| 1. |
- Andersson, M, et al.
(författare)
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A Novel Selection System for Potato Transformation Using a Mutated AHAS Gene.
- 2003
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Ingår i: Plant Cell Reports. - : Springer Science and Business Media LLC. - 1432-203X .- 0721-7714. ; 22:4, s. 261-267
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Tidskriftsartikel (refereegranskat)abstract
- Acetohydroxyacid synthase (AHAS) is the target enzyme for a number of herbicides. A S653N mutation in the AHAS gene results in an increased tolerance to imidazolinone herbicides. We have investigated the use of the mutated gene as selection gene for potato transformation. This resulted in a transformation system with a very high transformation frequency and low rate of escapes. The mutated AHAS gene was introduced into transformed potato together with a -glucuronidase (GUS) gene. Selection on 0.5 M Imazamox yielded GUS expression in 93–100% of regenerated shoots. Furthermore the mutated AHAS gene was used as selection gene for production of high-amylopectin potato lines. The high transformation frequency was verified and potato lines with the desirable starch quality were obtained.
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| 2. |
- Andersson, Mariette, et al.
(författare)
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Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts
- 2017
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Ingår i: Plant Cell Reports. - : Springer Science and Business Media LLC. - 0721-7714 .- 1432-203X. ; 36, s. 117-128
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Tidskriftsartikel (refereegranskat)abstract
- Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts.Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.
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| 3. |
- Borysiuk, Klaudia, et al.
(författare)
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Glyoxalase I activity affects Arabidopsis sensitivity to ammonium nutrition
- 2022
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Ingår i: Plant Cell Reports. - : Springer. - 0721-7714 .- 1432-203X. ; 41, s. 2393-2413
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Tidskriftsartikel (refereegranskat)abstract
- Key message: Elevated methylglyoxal levels contribute to ammonium-induced growth disorders in Arabidopsis thaliana. Methylglyoxal detoxification pathway limitation, mainly the glyoxalase I activity, leads to enhanced sensitivity of plants to ammonium nutrition.Abstract: Ammonium applied to plants as the exclusive source of nitrogen often triggers multiple phenotypic effects, with severe growth inhibition being the most prominent symptom. Glycolytic flux increase, leading to overproduction of its toxic by-product methylglyoxal (MG), is one of the major metabolic consequences of long-term ammonium nutrition. This study aimed to evaluate the influence of MG metabolism on ammonium-dependent growth restriction in Arabidopsis thaliana plants. As the level of MG in plant cells is maintained by the glyoxalase (GLX) system, we analyzed MG-related metabolism in plants with a dysfunctional glyoxalase pathway. We report that MG detoxification, based on glutathione-dependent glyoxalases, is crucial for plants exposed to ammonium nutrition, and its essential role in ammonium sensitivity relays on glyoxalase I (GLXI) activity. Our results indicated that the accumulation of MG-derived advanced glycation end products significantly contributes to the incidence of ammonium toxicity symptoms. Using A. thaliana frostbite1 as a model plant that overcomes growth repression on ammonium, we have shown that its resistance to enhanced MG levels is based on increased GLXI activity and tolerance to elevated MG-derived advanced glycation end-product (MAGE) levels. Furthermore, our results show that glyoxalase pathway activity strongly affects cellular antioxidative systems. Under stress conditions, the disruption of the MG detoxification pathway limits the functioning of antioxidant defense. However, under optimal growth conditions, a defect in the MG detoxification route results in the activation of antioxidative systems.
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| 4. |
- Brodelius, Peter, et al.
(författare)
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Permeabilization of Cultivated Plant Cells by Electroporation for Release of Intracellularly Stored Secondary Products
- 1988
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Ingår i: Plant Cell Reports. - 0721-7714 .- 1432-203X. ; 7:3, s. 186-188
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Tidskriftsartikel (refereegranskat)abstract
- Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.
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| 5. |
- Eriksson, Dennis
(författare)
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Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts
- 2016
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Ingår i: Plant Cell Reports. - : Springer Science and Business Media LLC. - 0721-7714 .- 1432-203X. ; 35, s. 1493-1506
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Tidskriftsartikel (refereegranskat)abstract
- Novel plant genome editing techniques call for an updated legislation regulating the use of plants produced by genetic engineering or genome editing, especially in the European Union. Established more than 25 years ago and based on a clear distinction between transgenic and conventionally bred plants, the current EU Directives fail to accommodate the new continuum between genetic engineering and conventional breeding. Despite the fact that the Directive 2001/18/EC contains both process- and product-related terms, it is commonly interpreted as a strictly process-based legislation. In view of several new emerging techniques which are closer to the conventional breeding than common genetic engineering, we argue that it should be actually interpreted more in relation to the resulting product. A legal guidance on how to define plants produced by exploring novel genome editing techniques in relation to the decade-old legislation is urgently needed, as private companies and public researchers are waiting impatiently with products and projects in the pipeline. We here outline the process in the EU to develop a legislation that properly matches the scientific progress. As the process is facing several hurdles, we also compare with existing frameworks in other countries and discuss ideas for an alternative regulatory system.
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| 6. |
- Hakman, Inger, et al.
(författare)
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An embryogenic cell-suspension culture of Picea glauca (White spruce)
- 1987
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Ingår i: Plant Cell Reports. - 0721-7714 .- 1432-203X. ; 6:1, s. 20-22
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Tidskriftsartikel (refereegranskat)abstract
- A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic cells and large irregular — shaped cells. The culture could be readily re-established on solid medium.
