bladkrøllevirus (strawberry crinkle virus, SCV). Det ble i 2017 påvist virus i prøver fra tre lokaliteter: SCV ble funnet på tre steder, mens SMYEV ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med SMYEV og SCV. Prøvene hadde ikke synlige symptomer. I 2018 ble det blant 156 prøver påvist virus i én prøve – det var SVBV i en prøve av ‘Sonata’ i Akershus. Denne prøven hadde heller ikke synlige symptomer.

Til dokument

Sammendrag

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 Javesella pellucida 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 (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), 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, Nicotiana benthamiana 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 N. benthamiana 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.

Sammendrag

Det ble sommeren 2017 gjennomført et kartleggingsprogram for å undersøke om importert plantemateriale av jordbær kunne være infisert av virus. Det ble tatt ut stikkprøver i jordbærfeltene hos utvalgte bærprodusenter som hadde baser sin produksjon på importerte planter, til sammen 150 prøver. Disse prøvene ble testet for fire bladlusoverførte virus som er relativt vanlige i Europa, men ikke i Norden: jordbær-nervebåndvirus (strawberry vein banding virus), jordbær-mildmosaikkvirus (strawberry mottle virus), jordbær-bladgulningvirus (strawberry mild yellow edge virus) og jordbærbladkrøllevirus (strawberry crinkle virus). Det ble påvist virus i prøver fra tre lokaliteter: strawberry crinkle virus ble funnet på tre steder, mens strawberry mild yellow edge virus ble funnet på ett sted i en prøve som hadde dobbeltinfeksjon med strawberry mild yellow edge virus og strawberry crinkle virus. Prøvene hadde ikke synlige symptomer.

Til dokument

Sammendrag

This study reports the findings of a distinct Potato virus Y (PVY) isolate found in Northeast China. One hundred and ten samples (leaves and tubers) were collected from potato plants showing mosaic symptoms around the city of Harbin in Heilongjiang province of China. The collected tubers were planted and let to grow in a greenhouse. New potato plants generated from these tubers showed similar symptoms, except for one plant. Subsequent serological analyses revealed PVY as the causing agent of the disease. A novel PVY isolate (referred to as HLJ-C-44 in this study) was isolated from this sample showing unique mild mosaic and crisped leaf margin symptoms. The complete genome of this isolate was analyzed and determined. The results showed that HLJ-C-44 is a typical PVY isolate. Phylogenetic analysis indicated that this isolate belongs to the N-Wi strain group of PVY recombinants (PVYN-Wi) and also shared the highest overall sequence identity (nucleotide and amino acid) with other members of this strain group. However, recombination analysis of isolate HLJ-C-44 revealed a recombination pattern that differed from that of other PVYN-Wi isolates. Moreover, biological assays in four different potato cultivars and in Nicotiana tabacum also revealed a different phenotypic response than that of a typical PVYN-Wi isolate. This data, combined, suggest that HLJ-C-44 is a novel PVY recombinant with distinct biological properties.

Sammendrag

Replication of all positive-sense single-stranded RNA viruses occurs in specific structures in close association with cellular membranes. Targeting of the viral replication complex (RC) to the site of replication is mediated by the interaction of viral-encoded proteins and host factors. Electron microscope studies have shown that Poinsettia mosaic virus (PnMV, family Tymoviridae) infection is associated with the presence of vesicular structures in the chloroplasts, which indicates that the replication of PnMV might occur in association with chloroplast-derived membranes. Using computer assisted homology search, we have identified that the coat protein (CP) of PnMV shows similarity to membrane bound proteins and contains a conserved amino acid sequence motif found in members of the Alb3/Oxa1/YidC protein family. This protein family is involved in the insertion of proteins into intracellular membranes. In this study we carried out localization studies combined with confocal laser microscopy to identify the cellular localization of the PnMV CP. Transient expression of red fluorescent protein (RFP)-tagged PnMV CP in Nicotiana benthamiana protoplast was shown to localize in the chloroplast.

Sammendrag

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.

