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Living Modified Organism
(LMO)
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.
Blight-tolerant Darling 54 American Chestnut
EN
Darling 54
Yes
ESF-DAR54-8
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- Organization: State University of New York College of Environmental Science and Forestry (ESF) | BCH-CON-SCBD-115754-1Organization:State University of New York College of Environmental Science and Forestry (ESF)Academic or research institute, American Chestnut Research and Restoration Project1 Forestry DriveSyracuse, New York
12310, United States of AmericaPhone: +1 315-470-6744,Fax:Email: chestnut@esf.edu,Website: 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.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
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BCH-ORGA-SCBD-115755-1 Organism Castanea dentata (American chestnut)Trees
EN
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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) | Resistance to antibiotics (Kanamycin), Resistance to diseases and pests (Fungi), Selectable marker genes and reporter genes
p35S-OxO
EN
- Agrobacterium-mediated DNA transfer
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0.220 kb
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0.839 kb
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1.309 kb
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Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)Promoter
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BCH-GENE-SCBD-115756-1 Oxalate oxidase | Triticum aestivum (Wheat)Protein coding sequence | Resistance to diseases and pests (Fungi)
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BCH-GENE-SCBD-115757-2 Actin 2 terminator | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Terminator
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BCH-GENE-SCBD-15001-5 Neomycin Phosphotransferase II | Escherichia coli (ECOLX)Protein coding sequence | Resistance to antibiotics (Kanamycin)
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BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)Terminator
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BCH-GENE-SCBD-104802-5 Polyubiquitin10 gene promoter | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Promoter
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.
- Research
- Other (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 ]
| Record type | Field | Record(s) | |
|---|---|---|---|
| Living Modified Organism | Related LMO(s) | 1 | |