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| 7. |
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| 8. |
- Hanson, Johannes, et al.
(författare)
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The expression pattern of the homeobox gene ATHB13 reveals a conservation of transcriptional regulatory mechanisms between Arabidopsis and hybrid aspen
- 2002
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Ingår i: Plant Cell Reports. - : SPRINGER-VERLAG. - 0721-7714 .- 1432-203X. ; 21:1, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- ATHB13 belongs to a family of homeodomain leucine zipper (HDZip) transcription factors in Arabidopsis thaliana. To understand the temporal and spatial distribution of ATHB13 gene expression, we examined the ATHB13 promoter activity by means of fusions to the uidA (GUS, beta-glucuronidase) reporter gene in transgenic plants. The strongest promoter activity was detected in the vasculature of the basal portion of petioles for both rosette leaves and cotyledons and at the base of cauline leaves. Activity was also detected in the stem at the base of the cauline leaf in an area corresponding to the leaf gap in the vasculature. In flowers, promoter activity was also present in the receptacle and in the stigma. Transformation of the same promoter-GUS construct into hybrid aspen (Populus tremula x P. tremuloides) resulted in an analogous expression pattern in the petioles of leaves. The similarity of these expression patterns indicates that the trans-acting factors responsible for ATHB13 expression are conserved between aspen and Arabidopsis. The conserved expression pattern of the highly specific Arabidopsis ATHB13 promoter in hybrid aspen demonstrates the potential utility of Arabidopsis promoters for tissue-specific expression in angiosperm trees.
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| 9. |
- Ivarson, Emelie, et al.
(författare)
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Significant increase of oleic acid level in the wild species Lepidium campestre through direct gene silencing
- 2016
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Ingår i: Plant Cell Reports. - : Springer Science and Business Media LLC. - 0721-7714 .- 1432-203X. ; 35, s. 2055-2063
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Tidskriftsartikel (refereegranskat)abstract
- Simultaneous RNAi silencing of the FAD2 and FAE1 genes in the wild species Lepidium campestre improved the oil quality with 80 % oleic acid content compared to 11 % in wildtype.Field cress (Lepidium campestre) is a wild biennial species within the Brassicaceae family with desirable agronomic traits, thus being a good candidate for domestication into a new oilseed and catch crop. However, it has agronomic traits that need to be improved before it can become an economically viable species. One of such traits is the seed oil composition, which is not desirable either for food use or for industrial applications. In this study, we have, through metabolic engineering, altered the seed oil composition in field cress into a premium oil for food processing, industrial, or chemical industrial applications. Through seed-specific RNAi silencing of the field cress fatty acid desaturase 2 (FAD2) and fatty acid elongase 1 (FAE1) genes, we have obtained transgenic lines with an oleic acid content increased from 11 % in the wildtype to over 80 %. Moreover, the oxidatively unstable linolenic acid was decreased from 40.4 to 2.6 %, and the unhealthy erucic acid was reduced from 20.3 to 0.1 %. The high oleic acid trait has been kept stable for three generations. This shows the possibility to use field cress as a platform for genetic engineering of oil compositions tailor-made for its end uses.
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| 10. |
- Kanagarajan, Selvaraju, et al.
(författare)
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Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana.
- 2012
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Ingår i: Plant Cell Reports. - : Springer Berlin/Heidelberg. - 0721-7714 .- 1432-203X. ; 31:7, s. 1309-1319
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Tidskriftsartikel (refereegranskat)abstract
- Artemisia annua L. produces a number of sesquiterpene synthases, which catalyze the conversion of farnesyl diphosphate to various sesquiterpenes. The cDNAs encoding amorpha-4,11-diene synthase (ADS), a key enzyme in the artemisinin biosynthesis, and epi-cedrol synthase (ECS), a complex sesquiterpene cyclization syn- thase, were cloned into Cowpea mosaic virus-based viral vector (pEAQ-HT) with Kozak consensus motif and C-terminal histidine tag. The plasmids were transformed into Agrobacterium LBA4404 and, agroinfiltrated into Nicotiana benthamiana leaves along with vector (pJL3:p19) containing Tomato bushy stunt virus post- transcriptional gene silencing suppressor. Quantitative PCR was carried out to measure the transcript levels at 0, 3, 6, 9, 12 and 15 days post-infiltration (dpi). The highest relative expression was observed at 9 dpi for both genes. Transiently expressed recombinant proteins of ADS and ECS were confirmed by SDS-PAGE and western blot. Recombinant proteins were extracted from 9 dpi leaves and purified by immobilized metal ion affinity chroma- tography using histidine tag, which produced yields of 90 and 96 mg kg-1 fresh weight of leaves for ADS and ECS, respectively. Activities of the purified enzymes were assayed using gas chromatography–mass spectrometry for product identification and quantification using valencene as internal standard. The recombinant ADS and ECS con- verted farnesyl diphosphate into amorpha-4,11-diene (97 %) and epi-cedrol (96 %) as the major products, respectively. The purified enzymes exhibited the specific activity of 0.002 and 0.01 mmol min-1 mg-1 protein for ADS and ECS, respectively. The apparent kcat values were 2.1 x 10-3 s-1 and 11 x 10-3 s-1 for ADS and ECS, respectively.
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