Editing American chestnut (section)

===Transgenic blight-resistant American chestnut===
Researchers at the [[State University of New York College of Environmental Science and Forestry]] (SUNY ESF) have developed the [[Darling 58]] chestnut cultivar. This cultivar expresses the gene for [[wheat]] [[oxalate oxidase]] enzyme, which breaks down the [[oxalic acid]] produced by the blight fungus, preventing the death of the tree. When pollen of transgenic fathers fertilizes an ovule of a native mother in the field, those resulting seedlings that express the oxalate oxidase enzyme show growth similar to non-transgenic full siblings, indicating that the [[transgene]] does not impede growth under field conditions.<ref>{{cite journal |last1=Riendeau |first1=Tyler |title=Advancing American Chestnut (Castanea Dentata) Restoration Through Science, GIS And Partnerships |url=https://dune.une.edu/theses/444/ |website=All Theses And Dissertations |date=1 July 2023}}</ref> The modified chestnut does not affect survival, pollen use, or reproduction of [[Bombus impatiens|bumble bees]].<ref>{{cite journal |last1=Newhouse |first1=Andrew E. |last2=Allwine |first2=Anastasia E. |last3=Oakes |first3=Allison D. |last4=Matthews |first4=Dakota F. |last5=McArt |first5=Scott H. |last6=Powell |first6=William A. |title=Bumble bee (Bombus impatiens) survival, pollen usage, and reproduction are not affected by oxalate oxidase at realistic concentrations in American chestnut (Castanea dentata) pollen |journal=Transgenic Research |date=10 June 2021 |volume=30 |issue=6 |pages=751–764 |doi=10.1007/s11248-021-00263-w|pmid=34110572 |pmc=8580921 |doi-access=free }}</ref> A deregulation petition for the Darling 58 variant has been submitted.<ref>[https://www.aphis.usda.gov/brs/aphisdocs/19-309-01p.pdf "Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut"]. https://www.aphis.usda.gov, Retrieved August 28, 2020.</ref><ref>[https://www.federalregister.gov/documents/2020/08/19/2020-18135/state-university-of-new-york-college-of-environmental-science-and-forestry-petition-for "State University of New York College of Environmental Science and Forestry; Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut"]. https://www.federalregister.gov, August 19, 2020. Retrieved August 28, 2020.</ref> If approved, these trees could be the first genetically modified forest trees released in the wild in the United States.<ref>{{cite journal |last1=Thompson |first1=Helen |year=2012 |title=Plant science: The chestnut resurrection |journal=[[Nature (journal)|Nature]] |volume=490 |issue=748|pages=22–23 |doi=10.1038/490022a |pmid=23038446 |bibcode=2012Natur.490...22T |doi-access=free }}</ref><ref>{{cite news|last=Wines|first=Michael|title=Like-Minded Rivals Race to Bring Back an American Icon|url=https://www.nytimes.com/2013/07/14/us/like-minded-rivals-race-to-bring-back-an-american-icon.html?ref=earth&_r=0|date=July 13, 2013|newspaper=New York Times|access-date=July 14, 2013}}</ref>

Unlike American chestnut, Japanese chestnut exhibits resistance to ''Phytophthora cinnamomi'', the fungal pathogen that causes ink disease. The mechanism of resistance of ''C.&nbsp;crenata'' to ''Phytophthora cinnamomi'' may derive from its expression of the Cast_[[ginkbilobin|Gnk2]]-like gene (99.6% identical with {{UniProt|A0A8J4V9V8}}).<ref>{{cite journal |vauthors=Santos C, Nelson CD, Zhebentyayeva T, Machado H, Gomes-Laranjo J, Costa RL |title=First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi |journal=PLOS ONE |volume=12 |issue=9 |pages=e0184381 |date=2017 |pmid=28880954 |pmc=5589223 |doi=10.1371/journal.pone.0184381 |bibcode=2017PLoSO..1284381S |doi-access=free }}</ref> Transgenic modification of ''C.&nbsp;dentata'' with the Cast_Gnk2-like gene may provide a mechanism for developing American chestnut trees resistant to ''Phytophthora cinnamomi''.<ref name=McGuigan20/> Stacking of the Cast_Gnk2-like gene and the oxalate oxidase gene may provide a means of developing genetically modified American chestnut trees resistant to both the chestnut blight and to ink disease.<ref name=McGuigan20>{{cite journal |last1=McGuigan |first1=Linda |last2=Fernandes |first2=Patrícia |last3=Oakes |first3=Allison |last4=Stewart |first4=Kristen |last5=Powell |first5=William |title=Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA Temporary Immersion Bioreactors and We Vitro Containers |journal=Forests |date=2020-11-13 |volume=11 |issue=11 |page=1196 |doi=10.3390/f11111196|doi-access=free }}</ref>