Sammendrag

Bladlusoverført virus er et problem i potet i Norge. Hvert år er det settepotetpartier i den sertifiserte avlen som ikke kan godkjennes fordi innholdet av PVY/PVA er for høye. For høyt virusinnhold i potetprodusentenes egen oppformering er også et problem. For årene 2008, 2009, 2010 og 2011 har det blitt sendt inn virusprøver til NAK (Nederland) fra egne oppformerte settepoteter hos potetdyrkerne. Resultatene viste høye innhold av både PVA og PVY. Dette kan fort gi store avlingstap. I Norge har vi ikke god nok kunnskap om hvilke lusarter som herjer i potetåkre. Med bakgrunn i problemstillingen skissert over ønsket Norsk Landbruksrådgiving at Bioforsk Plantehelse skulle opparbeider seg mer kunnskap om følgende: Hvilke bladlusarter er det i norske potetåkre? Prosjekt: Kartlegging av bladlusarter i potetåkre. Hvor aktive er de i overføring av virus? Hvilken skade gjør disse bladlusartene i potetåkre i Norge? Prosjekt: Forsøk i potetåker med bladlusproblemer. Hvordan kan lus i potet overvåkes, for dermed å sette inn riktig tiltak til rett tid, slik at skader ikke oppstår i avlinga. For eksempel få til en ”indeks” (terskel) som ut fra bladluspopulasjonen angir når riset skal fjernes for å redusere virusmengden i settepotetene. Prosjekt: Vurderes når kartlegging og resultater fra forsøk er ”på plass”

Sammendrag

Det ble våren 2011 igangsatt et overvåkingsprogram for viroider i prydplanter i søtvierfamilien. Det ble analysert i alt 158 prøver av ulike arter og sorter. Prøvene ble først analysert med en PCRmetode som påviser alle viroider i slekten pospiviroider. Hver prøve ble deretter identifisert til art ved hjelp av sekvensering. Det ble påvist viroider i 19 prøver (12 % av prøvene). Potato spindle tuber viroid (PSTVd) ble påvist i 1 prøve av Petunia. Tomato apical stunt viroid (TASVd) ble påvist i til sammen 15 prøver: 1 prøve av Brugmanisa, 11 prøver av Solanum jasminoides og 3 prøver av S. rantonetti. Citrus exocortis viroid (CEVd) ble påvist i 3 prøver av S. jasminoides.

Sammendrag

Tomato chlorotic dwarf viroid ble nylig påvist i et tomatgartneri i Rogaland. Påvisning av viroid som skadegjører i tomat representerer noe nytt for plantehelsesituasjonen i Norge. Til tross for at dette viroidet ikke står på karante¬nelister, har det blitt rapportert om store skader ved angrep i veksthus i andre europeiske land. De siste 10 årene har det blitt klart at mange viroid opptrer latent i prydplanter, og at mange av de samme viroidene kan forårsake sterke symptomer og skade i tomat. De to viktigste spredningsveiene inn til tomatveksthusene er kontaktsmitte fra prydplanter som ikke viser symptomer, og frøsmitte.

Til dokument

Sammendrag

Det er ikke registrert sammendrag

Sammendrag

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

An infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus (PnMV) was generated. It consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kDa. Sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from Germany. RNA transcripts derived from this cDNA were infectious in Nicotiana benthamiana. However, plants did not present typical PnMV symptoms. Furthermore, RNA transcripts from this cDNA clone were not infectious in poinsettia. Serial propagation of this cDNA clone in N. benthamiana plants restored symptom induction in this host but did not re-establish infectivity in poinsettia.

Sammendrag

Vi vil presentere strategier for kontroll og metoder for å unngå spredning av bladlusoverførte virus, med potetvirus Y som eksempel. Vi vil også legge fram de siste resultatene fra prosjekter som er i gang, potetmopptoppvirus (PMTV) prosjektet og prosjektet på rattelvirus (TRV) som er nytt fra i år.

Sammendrag

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus. PMTV is one of the most damaging potato infecting viruses due to the symptoms induced in the tubers. The symptoms caused by PMTV can be similar to the ones induced by another potato infecting virus, Tobacco rattle virus (TRV). Both viruses are known to generate great economic losses in Norway. Since the beginning of 2005, NCRI has as one of its objectives to enhance research on PMTV and will subsequently initiate studies on TRV as well.