GE American Chestnut Performance Failures

Summary of Findings with CBAN’s Preliminary Analysis
December 13, 2023

Introduction

On December 8, 2023, The American Chestnut Foundation (TACF) announced that it was discontinuing its development of its genetically engineered (GE or genetically modified) American chestnut tree called “Darling 58” while reporting “significant performance limitations” of the GE tree and a major “identity error” whereby the TACF has been using the wrong genetically engineered tree in its experiments.

TACF had been heavily promoting the use of the Darling 58 genetically engineered American chestnut tree as a blight tolerant tree for release into the wild, to “restore” this endangered species. However, TACF had little evidence that Darling 58 would perform well as a blight tolerant tree. TACF now reports that Darling 58 is unlikely to work and is “unsuitable as a restoration tree”, and they have withdrawn their support for its regulatory approval.

However, Darling 58 may still be approved for release into the wild by the US Department of Agriculture (USDA). TACF’s research partner SUNY-ESF (State University of New York College of Environmental Science and Forestry) submitted a request for approval to release the Darling 58 (petition for deregulation) on January 17, 2020. A final decision is close and SUNY-ESF has not yet withdrawn its request.

Summary of TACF findings and their implications

“…analysis indicated striking variability in Darling trees’ blight tolerance, significant losses in growth competitiveness, reduction in overall fitness including stunted growth, leaf browning and curling, and increased mortality.” – The American Chestnut Foundation, 2023

The genetically engineered American chestnut tree called “Darling 58” (D58) was genetically engineered by inserting genetic material from five different species including the OxO gene from wheat that codes for an enzyme that can detoxify a blight-associated toxin. Various cooperators were crossing pollen from D58 with non-GE trees in different locations to observe progeny that inherited D58 OxO (OxO+) with siblings that did not (OxO-), to evaluate growth rates, ability to survive blight, etc.

The GE American chestnut trees are not blight resistant, and the reasons are unknown.

Darling 58 progeny that have the OxO gene tend to have less severe cankering than OxO negative siblings, but there is a lot of variation in canker severity even within the OxO positive group. TACF reports that a subset of the OxO positive trees had large, severe cankers. On some of the GE trees that were selected as having smaller cankers, the smaller cankers continued to expand.

TACF does not have an explanation for this lack of blight tolerance but states two hypotheses:
1. That the OxO gene could become suppressed over time (stop working), leading to canker expansion later in the life of some trees or,
2. That oxalate detoxification by OxO is not the only mechanism of resistance or susceptibility to blight.

Implications:

– Researchers do not know how the OxO gene, over time, corresponds to relative canker expansion i.e. researchers do not know how blight tolerance works in the American chestnut:
– The researchers do not know the full functioning of the inserted OxO gene, and they do not fully understand the relationship between the OxO gene, other genes, and blight tolerance in the American chestnut. Disease resistance traits in trees and other plants may be highly complex and interactive, involving multiple genes and regulatory systems.
– The expression (behaviour) of inserted genetic material can change over time. In this case, an American chestnut tree that is successfully genetically engineered to have a measure of blight tolerance sees that tolerance diminish over time. Equally, other possible unexpected effects from the processes of genetically engineering may only become evident over time.

The GE American chestnut trees are significantly shorter and have a much lower survival rate than the non-GE trees.

The GE trees showed a significant reduction in growth and overall fitness.

At only 2 and 3 years of age, the OxO positive trees were 15-25% shorter, with ”stark visual differences” in height observed versus the progeny of D58 crosses that do not have OxO. This is evidence of a growth penalty that accompanies OxO inheritance.

The GE trees were also observed with increased leaf injury (curling, browning and stunting).

Only 5 out of 24 GE trees with the OxO gene were still alive at age 5, compared with 19 out of 24 OxO negative siblings.

Implications:
– Genetic engineering may impact an organism in unexpected ways. A single gene may influence more than one characteristic in an organism. Changing one gene can result in hampered growth or cost the tree important energy to fight disease, for example. In this case, the insertion of the OxO gene appears to hamper the growth of the tree (has a growth penalty), for reasons as yet unknown.

GE trees inheriting OxO from both parents will not survive

That research indicates that it is lethal to the tree when an individual plant inherits the OxO gene from both parents (the homozygous state).
TACF scientists do not know why it will kill the tree but have two hypotheses:
1. The constitutive expression of OxO is lethal in a homozygous state or,
2. The disruption of a native chestnut gene (SAL1) by OxO insertion (Darling 54) is lethal to the tree.

Implications:
– Researchers do not understand the impacts of the OxO gene on the tree.

The tree used in experiments was not the Darling 58 as assumed

Scientists accidentally discovered that researchers were not actually experimenting with Darling 58 as assumed.
Instead, the trees being used in tests were progeny of Darling 54 where the OxO gene is located on a different chromosome. This is significant because the OxO gene of Darling 54 had been inserted into a coding region, causing a deletion (or 1069 base pairs) in a salinity tolerance gene (SAL1).
The use of the wrong tree (wrong GE event) dates back to 2016.

Implications:
– Human error (a “mix-up of pollen”) resulted in a significant mistake that was only discovered once the science had progressed to make this testing more accessible. The research undertaken on Darling 58 was premature because it was not sufficiently supported by the necessary technologies and methodologies.
– Mistakes may remain undiscovered for a long time. In this case, the mistake was not discovered for seven years.
– The problems with the Darling 54 tree warn of the potential unexpected effects of the processes of genetic engineering on organisms. These effects need to be tested for and identified by product developers, and governments need to retain regulatory oversight over these genetically modified organisms (GMOs) to ensure that such testing is conducted.