Fast-track transformation and genome editing in Brachypodium distachyon
Abstract Background Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distach...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-03-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13007-023-01005-1 |
| _version_ | 1797853816285036544 |
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| author | Camille Soulhat Houssein Wehbi Yannick Fierlej Patrick Berquin Thomas Girin Pierre Hilson Oumaya Bouchabké-Coussa |
| author_facet | Camille Soulhat Houssein Wehbi Yannick Fierlej Patrick Berquin Thomas Girin Pierre Hilson Oumaya Bouchabké-Coussa |
| author_sort | Camille Soulhat |
| collection | DOAJ |
| description | Abstract Background Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. Results We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). Conclusions With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs. |
| first_indexed | 2024-04-09T19:56:55Z |
| format | Article |
| id | doaj.art-d6e4c75bc39c429b8d553bee048b1a61 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-04-09T19:56:55Z |
| publishDate | 2023-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-d6e4c75bc39c429b8d553bee048b1a612023-04-03T05:28:17ZengBMCPlant Methods1746-48112023-03-011911910.1186/s13007-023-01005-1Fast-track transformation and genome editing in Brachypodium distachyonCamille Soulhat0Houssein Wehbi1Yannick Fierlej2Patrick Berquin3Thomas Girin4Pierre Hilson5Oumaya Bouchabké-Coussa6Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB)Abstract Background Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. Results We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). Conclusions With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs.https://doi.org/10.1186/s13007-023-01005-1Brachypodium distachyonGenetic transformationGenome editingCRISPR/Cas9Nitrate reductase (NR) mutantsSomatic embryogenesis |
| spellingShingle | Camille Soulhat Houssein Wehbi Yannick Fierlej Patrick Berquin Thomas Girin Pierre Hilson Oumaya Bouchabké-Coussa Fast-track transformation and genome editing in Brachypodium distachyon Plant Methods Brachypodium distachyon Genetic transformation Genome editing CRISPR/Cas9 Nitrate reductase (NR) mutants Somatic embryogenesis |
| title | Fast-track transformation and genome editing in Brachypodium distachyon |
| title_full | Fast-track transformation and genome editing in Brachypodium distachyon |
| title_fullStr | Fast-track transformation and genome editing in Brachypodium distachyon |
| title_full_unstemmed | Fast-track transformation and genome editing in Brachypodium distachyon |
| title_short | Fast-track transformation and genome editing in Brachypodium distachyon |
| title_sort | fast track transformation and genome editing in brachypodium distachyon |
| topic | Brachypodium distachyon Genetic transformation Genome editing CRISPR/Cas9 Nitrate reductase (NR) mutants Somatic embryogenesis |
| url | https://doi.org/10.1186/s13007-023-01005-1 |
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