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Organisme vivant modifié
(LMO)
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Blight-tolerant Darling 54 American Chestnut
EN
Darling 54
Oui
ESF-DAR54-8
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- Organisation : State University of New York College of Environmental Science and Forestry (ESF) | BCH-CON-SCBD-115754-1Organisation :State University of New York College of Environmental Science and Forestry (ESF)Établissement d'enseignement ou de recherche, American Chestnut Research and Restoration Project1 Forestry DriveSyracuse, New York
12310, États-Unis d'AmériqueTéléphone : +1 315-470-6744,Télécopieur :E-mail : chestnut@esf.edu,Site Web : https://www.esf.edu/chestnut/,
The Darling 54 American chestnut (Castanea dentata) was modified for enhanced tolerance to chestnut blight caused by the chestnut blight fungus Cryphonectria parasitica through the expression of wheat (Triticum aestivum) oxalate oxidase (OxO). The OxO enzyme detoxifies oxalic acid produced by the fungus, reducing damage to host tissues and allowing the tree to tolerate infection. However, the tree is not resistant to blight infection. The modified tree additionally contains an Escherichia coli neomycin phosphotransferase II cassette for kanamycin selection during transformation.
Le terme « organisme récepteur » désigne un organisme (non modifié ou déjà modifié) ayant fait l'objet d'une modification génétique, tandis que les « organismes parentaux » désignent les organismes impliqués dans un croisement ou une fusion cellulaire pour générer un organisme modifié.
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BCH-ORGA-SCBD-115755-1 Organisme Castanea dentata (American chestnut)Arbres
FR
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ESF-DAR58-3 - Blight-tolerant Darling 58 American Chestnut| State University of New York College of Environmental Science and Forestry(ESF) | Résistance aux antibiotiques (Kanamycine), Résistance aux maladies et aux parasites (Champignons), Gènes marqueurs et gènes rapporteurs sélectables
p35S-OxO
EN
- Transgénèse par agrobacterium
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Certains de ces éléments génétiques peuvent être présents sous forme de fragments ou sous forme tronquée. Veuillez consulter les notes ci-dessous, s’il y a lieu.
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BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)Promoteur
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BCH-GENE-SCBD-115756-1 Oxalate oxidase | Triticum aestivum (Wheat)Séquence codante de protéines | Résistance aux maladies et aux parasites (Champignons)
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BCH-GENE-SCBD-115757-2 Actin 2 terminator | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Terminateur
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BCH-GENE-SCBD-15001-5 Neomycin Phosphotransferase II | Escherichia coli (ECOLX)Séquence codante de protéines | Résistance aux antibiotiques (Kanamycine)
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BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)Terminateur
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BCH-GENE-SCBD-104802-5 Polyubiquitin10 gene promoter | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Promoteur
Transgene expression:
The p35S-OxO vector contains two gene cassettes: Triticum aestivum oxalate oxidase (OxO) and Escherichia coli neomycin phosphotransferase II (nptII). Both cassettes are in a counterclockwise orientation.
(1) Expression of OxO is under control of the Cauliflower mosaic virus (CaMV) 35S promoter and the Arabidopsis thaliana actin II terminator. The CaMV promoter achieves high levels of transcription.
(2) Expression of nptII is under control of an A. thaliana ubiquitin 10 promoter and the Agrobacterium tumefaciens nopaline synthase terminator. The ubiquitin 10 promoter achieves high levels of expression throughout the tissues of the plant.
Note:
- qPCR analyses indicate that Darling 54 contains a single insertion of the p35S-OxO T-DNA.
The p35S-OxO vector contains two gene cassettes: Triticum aestivum oxalate oxidase (OxO) and Escherichia coli neomycin phosphotransferase II (nptII). Both cassettes are in a counterclockwise orientation.
(1) Expression of OxO is under control of the Cauliflower mosaic virus (CaMV) 35S promoter and the Arabidopsis thaliana actin II terminator. The CaMV promoter achieves high levels of transcription.
(2) Expression of nptII is under control of an A. thaliana ubiquitin 10 promoter and the Agrobacterium tumefaciens nopaline synthase terminator. The ubiquitin 10 promoter achieves high levels of expression throughout the tissues of the plant.
Note:
- qPCR analyses indicate that Darling 54 contains a single insertion of the p35S-OxO T-DNA.
