Darling 58 Performance

Darling Performance Results

This page outlines results and conclusions from lab and field trials of the Darling 58 transgenic American chestnut tree (now known to be Darling 54 progeny) conducted by The American Chestnut Foundation (TACF) and various research partners as of December 2023. For more information about the Darling 58 (D58) tree, please visit the Darling 58 page

In these results, trees that have inherited the oxalate oxidase (OxO) gene are referred to OxO positive and denoted as OxO+. Those that did not inherit the gene are referred to as OxO negative (OxO-).

Evidence of poor performance of Darling progeny

1. Variability of blight resistance among Darling progeny that inherited oxalate oxidase

Our partners from the Indiana chapter established field trials of Darling progeny in 2019 under permit from the USDA. In 2022, they inoculated about 150 Darling progeny with the chestnut blight fungus. Approximately half of the progeny inherited OxO and half did not inherit OxO. The progeny that inherited OxO initially had significantly smaller chestnut blight cankers than their siblings that did not inherit OxO; however, a subset of the OxO positive progeny had large, severe cankers similar to the OxO negative trees. Our Indiana collaborators kept the OxO positive trees that had the smallest cankers and cut down the trees that had larger cankers. One year after the inoculations, the cankers on the selections continued to expand on some of the trees. We observed similar phenomenon of expanding cankers on some of the oldest Darling progeny in existence. We hypothesize that delayed canker expansion is due to the expression of the OxO is being suppressed over time in some trees leading to delayed canker expansion. An alternative hypothesis is that there are additional important mechanisms of blight resistance besides oxalate detoxification by OxO. We are following up with studies of how expression of the OxO gene over time corresponds to relative canker expansion.

Variable resistance in Darling 58 progeny at Purdue University.

Variability in canker sizes observed in Darling 58 progeny at Purdue University, July 2023.

2. Growth and survival penalties associated with inheriting OxO among Darling progeny

At multiple stages of trees’ lifetimes and in multiple contexts, we have observed reduced growth and survival of Darling progeny that inherited oxalate oxidase calling into question the competitive ability of these trees in the forest. In Indiana field trials, OxO positive trees were 15% to 25% shorter on average than OxO negative siblings among at age 2 to 3 years. At a testing site in New York, we observed stark visual differences in the heights of OxO positive trees as compared to OxO negative siblings among trees that were at least six years old. At Meadowview Research Farms in 2021, the OxO positive trees exhibited reduced growth and increased leaf injury relative to OxO negative siblings. In a field trial at Virginia Tech, the survival of OxO positive trees at age 5 was much lower (5 OxO positive survivors of 24 planted) that OxO negative full siblings (19 survivors of 24 planted).

Evidence 1

Growth penalties associated with OxO inheritance, Purdue 2023.

Evidence 2

Cross-lab greenhouse trial (VA, PA, NY)2022. Significant growth penalties observed in OxO positive trees. Pooling across 3 replicate sites OxO inheritance predicts seedling height, with OxO positive siblings being significantly shorter than OxO negative siblings.

Evidence 3

Central NY. March 2022. Growth differential shown between OxO- trees and OxO+ trees.

Central NY. Growth differential between OxO- trees (left) and OxO+ trees (right)

Evidence 4

Virginia Tech Kentland BRAG plot, September 2022. Higher mortality rate in OxO positive seedlings. 19 of 24 OxO negative survived vs 5 of 24 OxO positive survived.

TACF staff observed blight cankering on some surviving OxO positive trees at the VT Kentland BRAG plot, September 2022

Evidence 5

Meadowview Research Farms nursery , Meadowview, VA 2023. D58 vs DarWin trial. Darling 58 positive seedlings had considerably lower first season survival than D58 negatives, DarWin positives, and DarWin negatives. All seedlings shared the same mother tree. Survived: 95% D58 neg, 65% D58 pos, 75% DW neg, 93% DW pos. This was a preliminary trial with 160 plants total.

3. Lethality in homozygous state and insertion of OxO into a native chestnut gene

Our partners at the University of New England began intercrossing flowering Darling trees indoors in their high light pollen production room. The expectation is that 25% of the progeny will inherit two copies of OxO, one from each parent, in a homozygous state. The UNE researchers enlisted a geneticist from U. Maine to genetically test the trees for homozygous inheritance. Through the process of testing for homozygosity they learned that the OxO gene was actually located on chromosome 4. That insertion deleted over 1000 base pairs in the SAL1 gene. In a review of over 40 progeny tested from the intercrosses at UNE, these results imply that either constitutive expression of OxO is lethal in a homozygous state or the disruption of a native chestnut gene (SAL1) by OxO insertion is lethal. Further research is required to disentangle these two possibilities.

4. Leaf curling, browning, stunting

Evidence 1

MRF nursery 2021 leaf injury data. Leaf scorch injury was observed on average at 8.4X greater intensity on OxO positive than on OxO negative seedlings. Leaf curl injury was observed on average at 4.1X greater intensity on OxO positive than on OxO negative seedlings. OxO positive seedlings grew 17% shorter in a season than their OxO negative seedlings.

Characteristic suite of symptoms on an OxO+ seedling

Bar graphs of seedling symptoms, data collected July 2021. N = 628 OxO-, 314 OxO+.

Evidence 2

Purdue nursery 2021. OxO positive seedlings showed considerably more leaf margin scorching compared to OxO negative seedlings. Photos from July 2021.

Purdue 2021 OxO+ (OxO positive) seedlings. Photo credit: Caleb Kell.

Purdue 2021 OxO- (OxO negative) seedlings. Photo credit: Caleb Kell.

 Evidence 3

Cross-lab greenhouse trial (VA, IN, NY) 2022 leaf injury data. Pooling across 3 replicate sites, OxO inheritance predicts leaf scorch and leaf curl injuries, with greater leaf scorch and leaf curl injuries in trees that inherit OxO.

Photo of seedlings that match the mean height and leaf injury values at each replicated location, showing decreased height and increased leaf scorch and curl in OxO positive (OxO+) seedlings.

Evidence 4

Greenhouse NY Chapter seedlings, May 2022.

TACF NY Chapter OxO+ seedlings beginning to manifest characteristic leaf injury symptoms, May 2022.