| 1. |
- Andreasson, Erik, et al.
(författare)
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Utilization of protoplasts to facilitate gene editing in plants: schemes for in vitro shoot regeneration from tissues and protoplasts of potato and rapeseed: implications of bioengineering such as gene editing of broad-leaved plants
- 2022
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Ingår i: Frontiers in Genome Editing. - : Frontiers Media SA. - 2673-3439. ; 4
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Forskningsöversikt (refereegranskat)abstract
- Schemes for efficient regeneration and recovery of shoots from in vitro tissues or single cells, such as protoplasts, are only available for limited numbers of plant species and genotypes and are crucial for establishing gene editing tools on a broader scale in agriculture and plant biology. Growth conditions, including hormone and nutrient composition as well as light regimes in key steps of known regeneration protocols, display significant variations, even between the genotypes within the same species, e.g., potato (Solanum tuberosum). As fresh plant material is a prerequisite for successful shoot regeneration, the plant material often needs to be refreshed for optimizing the growth and physiological state prior to genetic transformation. Utilization of protoplasts has become a more important approach for obtaining transgene-free edited plants by genome editing, CRISPR/Cas9. In this approach, callus formation from protoplasts is induced by one set of hormones, followed by organogenesis, i.e., shoot formation, which is induced by a second set of hormones. The requirements on culture conditions at these key steps vary considerably between the species and genotypes, which often require quantitative adjustments of medium compositions. In this mini-review, we outline the protocols and notes for clonal regeneration and cultivation from single cells, particularly protoplasts in potato and rapeseed. We focus mainly on different hormone treatment schemes and highlight the importance of medium compositions, e.g., sugar, nutrient, and light regimes as well as culture durations at the key regeneration steps. We believe that this review would provide important information and hints for establishing efficient regeneration strategies from other closely related and broad-leaved plant species in general.
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| 2. |
- Li, Xue-Yuan, et al.
(författare)
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Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)
- 2021
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3-4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l(-1)) and 2,4-D (0.5 mg l(-1)) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l(-1) TDZ in combination with auxin 0.5 mg l(-1) NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.
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| 3. |
- Sandgrind, Sjur, et al.
(författare)
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Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
- 2021
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Ingår i: Frontiers in Genome Editing. - : Frontiers Media SA. - 2673-3439. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.
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| 4. |
- Sandgrind, Sjur
(författare)
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Genome editing of oilseed species by CRISPR/Cas9 for trait improvement
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The demand for vegetable oils for food and other applications is rapidly increasing due to a growing population and an urgent need to replace fossil oils. Furthermore, there are several challenges facing agricultural production, such as a shortage of arable land, climate change, and water pollution due to nutrient leaching from extensive use of fertilizers and tillage. More sustainable agricultural systems, better use of side-streams, domestication of novel oil crops, and improvement of existing oil crops are important for global food security and the supply of raw materials for different industries. In Sweden, rapeseed is the main oil crop, but is mainly cultivated in the southern region of Sweden due to its weak cold tolerance. The seed cake is rich in protein, but is underutilized due to high levels of anti-nutritional compounds such as glucosinolates. Field cress (Lepidium campestre) is a novel cold-hardy and high-yielding oilseed crop that can be undersown with a spring cereal in cool climates as a cover crop for reducing nutrient leaching and soil erosion, while producing a valuable seed oil and improving the cereal yield. Development of the CRISPR/Cas9 genome editing system has revolutionized the field of biology and led to major advances in plant science. This novel breeding tool offers great opportunities for accelerated trait improvement of crops with high precision. In this thesis, we have developed efficient protocols for protoplast transfection and regeneration of field cress and rapeseed, and demonstrated their usefulness in genome editing via CRISPR/Cas9 by mutating multiple glucosinolate transporter genes in rapeseed and field cress for reducing seed glucosinolate contents, and by mutating key genes for improving the oil quality of field cress.
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| 5. |
- Sandgrind, Sjur, et al.
(författare)
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Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing
- 2023
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- The wild species field cress (Lepidium campestre) has the potential to become a novel cover and oilseed crop for the Nordic climate. Its seed oil is however currently unsuitable for most food, feed, and industrial applications, due to the high contents of polyunsaturated fatty acids (PUFAs) and erucic acid (C22:1). As the biosynthesis of these undesirable fatty acids is controlled by a few well-known major dominant genes, knockout of these genes using CRISPR/Cas9 would thus be more effective in improving the seed oil quality. In order to increase the level of the desirable oleic acid (C18:1), and reduce the contents of PUFAs and C22:1, we targeted three important genes FATTY ACID ELONGASE1 (FAE1), FATTY ACID DESATURASE2 (FAD2), and REDUCED OLEATE DESATURASE1 (ROD1) using a protoplast-based CRISPR/Cas9 gene knockout system. By knocking out FAE1, we obtained a mutated line with almost no C22:1, but an increase in C18:1 to 30% compared with 13% in the wild type. Knocking out ROD1 resulted in an increase of C18:1 to 23%, and a moderate, but significant, reduction of PUFAs. Knockout of FAD2, in combination with heterozygous FAE1fae1 genotype, resulted in mutated lines with up to 66% C18:1, very low contents of PUFAs, and a significant reduction of C22:1. Our results clearly show the potential of CRISPR/Cas9 for rapid trait improvement of field cress which would speed up its domestication process. The mutated lines produced in this study can be used for further breeding to develop field cress into a viable crop.
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