- Sequence analysis identified the insertion on chromosome 4 of the Castanea dentata ‘Ellis’ genome. The insertion interrupts an endogenous gene sequence identified as Caden.04G062600, which is similar to the SAL1 family of genes characterized in other plants.
- Analysis of T1 and T2 offspring did not identify additional insertions of Agrobacterium tumefaciens binary-vector backbone sequences.
- PCR and sequencing confirmed the presence and sequence identity of the OxO coding sequence.
- RT-qPCR demonstrated transcription of OxO, and histochemical and quantitative enzyme assays demonstrated OxO activity.
- The NOS terminator sequence present in the integrated T-DNA is truncated relative to the sequence in the original plasmid vector.
The T-DNA insertion in Darling 54 disrupts the endogenous SAL1-like gene (Caden.04G062600) on chromosome 4. SAL1 homologues have been characterized in other plant species, including Arabidopsis thaliana, where SAL1 is involved in responses to abiotic stresses. The specific function of Caden.04G062600 in Castanea dentata and the effect of the insertion on its expression have not been directly characterized.
- Recherche
- Autres (Restoration)
PCR detection of the OxO transgene can be performed using the LN primers described in the revised petition (forward: CAGACCCTCTCCAGGACT; reverse: GGCAAACTTGGACTTGAG), producing a 586-bp amplicon. Additional qPCR primers for OxO and NPTII are also provided in the petition. These transgene-targeting assays are not event-specific. Sequence information for the Darling 54 insertion site and chromosome 4 flanking regions is provided in Appendix III of the revised petition.
The American chestnut is critically endangered due to the introduction of the chestnut blight fungus (Cryphonectria parasitica (Murr.) Barr) from Asia. The disease was first discovered in the Bronx Zoological Park (New York, USA). The pathogen infects the trees through the stem through wounds or cracks in the bark. Symptoms of the disease include the formation of crankers, mycelial growth in the inner bark and appearance of orange fruiting bodies, which disperse spores by the wind. The fungus can also persist as a saprophyte. In addition to chestnut blight, the wild-type trees are also susceptible to root rot caused by Phytophthora cinnamomi.
The fungus invades the tree by secreting oxalic acid into kill living tissue. Oxalic acid acidifies host tissues to toxic levels, chelates calcium ions from cell wall pectin, suppresses reactive oxygen burst and promotes programmed cell death. The pathogen does not exclusively rely on oxalic acid to infect the tree. Thus, in trees containing a transgene or endogenous OxO gene, the fungal damage is lessened and the lifecycle is more saprophytic (not necrotrophic).
The original petition for the blight-tolerant American chestnut submitted to APHIS in 2020, identified the event as Darling 58. The petition was revised in August 2024 to correct the event name to Darling 54 after analyses determined that offspring from the initial crosses were derived from Darling 54.
The fungus invades the tree by secreting oxalic acid into kill living tissue. Oxalic acid acidifies host tissues to toxic levels, chelates calcium ions from cell wall pectin, suppresses reactive oxygen burst and promotes programmed cell death. The pathogen does not exclusively rely on oxalic acid to infect the tree. Thus, in trees containing a transgene or endogenous OxO gene, the fungal damage is lessened and the lifecycle is more saprophytic (not necrotrophic).
The original petition for the blight-tolerant American chestnut submitted to APHIS in 2020, identified the event as Darling 58. The petition was revised in August 2024 to correct the event name to Darling 54 after analyses determined that offspring from the initial crosses were derived from Darling 54.
- Transgenic American Chestnuts Do Not Inhibit Germination of Native Seeds or Colonization of Mycorrhizal Fungi.pdf [ English ]
- Developing Blight-Tolerant American Chestnut Trees.pdf [ English ]
- Rescue of American chestnut with extraspecific genes following its destruction by a naturalized pathogen.pdf [ English ]
- PETITION NONREGULATED STATUS FOR DARLING 54 CHESTNUT.pdf ( Chestnut, Castanea, Blight-tolerant, Darling 54, OxO+ ) [ English ]
- Two-year field testing of genetically engineered American chestnut reveals fungal blight tolerance [ English ]
- Comments of the American Chestnut Foundation on Draft Environmental Impact Statement.pdf ( Blight-Tolerant, Darling 54, American Chestnut, Castanea, Risk assessment ) [ English ]
- Speed Breeding Transgenic American Chestnut Trees Toward Restoration.pdf ( tree breeding, American chestnut, Castanea, restoration ) [ English ]
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| Organisme vivant modifié | OVM connexe(s) | 1 | |