The American Chestnut Research and Restoration Project at SUNY-ESF is not pursuing [[intellectual property]] protection through patents. [[William Powell (biologist)|Dr William Powell]], the project's co-director, states that the decision to not pursue a patent on the project's transgenic lines was to allow the plant to be more accessible for conservationists and members of the public.<ref name="Barnes-Delborne-2021" /> Powell posits that a patent would constrain the spread of the oxalate oxidase transgene into American chestnut populations by limiting the ability to freely plant transgenic trees and cross the trees with surviving American chestnuts or the hybrids produced in the backcross program. Powell states that patents would be a barrier to chestnut restoration and in direct opposition to the program's goals of collaboration.<ref name="Barnes-Delborne-2021" /> While patent protection is not sought, the non-profit [[The American Chestnut Foundation|American Chestnut Foundation]] (TACF) maintains control through a [[Germplasm]] Agreement, ensuring authorized use of chestnut germplasm. The agreement safeguards TACF's rights and aligns with the organization's restoration goals.

A laboratory error resulted in mistaken use of the Darling 54 cultivar instead of the Darling 58 cultivar in some field trials.<ref>{{cite web |last1=Todd |first1=Roxy |title=After GMO program hits snag, what's the future of restoring American chestnuts? |url=https://www.alleghenyfront.org/american-chestnut-blight-genetically-modified/ |website=The Allegheny Front |date=8 March 2024}}</ref> TACF and colleagues have also reported decreased growth rates, and poor heritability of resistance of the Darling 58 cultivar.<ref>{{cite web |title=TACF Chestnut Chat |url=https://www.youtube.com/watch?v=9w_ehgYyxGY |website=YouTube | date=September 15, 2023 |publisher=The American Chestnut Foundation |access-date=8 June 2024}}</ref> In response, the American Chestnut Foundation withdrew its support of development of the Darling 58 cultivar in December 2023.<ref>{{Cite web |last=Brindley |first=Hal |date=2023-12-08 |title=Press Release: TACF Discontinues Development of Darling 58 |url=https://tacf.org/tacf-discontinues-development-of-darling-58/ |access-date=2024-06-05 |website=The American Chestnut Foundation |language=en-US}}</ref> The American Chestnut Research & Restoration Program at SUNY ESF is continuing to pursue deregulation.<ref>{{Cite news |last=Grandoni |first=Dino |date=2023-12-27 |title=Genetic engineering was meant to save chestnut trees. Then there was a mistake. |url=https://www.washingtonpost.com/climate-environment/2023/12/24/chestnut-tree-genetic-engineering-mistake/ |access-date=2024-02-05 |newspaper=Washington Post |language=en-US |issn=0190-8286}}</ref><ref>{{Cite web |title=Progress Update 2023 |url=https://www.esf.edu/chestnut/progress-report/2023.php |access-date=2024-02-05 |website=www.esf.edu |language=en}}</ref><ref>{{Cite web |title=The American Chestnut Research & Restoration Project at ESF |url=https://www.esf.edu/chestnut/index.php |access-date=2024-06-05 |website=www.esf.edu |language=en}}</ref><ref>{{Cite web |last=Brindley |first=Hal |date=2023-12-08 |title=Press Release: TACF Discontinues Development of Darling 58 |url=https://tacf.org/tacf-discontinues-development-of-darling-58/ |access-date=2024-02-05 |website=The American Chestnut Foundation |language=en-US}}</ref><ref>{{Cite web |title=Darling 58 |url=https://tacf.org/darling-58/ |access-date=2024-02-05 |website=The American Chestnut Foundation |language=en-US}}</ref>

In 2022, the SUNY ESF group developed transgenic American chestnut trees incorporating both the oxalate oxidase transgene from wheat and the win3.12 promoter transgene from the [[Populus deltoides|necklace poplar]]. Unlike the CAMV 35S promoter which acts at all times, this poplar promoter drives OxO expression at a low level under basal conditions, but elevates to high levels under conditions of wounding or tissue infection. In laboratory bioassays, win3.12-OxO lines showed elevated disease tolerance similar to that exhibited by blight-resistant Chinese chestnut.<ref>{{cite journal |last1=Carlson |first1=Erik |last2=Stewart |first2=Kristen |last3=Baier |first3=Kathleen |last4=McGuigan |first4=Linda |last5=Culpepper |first5=Tobi |last6=Powell |first6=William |title=Pathogen-induced expression of a blight tolerance transgene in American chestnut |journal=Molecular Plant Pathology |date=March 2022 |volume=23 |issue=3 |pages=370–382 |doi=10.1111/mpp.13165|pmid=34841616 |pmc=8828690 |bibcode=2022MolPP..23..370C }}</ref> Limiting expression of the OxO gene to blight infected tissues is expected to reduce the metabolic cost of gene expression, resulting in increased competitiveness of these new cultivars. As of January 2025, the win3.12 OxO chestnut is still in the research and development phase and has not been deployed for restoration purposes.<ref>{{cite web |title=Darling 58 /54 {{!}} The American Chestnut Foundation |url=https://tacf.org/darling-58/}}</ref>