ESF-DAR58-3 - Blight-tolerant Darling 58 American Chestnut | BCH-LMO-SCBD-115759 | Living Modified Organism | Biosafety Clearing-House


Living Modified Organism (LMO)

Decisions on the LMO Risk Assessments  
published: 17 Nov 2020 last updated: 18 Nov 2020
Living Modified Organism identity
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 58 American Chestnut
Darling 58
The Darling 58 American chestnut (Castanea dentata) was modified for blight resistance through the inclusion of  wheat (Triticum aestivum) oxalate oxidase. The enzyme detoxifies oxalic acid produced by the chestnut blight fungus (Cryphonectria parasitica) and allows 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.
Characteristics of the modification process
  • Agrobacterium-mediated DNA transfer
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
  • BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)
  • BCH-GENE-SCBD-115756-1 Oxalate oxidase | Triticum aestivum (Wheat)
    Protein coding sequence | Resistance to diseases and pests (Fungi)
  • BCH-GENE-SCBD-115757-2 Actin 2 terminator | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)
  • BCH-GENE-SCBD-15001-5 Neomycin Phosphotransferase II | Escherichia coli (ECOLX)
    Protein coding sequence | Resistance to antibiotics (Kanamycin)
  • BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)
  • BCH-GENE-SCBD-104802-5 Polyubiquitin10 gene promoter | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)
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.

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.

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.

- qPCR analysis indicated the integration of a single copy for OxO in the American chestnut genome.
- Sequencing of the flanking sequences at the insertion site  indicated the T-DNA was inserted into a non-coding sequence of Chromosome 7 .
- qPCR analysis indicated transcription of OxO.
- Histochemical staining indicated OxO activity from Darling 58 tisues.
LMO characteristics
  • Research
  • Other (Restoration)
Detection method(s)
For DNA (PCR) detection methods, kindly refer to the APHIS submission document.
Additional Information
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).