| 1. |
- Andersson, Mariette
(författare)
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Colour change in potato (Solanum tuberosum L.) tubers by disruption of the anthocyanin pathway via ribonucleoprotein complex delivery of the CRISPR/Cas9 system
- 2024
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Ingår i: Plant Cell, Tissue and Organ Culture. - 0167-6857 .- 1573-5044. ; 157
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Tidskriftsartikel (refereegranskat)abstract
- Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, 'Desiree' and 'Nansen' from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40oC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle.
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| 2. |
- Andersson, Mariette, et al.
(författare)
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Comparative potato genome editing: Agrobacterium tumefaciens-mediated transformation and protoplasts transfection delivery of CRISPR/Cas9 components directed to StPPO2 gene
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 145, s. 291-305
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Tidskriftsartikel (refereegranskat)abstract
- Key message We compared the StPPO2 gene editing outcomes and efficiencies through Agrobacterium-mediated transformation and protoplasts transfection with DNA or RNPs, and demonstrated that genome editing efficiency depends on the CRISPR/Cas9 delivery approach in potato.Delivery of the CRISPR/Cas9 components to the plant cells is a key step in its application as a genome editing tool. Here, we compared Agrobacterium-mediated transformation and protoplast transfection with CRISPR/Cas9 components for potato genome editing. Two sgRNAs were designed to simultaneously direct Cas9 to the StPPO2 gene encoding for a tuber polyphenol oxidase (PPO). A binary vector (CR-PPO) was utilized for either Agrobacterium-mediated transformation or for transient expression in protoplasts, while ribonucleoprotein complexes (RNP-PPO) were additionally assayed in protoplasts. Editing efficiency varied, yielding 9.6%, 18.4% and 31.9% of edited lines from Agrobacterium-mediated transformation, RNP-PPO and CR-PPO transient expression in protoplasts, respectively. Furthermore, only the CR-PPO transient expression resulted in lines edited in all four StPPO2 alleles, observed in 46% of the edited lines and confirmed by tuber PPO activity and enzymatic browning analysis. Lines with on-target DNA insertions were found from all three approaches and characterized by sequencing. The dual-sgRNA strategy resulted in a low incidence of the targeted deletion, likely due to contrasting efficiencies between sgRNAs, that was partially evident in the in silico analysis. Our results demonstrate that gene editing efficiency in potato depends on the CRISPR/Cas9 delivery strategy and provide insights to consider when selecting the appropriate methodology.
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| 3. |
- Andersson, Mariette, et al.
(författare)
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Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 144, s. 463-467
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Tidskriftsartikel (refereegranskat)abstract
- The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency.Key message RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts.
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| 4. |
- Kianersi, Farzad, et al.
(författare)
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Biosynthesis of rutin changes in Capparis spinosa due to altered expression of its pathway genes under elicitors' supplementation
- 2020
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Ingår i: Plant Cell Tissue and Organ Culture. - : Springer Nature. - 0167-6857 .- 1573-5044. ; 141:3, s. 619-631
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Tidskriftsartikel (refereegranskat)abstract
- Caper plant is (Capparis spinosa L.) a good source of rutin which plays a key role in the human diet. In this study, the effect of different concentrations of salicylic acid (SA) and methyl jasmonate (MeJA) on the weight of anther-derived calli and their rutin contents were assessed in caper plants. Also, we investigated the rutin content and expression pattern of some rutin related genes in leaves of caper plants at vegetative and fresh fruiting growth stages under SA and MeJA treatments. In the first experiment, the highest rutin contents were observed in anther-derived calli treated with 10 mu M MeJA and 100 mg L-1 SA after 2 weeks from initial treatments, which were 2.44 and 2.22-fold higher than control. Also, the treatment of caper plants with150 mu M MeJA and 100 mg L-1 SA resulted in a higher increase in the rutin content of leaves at the fresh fruiting stage (61.46 and 9.99 mg g(-1) DW, respectively), in the second experiment. Among the studied genes, the FLS gene showed the highest expression in the leaves of the MeJA- and SA-treated plants at vegetative growth stage, while in the fresh fruiting stage the highest expression was related to the RT gene. Use of 150 mu M MeJA and 100 mg L-1 SA enhanced the expression levels of the RT gene up to 7.36 and 2.89 times of the control, respectively. These results suggest that rutin content and the expression patterns of rutin biosynthesis genes in caper can be significantly enhanced by the SA and MeJA treatments in a growth stage-dependent manner. Key message Methyl jasmonate and salicylic acid treatments enhance the rutin contents of Capparis spinosa in vitro and in vivo and up-regulate the rutin biosynthetic related genes at two different growth stages.
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| 5. |
- Le, Kim-Cuong
(författare)
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Culture condition optimization and FT-IR analysis of Polygonum multiflorum Thunb. adventitious root cultures grown in an air-lift bioreactor system
- 2021
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Ingår i: Plant Cell, Tissue and Organ Culture. - : Springer Science and Business Media LLC. - 0167-6857 .- 1573-5044. ; 144, s. 371-381
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Tidskriftsartikel (refereegranskat)abstract
- Key message The optimum condition for bioreactor culture of Polygonum multiflorum is full-strength MS medium supplemented with 2 mg center dot L-1 IBA and 5% sucrose after 4 weeks of culture. FT-IR analysis can rapidly discriminate among root samples based on total metabolite equivalence.Bioreactor cultures have been used for biomass production and bioactive compounds accumulation in adventitious root cultures of medicinal plants. In this study, we evaluated the effects of different auxin types [indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA)] and concentrations (0.5, 1.0, 2.0 and 4.0 mg center dot L-1), Murashige and Skoog (MS) medium salt strength (0.25, 0.5, 0.75, 1.0, 1.5 and 2X), and sucrose concentrations (0, 1.5, 3, 5, 7 and 10%) on Polygonum multiflorum Thunb. adventitious root cultures in a 3-L balloon-type bubble bioreactor (BTBB). IBA (1, 2, and 4 mg center dot L-1) was more effective than NAA in promoting root growth. Additionally, low MS salt strength (0.25 and 0.5X MS) increased the accumulation of total phenolics and flavonoids but reduced biomass accumulation. Four weeks of culture in full-strength MS medium supplemented with 2 mg center dot L-1 IBA and 5% sucrose resulted in the highest root biomass [98.46 g center dot L-1 fresh weight (FW); 13.46 g center dot L-1 dry weight (DW)] and bioactive compounds accumulation (total phenolics compounds, 53.08 mg center dot g(-1) DW; total flavonoids, 25.10 mg center dot g(-1) DW). To determine whether metabolic fingerprinting of whole-cell extracts could be used to compare metabolic equivalence of P. multiflorum root samples, we treated adventitious roots with different culture conditions, and analyzed the treated adventitious roots and natural roots by Fourier transform infrared (FT-IR) spectroscopy. The results showed that the metabolic pattern of adventitious root samples was similar under different culture conditions; however, these samples could be discriminated from each other in pilot-scale bioreactors. Overall, our study provides useful information for industrial-scale cultivation of P. multiflorum adventitious roots.
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